Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 1 of 165

EXHIBIT 1
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 2 of 165

IN THE UNITED STATES DISTRICT COURT

FOR THE DISTRICT OF COLORADO

Civil Action No. ___________________

A.


ǡ‘…–‘” McCulloughǡǡ ǡ „‡‹‰†—Ž›•™‘”ǡ†‡’‘•‡ƒ†•–ƒ–‡ƒ• ˆ‘ŽŽ‘™•ǣ

ͳǤ ƒ‡–Š‹•ƒˆˆ‹†ƒ˜‹–‹•—’’‘”–‘ˆ–Š‡ƒ„‘˜‡”‡ˆ‡”‡…‡†‘’Žƒ‹–ƒ•‡š’‡”–
–‡•–‹‘›‹•—’’‘”––Š‡”‡‘ˆǤ
ʹǤ Š‡ ‡š’‡”– ‘’‹‹‘• ‡š’”‡••‡† Š‡”‡ ƒ”‡ › ‘™ ƒ† ƒ””‹˜‡† ƒ– ˆ”‘ ›
’‡”•‘•ǡ’”‘ˆ‡••‹‘ƒŽƒ†‡†—…ƒ–‹‘ƒŽ‡š’‡”‹‡…‡•–ƒ‡‹…‘–‡š–ǡ™Š‡”‡ƒ’’”‘’”‹ƒ–‡ǡ„›
•…‹‡–‹ˆ‹…†ƒ–ƒǡ’—„Ž‹…ƒ–‹‘•ǡ–”‡ƒ–‹•‡•ǡ‘’‹‹‘•ǡ†‘…—‡–•ǡ”‡’‘”–•ƒ†‘–Š‡”‹ˆ‘”ƒ–‹‘
”‡Ž‡˜ƒ––‘–Š‡•—„Œ‡…–ƒ––‡”Ǥ

͵Ǥ ƒ…‘’‡–‡––‘–‡•–‹ˆ›–‘–Š‡ˆƒ…–•ƒ†ƒ––‡”••‡–ˆ‘”–ŠŠ‡”‡‹Ǥ–”—‡ƒ†
ƒ……—”ƒ–‡…‘’›‘ˆ› ‹•ƒ––ƒ…Š‡†Š‡”‡–‘ƒ• Ǥ

ͶǤ ˆ–‡” ”‡…‡‹˜‹‰ ƒ „ƒ…Š‡Ž‘”ǯ• †‡‰”‡‡ ˆ”‘ ƒ›Ž‘” ‹˜‡”•‹–›ǡ  …‘’Ž‡–‡† ›
‡†‹…ƒŽ †‡‰”‡‡ ƒ• ƒ Ž’Šƒ ‡‰ƒ Ž’Šƒ ‰”ƒ†—ƒ–‡ ˆ”‘ –Š‡ ‹˜‡”•‹–› ‘ˆ ‡šƒ•
‘—–Š™‡•–‡” ‡†‹…ƒŽ …Š‘‘Ž ‹ ƒŽŽƒ•Ǥ  ™‡– ‘ –‘ …‘’Ž‡–‡ › ‹–‡”ƒŽ ‡†‹…‹‡
”‡•‹†‡…› ƒ– –Š‡ ‹˜‡”•‹–› ‘ˆ ƒ•Š‹‰–‘ ‹ ‡ƒ––Ž‡ǡ ƒ …ƒ”†‹‘Ž‘‰› ˆ‡ŽŽ‘™•Š‹’ ‹…Ž—†‹‰
•‡”˜‹…‡ƒ•Š‹‡ˆ ‡ŽŽ‘™ƒ–‹ŽŽ‹ƒ‡ƒ—‘–‘•’‹–ƒŽǡƒ†ƒƒ•–‡”ǯ•†‡‰”‡‡‹’—„Ž‹…Š‡ƒŽ–Š
ƒ––Š‡‹˜‡”•‹–›‘ˆ‹…Š‹‰ƒǤ

ͷǤ ƒ„‘ƒ”†…‡”–‹ˆ‹‡†‹‹–‡”ƒŽ‡†‹…‹‡ƒ†…ƒ”†‹‘˜ƒ•…—Žƒ”†‹•‡ƒ•‡ƒ†Š‘Ž†
ƒƒ††‹–‹‘ƒŽ…‡”–‹ˆ‹…ƒ–‹‘‹…Ž‹‹…ƒŽŽ‹’‹†‘Ž‘‰›ǡƒ†’”‡˜‹‘—•Ž›‡…Š‘…ƒ”†‹‘‰”ƒ’Š›Ǥ ƒ‘
–Š‡ ƒ…–‹˜‡ ‡†‹…ƒŽ •–ƒˆˆ ƒ– ƒ›Ž‘” ‹˜‡”•‹–› ‡†‹…ƒŽ ‡–‡” ƒ† ƒ›Ž‘” ƒ… ƒ† ƒ‡
ƒ‹Ž–‘‡ƒ”–ƒ†ƒ•…—Žƒ”‘•’‹–ƒŽǡ‹ƒŽŽƒ•ǡ‡šƒ•Ǥ ƒƒŽ•‘‘•–ƒˆˆƒ–ƒ›Ž‘”‡ƒ”–ƒ†
ƒ•…—Žƒ” •–‹–—–‡ǡ ™Š‹…Š ’”‘‘–‡• …ƒ”†‹‘˜ƒ•…—Žƒ” ”‡•‡ƒ”…Š ƒ† ‡†—…ƒ–‹‘Ǥ  ’”ƒ…–‹…‡
‹–‡”ƒŽ‡†‹…‹‡ƒ†…Ž‹‹…ƒŽ…ƒ”†‹‘Ž‘‰›ƒ•™‡ŽŽƒ•–‡ƒ…Šƒ†…‘†—…–”‡•‡ƒ”…Šǡƒ† ƒƒ
ƒ…–‹˜‡•…Š‘Žƒ”‹‡†‹…‹‡™‹–Š”‘Ž‡•ƒ•ƒƒ—–Š‘”ǡ‡†‹–‘”‹ƒŽ‹•–ǡƒ†”‡˜‹‡™‡”ˆ‘”†‘œ‡•‘ˆ
ƒŒ‘” ‡†‹…ƒŽ Œ‘—”ƒŽ• ƒ† –‡š–„‘‘•Ǥ   ƒ ”‘ˆ‡••‘” ‘ˆ ‡†‹…‹‡ ƒ– ‡šƒ• Š”‹•–‹ƒ
‹˜‡”•‹–›ƒ†–Š‡‹˜‡”•‹–›‘ˆ‘”–Š‡šƒ•‡ƒŽ–Š…‹‡…‡•‡–‡”…Š‘‘Ž‘ˆ‡†‹…‹‡Ǥ

͸Ǥ  Šƒ˜‡ Ž‡† …Ž‹‹…ƒŽǡ ‡†—…ƒ–‹‘ǡ ”‡•‡ƒ”…Šǡ ƒ† ’”‘‰”ƒ ‘’‡”ƒ–‹‘• ƒ– ƒŒ‘”
ƒ…ƒ†‡‹… …‡–‡”• ȋ‡”› ‘”† ‘•’‹–ƒŽǡ ƒŽƒ† ‹˜‡”•‹–› ‹ŽŽ‹ƒ ‡ƒ—‘– …Š‘‘Ž ‘ˆ
‡†‹…‹‡Ȍƒ•™‡ŽŽƒ•ƒ…ƒ†‡‹…ƒŽŽ›‘”‹‡–‡†…‘—‹–›Š‡ƒŽ–Š•›•–‡•Ǥ •’‡ƒ”Š‡ƒ†‡†–Š‡
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 3 of 165

…Ž‹‹…ƒŽ †‡˜‡Ž‘’‡– ‘ˆ ƒ–”‹—”‡–‹… ’‡’–‹†‡ ƒ† ‡—–”‘’Š‹Ž ‰‡Žƒ–‹ƒ•‡ ƒ••‘…‹ƒ–‡†

Ž‹’‘…ƒŽ‹ƒ••ƒ›•‹†‹ƒ‰‘•‹•ǡ’”‘‰‘•‹•ǡƒ†ƒƒ‰‡‡–‘ˆŠ‡ƒ”–ƒ†‹†‡›†‹•‡ƒ•‡‘™
—•‡†™‘”Ž†™‹†‡Ǥ ƒŽ•‘Ž‡†–Š‡ˆ‹”•–…Ž‹‹…ƒŽ•–—†›†‡‘•–”ƒ–‹‰–Š‡”‡Žƒ–‹‘•Š‹’„‡–™‡‡
•‡˜‡”‹–›‘ˆƒ…—–‡‹†‡›‹Œ—”›ƒ†‘”–ƒŽ‹–›ƒˆ–‡”›‘…ƒ”†‹ƒŽ‹ˆƒ”…–‹‘Ǥ Šƒ˜‡…‘–”‹„—–‡†
–‘–Š‡—†‡”•–ƒ†‹‰‘ˆ–Š‡‡’‹†‡‹‘Ž‘‰›‘ˆ…Š”‘‹…Š‡ƒ”–ƒ†‹†‡›†‹•‡ƒ•‡–Š”‘—‰Šƒ›
ƒ—•…”‹’–•ˆ”‘–Š‡‹†‡›ƒ”Ž›˜ƒŽ—ƒ–‹‘”‘‰”ƒ—ƒŽƒ–ƒ‡’‘”–’—„Ž‹•Š‡†‹–Š‡
‡”‹…ƒ ‘—”ƒŽ‘ˆ‹†‡›‹•‡ƒ•‡ƒ†’ƒ”–‹…‹’ƒ–‡†‹…Ž‹‹…ƒŽ–”‹ƒŽ†‡•‹‰ƒ†‡š‡…—–‹‘‹
…ƒ”†‹‘”‡ƒŽ ƒ’’Ž‹…ƒ–‹‘• ‘ˆ ƒ…—–‡ ‹†‡›‹Œ—”›ǡ Š›’‡”–‡•‹‘ǡ ƒ…—–‡ …‘”‘ƒ”›•›†”‘‡•ǡ
Š‡ƒ”– ˆƒ‹Ž—”‡ǡ ƒ† …Š”‘‹… …ƒ”†‹‘”‡ƒŽ •›†”‘‡•Ǥ  ’ƒ”–‹…‹’ƒ–‡† ‹ ‡˜‡– ƒ†Œ—†‹…ƒ–‹‘
ȋ‹˜‘Ž˜‡†ƒ––”‹„—–‹‘‘ˆ…ƒ—•‡‘ˆ†‡ƒ–ŠȌ‹–”‹ƒŽ•‘ˆƒ…—–‡…‘”‘ƒ”›•›†”‘‡•ǡ…Š”‘‹…‹†‡›
†‹•‡ƒ•‡ǡ Š‡ƒ”– ˆƒ‹Ž—”‡ǡ ƒ† †ƒ–ƒ •ƒˆ‡–› ƒ† ‘‹–‘”‹‰ ‘ˆ ƒ–‹Ǧ†‹ƒ„‡–‹… ƒ‰‡–•ǡ ”‡ƒŽ
–Š‡”ƒ’‡—–‹…•ǡ Š‡ƒ–‘Ž‘‰› ’”‘†—…–•ǡ ƒ† ‰ƒ•–”‘‹–‡•–‹ƒŽ–”‡ƒ–‡–•Ǥ  Šƒ˜‡ •‡”˜‡† ƒ• –Š‡
…Šƒ‹”ƒ‘”ƒ•ƒ‡„‡”‘ˆ‘˜‡”–™‡–›”ƒ†‘‹œ‡† –”‹ƒŽ•‘ˆ†”—‰•ǡ†‡˜‹…‡•ǡƒ†…Ž‹‹…ƒŽ
•–”ƒ–‡‰‹‡•Ǥ ’‘•‘”• Šƒ˜‡ ‹…Ž—†‡† ’Šƒ”ƒ…‡—–‹…ƒŽ ƒ—ˆƒ…–—”‡”•ǡ „‹‘–‡…Š‘Ž‘‰›
…‘’ƒ‹‡•ǡƒ†–Š‡ƒ–‹‘ƒŽ •–‹–—–‡•‘ˆ‡ƒŽ–ŠǤ

͹Ǥ  ˆ”‡“—‡–Ž› Ž‡…–—”‡ ƒ† ƒ†˜‹•‡ ‘ ‹–‡”ƒŽ ‡†‹…‹‡ǡ ‡’Š”‘Ž‘‰›ǡ ƒ†
…ƒ”†‹‘Ž‘‰›ˆ‘”Ž‡ƒ†‹‰‹•–‹–—–‹‘•™‘”Ž†™‹†‡Ǥ ƒ”‡…‘‰‹œ‡†„››’‡‡”•ˆ‘”›™‘”‘
–Š‡”‘Ž‡‘ˆ…Š”‘‹…‹†‡›†‹•‡ƒ•‡ƒ•ƒ…ƒ”†‹‘˜ƒ•…—Žƒ””‹••–ƒ–‡Ǥ Šƒ˜‡‘˜‡”ͳǡͲͲͲ”‡Žƒ–‡†
•…‹‡–‹ˆ‹…’—„Ž‹…ƒ–‹‘•ǡ‹…Ž—†‹‰–Š‡Dz –‡”ˆƒ…‡„‡–™‡‡‡ƒŽ‹•‡ƒ•‡ƒ†ƒ”†‹‘˜ƒ•…—Žƒ”
ŽŽ‡••dz ‹ ”ƒ—™ƒŽ†ǯ• ‡ƒ”– ‹•‡ƒ•‡ ‡š–„‘‘Ǥ  › ™‘”• Šƒ˜‡ ƒ’’‡ƒ”‡† ‹ –Š‡ ‡™
‰Žƒ† ‘—”ƒŽ‘ˆ‡†‹…‹‡ǡ ‘—”ƒŽ‘ˆ–Š‡‡”‹…ƒ‡†‹…ƒŽ••‘…‹ƒ–‹‘ǡƒ†‘–Š‡”–‘’Ǧ–‹‡”
Œ‘—”ƒŽ•™‘”Ž†™‹†‡Ǥ ƒƒƒ••‘…‹ƒ–‡‡†‹–‘”‘ˆ–Š‡‡”‹…ƒ ‘—”ƒŽ‘ˆƒ”†‹‘Ž‘‰›ƒ†–Š‡
‡”‹…ƒ ‘—”ƒŽ‘ˆ‹†‡›‹•‡ƒ•‡•Ǥ Šƒ˜‡–‡•–‹ˆ‹‡†„‡ˆ‘”‡–Š‡ǤǤ‡ƒ–‡‘‹––‡‡‘
‘‡Žƒ† ‡…—”‹–› ƒ†
‘˜‡”‡–ƒŽ ˆˆƒ‹”•ǡ –Š‡ ǤǤ ‘‘† ƒ† ”—‰ †‹‹•–”ƒ–‹‘
ƒ”†‹‘”‡ƒŽ†˜‹•‘”›ƒ‡Žƒ†‹–•ǤǤ‘‰”‡••‹‘ƒŽ˜‡”•‹‰Š–‘‹––‡‡ǡƒ†–Š‡‡šƒ•
‡ƒ–‡‘‹––‡‡‘‡ƒŽ–Šƒ†—ƒ‡”˜‹…‡•Ǥ

ͺǤ  ƒ ƒ ‡ŽŽ‘™ ‘ˆ –Š‡ ‡”‹…ƒ ‘ŽŽ‡‰‡ ‘ˆ ƒ”†‹‘Ž‘‰›ǡ –Š‡ ‡”‹…ƒ ‡ƒ”–
••‘…‹ƒ–‹‘ǡ–Š‡‡”‹…ƒ‘ŽŽ‡‰‡‘ˆŠ›•‹…‹ƒ•ǡ–Š‡‡”‹…ƒ‘ŽŽ‡‰‡‘ˆŠ‡•–Š›•‹…‹ƒ•ǡ
–Š‡ƒ–‹‘ƒŽ‹’‹†••‘…‹ƒ–‹‘ǡƒ†–Š‡ƒ–‹‘ƒŽ‹†‡› ‘—†ƒ–‹‘Ǥ ƒƒŽ•‘ƒ‹’Ž‘ƒ–‡
‘ˆ–Š‡‡”‹…ƒ‘ƒ”†‘ˆŽ‹‹…ƒŽ‹’‹†‘Ž‘‰›Ǥ

9. ʹͲͳ͵ǡ ™ƒ•Š‘‘”‡†™‹–Š–Š‡ –‡”ƒ–‹‘ƒŽ‹…‡œƒ™ƒ”†ˆ‘””‹–‹…ƒŽƒ”‡
‡’Š”‘Ž‘‰› ˆ‘” › …‘–”‹„—–‹‘ ƒ† †‡†‹…ƒ–‹‘ –‘ –Š‡ ‡‡”‰‹‰ ’”‘„Ž‡ ‘ˆ …ƒ”†‹‘”‡ƒŽ
•›†”‘‡•Ǥ  ƒ –Š‡ ”‡•‹†‡– ‘ˆ –Š‡ ƒ”†‹‘”‡ƒŽ ‘…‹‡–› ‘ˆ ‡”‹…ƒǡ ƒ ‘”‰ƒ‹œƒ–‹‘
†‡†‹…ƒ–‡† –‘ „”‹‰‹‰ –‘‰‡–Š‡” …ƒ”†‹‘Ž‘‰‹•–• ƒ† ‡’Š”‘Ž‘‰‹•–•ǡ ƒ†  ‡‰ƒ‰‡ ‹ ”‡•‡ƒ”…Šǡ
‹’”‘˜‡†“—ƒŽ‹–›‘ˆ…ƒ”‡ǡƒ†…‘—‹–›‘—–”‡ƒ…Š–‘’ƒ–‹‡–•™‹–Š„‘–ŠŠ‡ƒ”–ƒ†‹†‡›
†‹•‡ƒ•‡Ǥ ͳ

10.  ƒ –Š‡ …—””‡– ”‡•‹†‡– ‘ˆ –Š‡ ƒ”†‹‘”‡ƒŽ ‘…‹‡–› ‘ˆ ‡”‹…ƒǡ ƒ
’”‘ˆ‡••‹‘ƒŽ‘”‰ƒ‹œƒ–‹‘†‡†‹…ƒ–‡†–‘ƒ†˜ƒ…‹‰”‡•‡ƒ”…Šƒ†…Ž‹‹…ƒŽ…ƒ”‡ˆ‘”’ƒ–‹‡–•™Š‘
Šƒ˜‡…‘„‹‡†Š‡ƒ”–ƒ†‹†‡›†‹•‡ƒ•‡Ǥ ƒ–Š‡†‹–‘”Ǧ‹ǦŠ‹‡ˆ‘ˆ ǡƒ

1
See http://www.cardiorenalsociety.org/.
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’”‹ƒ”› ”‡•‡ƒ”…Š Œ‘—”ƒŽ Ž‹•–‡† „› –Š‡ ƒ–‹‘ƒŽ ‹„”ƒ”› ‘ˆ ‡†‹…‹‡ ™Š‹…Š ‹• –Š‡ ‘Ž›
’—„Ž‹…ƒ–‹‘™‹–Šƒ’”‹ƒ”›ˆ‘…—•‘”‡•‡ƒ”…Š…‘…‡”‹‰’ƒ–‹‡–•™‹–Š…‘„‹‡†Š‡ƒ”–ƒ†
‹†‡› †‹•‡ƒ•‡Ǥ ‹ƒŽŽ›ǡ  ƒ –Š‡ †‹–‘”Ǧ‹ǦŠ‹‡ˆ ‘ˆ cineǡ ƒ
™‹†‡Ž›”‡ƒ†Œ‘—”ƒŽ–Šƒ–’—„Ž‹•Š‡•”‡˜‹‡™•‘…‘–‡’‘”ƒ”›–‘’‹…•‹…ƒ”†‹‘Ž‘‰›ƒ†‹•ƒŽ•‘
Ž‹•–‡†„›–Š‡ƒ–‹‘ƒŽ‹„”ƒ”›‘ˆ‡†‹…‹‡Ǥ

ͳͳǤ › ƒ’’‡†‡† ˆ—”–Š‡” †‡‘•–”ƒ–‡• › ƒ…ƒ†‡‹… ƒ†

•…‹‡–‹ˆ‹… ƒ…Š‹‡˜‡‡–• ƒ† ’”‘˜‹†‡• ƒ Ž‹•– ‘ˆ ’—„Ž‹…ƒ–‹‘• ƒ—–Š‘”‡† „› ‡ ‘˜‡” –Š‡ ’ƒ•–
–Š‹”–››‡ƒ”•Ǥ

ͳʹǤ ‹…‡–Š‡‘—–•‡–‘ˆ–Š‡’ƒ†‡‹…ǡ Šƒ˜‡„‡‡ƒŽ‡ƒ†‡”‹–Š‡‡†‹…ƒŽ”‡•’‘•‡
–‘ –Š‡  Ǧͳͻ †‹•ƒ•–‡” ƒ† Šƒ˜‡’—„Ž‹•Š‡†Dzƒ–Š‘’Š›•‹‘Ž‘‰‹…ƒŽ ƒ•‹• ƒ† ƒ–‹‘ƒŽ‡ ˆ‘”
ƒ”Ž› —–’ƒ–‹‡– ”‡ƒ–‡– ‘ˆ Ǧ‘Ǧʹ ȋ ǦͳͻȌ ˆ‡…–‹‘ǡdz –Š‡ ˆ‹”•– •›–Š‡•‹• ‘ˆ
•‡“—‡…‡† —Ž–‹†”—‰ –”‡ƒ–‡– ‘ˆ ƒ„—Žƒ–‘”› ’ƒ–‹‡–• ‹ˆ‡…–‡† ™‹–Š Ǧ‘Ǧʹ ‹ –Š‡
ƒ† —’†ƒ–‡† ‹ Ǥ ʹ Šƒ˜‡
ˆ‘”–›Ǧ•‡˜‡ ’‡‡”Ǧ”‡˜‹‡™‡† ’—„Ž‹…ƒ–‹‘• ‘ –Š‡  Ǧͳͻ ‹ˆ‡…–‹‘ …‹–‡† ‹ –Š‡ ƒ–‹‘ƒŽ
‹„”ƒ”› ‘ˆ ‡†‹…‹‡Ǥ Š”‘—‰Š ƒ ™‹†‘™ –‘ ’—„Ž‹… ’‘Ž‹…›ƒ‡”•ǡ  Šƒ˜‡ …‘–”‹„—–‡†
‡š–‡•‹˜‡Ž›‘‹••—‡••—””‘—†‹‰–Š‡ Ǧͳͻ…”‹•‹•‹ƒ•‡”‹‡•‘ˆǯ•ˆ‘” 
–‡•–‹ˆ‹‡†‘–Š‡Ǧ‘Ǧʹ‘—–„”‡ƒ‹ –Š‡ǤǤ‡ƒ–‡‘‹––‡‡ ‘‘‡Žƒ† ‡…—”‹–›
ƒ†
‘˜‡”‡–ƒŽˆˆƒ‹”•‘ ‘˜‡„‡”ͳͻǡʹͲʹͲǤ  –‡•–‹ˆ‹‡†‘Ž‡••‘•Ž‡ƒ”‡†ˆ”‘ –Š‡
’ƒ†‡‹…”‡•’‘•‡‹–Š‡‡šƒ•‡ƒ–‡‘‹––‡‡‘‡ƒŽ–Šƒ†—ƒ‡”˜‹…‡•‘ƒ”…Š
ͳͲǡʹͲʹͳǡƒ†‘‡ƒ”Ž›–”‡ƒ–‡–‘ˆ Ǧͳͻˆ‘”–Š‡‘Ž‘”ƒ†‘
‡‡”ƒŽ••‡„Ž›‘ƒ”…Š
͵ͳǡʹͲʹͳǤ††‹–‹‘ƒŽŽ›ǡ –‡•–‹ˆ‹‡†‹–Š‡‡™ƒ’•Š‹”‡‡ƒ–‡‘Ž‡‰‹•Žƒ–‹‘…‘…‡”‹‰–Š‡
‹˜‡•–‹‰ƒ–‹‘ƒŽ  Ǧͳͻ ˜ƒ……‹‡ ‘ ’”‹Ž ͳͶǡ ʹͲʹͲǤ › ‡š’‡”–‹•‡ ‘ –Š‡ Ǧ‘Ǧʹ
‹ˆ‡…–‹‘ƒ† Ǧͳͻ•›†”‘‡ǡŽ‹‡–Šƒ–‘ˆ‹ˆ‡…–‹‘—•†‹•‡ƒ•‡•’‡…‹ƒŽ‹•–•ǡ‹•ƒ’’”‘š‹ƒ–‡Ž›
‡‹‰Š–‡‡‘–Š•‘Ž†Ǥ Šƒ˜‡ˆ‘”‡†›‘’‹‹‘•„ƒ•‡†—’‘›†‹”‡…–…Ž‹‹…ƒŽ‡š’‡”‹‡…‡
™‹–Šƒ…—–‡ƒ†…‘˜ƒŽ‡•…‡– Ǧͳͻ…ƒ•‡•ƒ•™‡ŽŽƒ•‘…Ž‘•‡Ž›ˆ‘ŽŽ‘™‹‰–Š‡’”‡’”‹–ƒ†
’—„Ž‹•Š‡†Ž‹–‡”ƒ–—”‡‘–Š‡‘—–„”‡ƒǤ Šƒ˜‡ƒ††‹–‹‘ƒŽŽ›ǡ•’‡…‹ˆ‹…ƒŽŽ›”‡˜‹‡™‡†ƒŽŽ‘ˆ–Š‡‡›
’—„Ž‹•Š‡†”ƒ”‡…ƒ•‡•ƒ†”‡’‘”–•…‘…‡”‹‰’‘••‹„Ž‡”‡…—””‡…‡‘ˆǦ‘ǦʹǤ


Opinion

2
McCullough PA, Kelly RJ, Ruocco G, Lerma E, Tumlin J, Wheelan KR, Katz N, Lepor NE, Vijay K, Carter H,
Singh B, McCullough SP, Bhambi BK, Palazzuoli A, De Ferrari GM, Milligan GP, Safder T, Tecson KM, Wang DD,
McKinnon JE, O'Neill WW, Zervos M, Risch HA. Pathophysiological Basis and Rationale for Early Outpatient
Treatment of SARS-CoV-2 (COVID-19) Infection. Am J Med. 2021 Jan;134(1):16-22. doi:
10.1016/j.amjmed.2020.07.003. Epub 2020 Aug 7. PMID: 32771461; PMCID: PMC7410805 available at
https://pubmed.ncbi.nlm.nih.gov/32771461/; McCullough PA, Alexander PE, Armstrong R, Arvinte C, Bain AF,
Bartlett RP, Berkowitz RL, Berry AC, Borody TJ, Brewer JH, Brufsky AM, Clarke T, Derwand R, Eck A, Eck J,
Eisner RA, Fareed GC, Farella A, Fonseca SNS, Geyer CE Jr, Gonnering RS, Graves KE, Gross KBV, Hazan S, Held
KS, Hight HT, Immanuel S, Jacobs MM, Ladapo JA, Lee LH, Littell J, Lozano I, Mangat HS, Marble B, McKinnon
JE, Merritt LD, Orient JM, Oskoui R, Pompan DC, Procter BC, Prodromos C, Rajter JC, Rajter JJ, Ram CVS, Rios
SS, Risch HA, Robb MJA, Rutherford M, Scholz M, Singleton MM, Tumlin JA, Tyson BM, Urso RG, Victory K,
Vliet EL, Wax CM, Wolkoff AG, Wooll V, Zelenko V. Multifaceted highly targeted sequential multidrug treatment
of early ambulatory high-risk SARS-CoV-2 infection (COVID-19). Rev Cardiovasc Med. 2020 Dec 30;21(4):517-
530. doi: 10.31083/j.rcm.2020.04.264. PMID: 33387997 available at https://pubmed.ncbi.nlm.nih.gov/33387997/.
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ͳ͵Ǥ Šƒ˜‡”‡˜‹‡™‡†–Š‡‘’Žƒ‹–ˆ‘”‡…Žƒ”ƒ–‘”› —†‰‡– ™Š‹…Š†‡Ž‹‡ƒ–‡•–Š‡

•—„Œ‡…– ƒ––‡” ”‡Žƒ–‹‰ –‘ ’”‘’‘•‡† ƒ…–• „› –Š‡ ‹–‡† –ƒ–‡• ‡’ƒ”–‡– ‘ˆ ‡ˆ‡•‡ –‘
…‘’‡Ž ˜‡•–‹‰ƒ–‹˜‡‡™”—‰•—†‡”–Š‡‡”‰‡…›•‡—–Š‘”‹œƒ–‹‘–‘–Š‡‡„‡”•‘ˆ
–Š‡”‡†ˆ‘”…‡•–Šƒ–Šƒ˜‡…‘–”ƒ…–‡†ƒ†”‡…‘˜‡”‡†ˆ”‘–Š‡‘˜‹†Ǧͳͻ˜‹”—•Ǥ

ͳͶǤ  ƒ …‘’‡–‡– –‘ ‘’‹‡ ‘ –Š‡ ‡†‹…ƒŽ ƒ•’‡…–• ‘ˆ –Š‡•‡ ƒŽŽ‡‰ƒ–‹‘• „ƒ•‡†
—’‘›ƒ„‘˜‡Ǧ”‡ˆ‡”‡…‡†‡†—…ƒ–‹‘ƒ†’”‘ˆ‡••‹‘ƒŽ‡†‹…ƒŽ‡š’‡”‹‡…‡ƒ†–Š‡„ƒ•‹•‘ˆ
›‘’‹‹‘•ƒ”‡ˆ‘”‡†ƒ•ƒ”‡•—Ž–‘ˆ›‡†—…ƒ–‹‘ƒ†‡š’‡”‹‡…‡Ǥ

ͳͷǤ • ƒ ‡†‹…ƒŽ ‘…–‘” ƒ† •…‹‡–‹•– ‹ –Š‡ „‹‘Ž‘‰‹…ƒŽ Š‡ƒŽ–Š ƒ† –”‡ƒ–‡– ‘ˆ
Š—ƒ„‡‹‰•ǡ …‘ˆ‹”ƒ†ƒ––‡•––Šƒ–ǣ

ƒȌ –Š‡ˆƒ–ƒŽ‹–›”ƒ–‡‘ˆ–Š‡‘˜‹†Ǧͳͻ˜‹”—•ǡ‘”ƒ›˜ƒ”‹ƒ–‹‘–Š‡”‡‘ˆǡ‹…Ž—†‹‰–Š‡•‘
…ƒŽŽ‡†Dz‡Ž–ƒdz‘”Dzƒ„†ƒdzȋŠ‡”‡‹ƒˆ–‡”Dz‹”—•dzȌ‹•ǤͲͲͶ͸Ψƒ…”‘••…‘„‹‡†ƒ‰‡
‰”‘—’•‹–Š‡‹–‡†–ƒ–‡•Ǣ

„Ȍ –Š‡ ˆƒ–ƒŽ‹–› ”ƒ–‡ ƒ••‘…‹ƒ–‡† ™‹–Š –Š‡ ‹”—• †‘‡• ‘– ‡‡– –Š‡ †‡ˆ‹‹–‹‘ ‘ˆ ƒ
Dzƒ†‡‹…dzƒ•†‡…Žƒ”‡†„›–Š‡‡ˆ‡†ƒ–•‘”Ž†‡ƒŽ–Š”‰ƒ‹œƒ–‹‘ǡ‡–‡”•ˆ‘”
‹•‡ƒ•‡ ‘–”‘Ž ƒ† ”‡˜‡–‹‘ǡ Š‡ ƒ–‹‘ƒŽ •–‹–—–‡• ‘ˆ‡ƒŽ–Š ‘” ƒ› ‘–Š‡”
‰‘˜‡”‡–ƒŽ ‘” ‘Ǧ‰‘˜‡”‡–ƒŽ ƒ‰‡…› ‘” ‡–‹–› ƒ…–‹‰ ™‹–Š ƒ—–Š‘”‹–› –‘
ƒ‡•—…Šƒ†‡…Žƒ”ƒ–‹‘Ǣ

…Ȍ –Š‡”‡ƒ”‡™‹†‡Ž›ƒ˜ƒ‹Žƒ„Ž‡‡–Š‘†•–‘”‡†—…‡–Š‡”‹•‘ˆ‹ˆ‡…–‹‘™‹–ŠǦ‘Ǧ
ʹ‹…Ž—†‹‰ƒ–‹Ǧ‹ˆ‡…–‹˜‡‘”ƒŽƒ†ƒ•ƒŽ•’”ƒ›•ƒ†™ƒ•Š‡•ǡ‘”ƒŽ‡†‹…ƒ–‹‘•ǡ ƒ†
‘—–’ƒ–‹‡–‘‘…Ž‘ƒŽƒ–‹„‘†‹‡•ǡ™Š‹…Šƒ”‡Dzƒ’’”‘˜‡†dz†”—‰•„›–Š‡ ‘‘†ƒ†
”—‰ †‹‹•–”ƒ–‹‘ ƒ† Š‹‰ŠŽ› ‡ˆˆ‡…–‹˜‡ ‹ ’”‡˜‡–‹‰ ƒ† –”‡ƒ–‹‰ –Š‡ ‹”—•Ǣ
ȋ”‡ˆ‡”‡…‡•ǣ Š––’•ǣȀȀ™™™ǤƒŒ‡†Ǥ…‘Ȁƒ”–‹…Ž‡ȀͲͲͲʹǦͻ͵Ͷ͵ȋʹͲȌ͵Ͳ͸͹͵Ǧ
ʹȀˆ—ŽŽ–‡š–Ǣ Ƭ Š––’•ǣȀȀ”…Ǥ‹”’”‡••Ǥ…‘ȀȀͳͲǤ͵ͳͲͺ͵ȀŒǤ”…ǤʹͲʹͲǤͲͶǤʹ͸Ͷǡ Ƭ
Š––’•ǣȀȀ†‘‹Ǥ‘”‰ȀͳͲǤʹ͵ͻͷͺȀ‹Œ‹”•Ȁ˜‘ŽͲ͸Ǧ‹Ͳ͵ȀͳͳͲͲǢ

†Ȍ ˆ–Š‘•‡’‡‘’Ž‡–Šƒ–Šƒ˜‡ƒŽ”‡ƒ†›…‘–”ƒ…–‡†ƒ†”‡…‘˜‡”‡†ˆ”‘–Š‡‹”—•–Š‡”‡
‹• ‘ „‡––‡” ’”‘–‡…–‹‘ ƒ‰ƒ‹•– ”‡‹ˆ‡…–‹‘ǡ ‹…Ž—†‹‰ –Š‡ ˆ‘”‡‰‘‹‰ ƒ‡†
–Š‡”ƒ’‡—–‹…•Ǥ

‡Ȍ ˆƒ…–ǡ‘”‡–Šƒ–Š‹”–›’‡”…‡–ȋΪ͵ͲΨȌ‘ˆ–Š‡‰Ž‘„ƒŽ’‘’—Žƒ–‹‘Šƒ˜‡ƒŽ”‡ƒ†›Šƒ†
ƒ† ”‡…‘˜‡”‡† ˆ”‘ –Š‡ ‹”—• †—‡ –‘ ‹–• Š‹‰ŠŽ› …‘–ƒ‰‹‘—• ƒ† ‡š–”‡‡Ž› Ž‘™
‘”–ƒŽ‹–›”ƒ–‡Ǥ †‡‡†ǡ–Š‡‹”—•ƒ•ƒ‹•‘Žƒ–‡†‹ˆ‡…–‹‘‹••—„•–ƒ–‹ƒŽŽ›Ž‡••ˆƒ–ƒŽ
–Šƒƒ››‡ƒ”ǯ•‘”ƒŽˆŽ—‘”‘”‘ƒ‹”—•Ǥ

ˆȌ —Ž–‹’Ž‡•–—†‹‡•ǡ‹…Ž—†‹‰„—–‘–Ž‹‹–‡†–‘ǣDz —‘Ž‘‰‹…ƒŽ‡‘”›–‘Ǧ
‘Ǧʹƒ••‡••‡†ˆ‘”‰”‡ƒ–‡”–Šƒ•‹š ‘–Š•ƒˆ–‡”‹ˆ‡…–‹‘Ǣdz “Ǧ‘Ǧʹ
‹ˆ‡…–‹‘‹†—…‡•Ž‘‰ǦŽ‹˜‡†„‘‡ƒ””‘™’Žƒ•ƒ…‡ŽŽ•‹Š—ƒ•ǢdzDzš’‘•—”‡–‘
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EXHIBIT 2
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June 5, 2021. Page
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It is made available under a CC-BY-NC-ND 4.0 International license .

Nabin K. Shrestha,1 Patrick C. Burke,2 Amy S. Nowacki,3 Paul Terpeluk,4 Steven M. Gordon1

From the Departments of 1Infectious Diseases, 2Infection Prevention, 3Quantitative Health Sciences, and
4
Occupational Health, Cleveland Clinic, Cleveland, Ohio.

Keywords: SARS-CoV-2; COVID-19; Incidence; Vaccines; Immunity;

Running Title: COVID-19 vaccination if already infected

Corresponding author:

Nabin K. Shrestha, MD, MPH

9500 Euclid Avenue / G-21

Cleveland, OH 44195

Phone: 216-636-1873 / Fax: 216-445-9446 / Email: shrestn@ccf.org

Summary: Cumulative incidence of COVID-19 was examined among 52238 employees in an American

healthcare system. COVID-19 did not occur in anyone over the five months of the study among 2579

individuals previously infected with COVID-19, including 1359 who did not take the vaccine.
vaccine

1
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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ABSTRACT

Background. The purpose of this study was to evaluate the necessity of COVID-19 vaccination in

persons previously infected with SARS-CoV-2.

Methods. Employees of the Cleveland Clinic Health System working in Ohio on Dec 16, 2020, the

day COVID-19 vaccination was started, were included. Any subject who tested positive for SARS-CoV-2

at least 42 days earlier was considered previously infected. One was considered vaccinated 14 days after

receipt of the second dose of a SARS-CoV-2 mRNA vaccine. The cumulative incidence of SARS-CoV-2

infection over the next five months, among previously infected subjects who received the vaccine, was

compared with those of previously infected subjects who remained unvaccinated, previously uninfected

subjects who received the vaccine, and previously uninfected subjects who remained unvaccinated.

Results. Among the 52238 included employees, 1359 (53%) of 2579 previously infected subjects

remained unvaccinated, compared with 22777 (41%) of 49659 not previously infected. The cumulative

incidence of SARS-CoV-2 infection remained almost zero among previously infected unvaccinated

subjects, previously infected subjects who were vaccinated, and previously uninfected subjects who were

vaccinated, compared with a steady increase in cumulative incidence among previously uninfected

subjects who remained unvaccinated. Not one of the 1359 previously infected subjects who remained

unvaccinated had a SARS-CoV-2 infection over the duration of the study

study. In a Cox proportional hazards

regression model, after adjusting for the phase of the epidemic, vaccination was associated with a

significantly lower risk of SARS-CoV-2 infection among those not previously infected (HR 0.031, 95%

CI 0.015 to 0.061) but not among those previously infected (HR 0.313, 95% CI 0 to Infinity).

Conclusions. Individuals who have had SARS-CoV-2 infection are unlikely to benefit from COVID-19

vaccination, and vaccines can be safely prioritized to those who have not been infected before.

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INTRODUCTION

The two FDA-approved (BNT162b2 mRNA [Pfizer-BioNTech] and mRNA-1273 [Moderna])

mRNA vaccines have been shown to be very efficacious in protecting against Severe Acute Respiratory

Syndrome (SARS) – associated Coronavirus-2 (SARS-CoV-2) infection [1,2]. The effectiveness of the

Pfizer-BioNTech vaccine in a real-world setting has also been shown to be comparable to the efficacy

demonstrated in clinical trials [3,4]. Given these, there has been an understandable desire to vaccinate as

many people as possible.

The ability to vaccinate a large part of the population is limited by the supply of vaccine. As of

March 21, 2021, 78% of 447 million doses of the coronavirus disease 2019 (COVID-19) vaccines that

had been deployed had gone to only ten countries [5]. The COVAX initiative was borne out of the

recognition that equitable distribution of vaccines worldwide was essential for effective control of the

COVID-19 pandemic. However, the reality is that there is great disparity in the availability of vaccines

across countries. Countries with limited supplies of vaccine have to prioritize how their supply of

vaccines will be allocated within their populations. Criteria used for such prioritization have included

profession, age, and comorbid conditions. Data that inform prioritization criteria with help maximize the

benefits of whatever vaccine is available.

Observational studies have found very low rates of reinfection among individuals with prior

SARS-CoV-2 infection [6–8]. This brings up the question about whether it is necessary to vaccinate

previously infected individuals. These studies notwithstanding, there remains a theoretical possibility that

the vaccine may still provide some benefit in previously infected persons. A prior large observational

study concluded that immunity from natural infection cannot be relied on to provide adequate protection

and advocated for vaccination of previously infected individuals [9]. The CDC website recommends that

persons previously infected with SARS-CoV-2 still get the vaccine [10]. Despite these recommendations,

credible reports of previously infected persons getting COVID-19 are rare. The rationale often provided

for getting the COVID-19 vaccine is that it is safer to get vaccinated than to get the disease. This is

3
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certainly true, but it is not an explanation for why people who have already had the disease need to be

vaccinated. A strong case for vaccinating previously infected persons can be made if it can be shown that

previously infected persons who are vaccinated have a lower incidence of COVID-19 than previously

infected persons who did not receive the vaccine.

The purpose of this study was to attempt to do just that, and thereby evaluate the necessity of the

COVID-19 vaccine in persons who were previously infected with SARS-CoV-2.

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METHODS

Study design

This was a retrospective cohort study conducted at the Cleveland Clinic Health System in Ohio,

USA. The study was approved by the Cleveland Clinic Institutional Review Board. A waiver of informed

consent and waiver of HIPAA authorization were approved to allow access to personal health information

by the research team, with the understanding that sharing or releasing identifiable data to anyone other

than the study team was not permitted without additional IRB approval.

Setting

PCR testing for SARS-CoV-2 at Cleveland Clinic began on March 12, 2020, and a streamlined

process dedicated to the testing of health care personnel (HCP) was begun shortly thereafter. All

employees with a positive SARS-CoV-2 test were interviewed by Occupational Health, with date of onset

of symptoms of COVID-19 being one of the questions asked. Vaccination for COVID-19 began at

Cleveland Clinic on December 16, 2020. When initially started it was the Pfizer-BioNTech vaccine that

was administered, until the Moderna vaccine became available, from which time employees received one

or the other. All employees were scheduled to receive their second vaccine dose 28 days after the first

one, regardless of which vaccine was given. The employee cohort was chosen for this study because of

documentation of their COVID-19 vaccination and of any SARS-CoV-2 infection in the Occupational

Health database.

Participants

All employees of the Cleveland Clinic Health System, working in Ohio, on Dec 16, 2020, were

screened for inclusion in the study. Those who were in employment on December 16, 2020, were

included.

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Variables

SARS-CoV-2 infection was defined as a positive nucleic acid amplification test. The date of

infection was taken to be the date of onset of symptoms when available, and the date of specimen

collection when not. A person was considered vaccinated 14 days after receipt of the second dose of the

vaccine (which would have been 42 days after receipt of the first dose of the vaccine for most subjects).

For the sake of consistency in the duration assumed for development of natural and vaccine immunity,

any person who tested positive for SARS-CoV-2 at least 42 days before the vaccine rollout date, was

considered previously infected.Other covariates collected were age, job location, job type (patient-facing

or non-patient facing), and job category. The job location variable could be one of the following:

Cleveland Clinic Main Campus, regional hospital (within Ohio), ambulatory center, administrative center,

or remote location. The job category was one of the following: professional staff, residents/fellows,

advance practice practitioners, nursing, pharmacy, clinical support, research, administration, and

administration support.

Outcome

The study outcome was time to SARS-CoV-2 infection, the latter defined as a positive nucleic

acid amplification test for SARS-CoV-2 on or after December 16, 2020. Time to SARS-CoV-2 infection

was calculated as number of days from December 16, 2020 (vaccine rollout date) to SARS-CoV-2

infection. Employees that had not developed a SARS-CoV-2 infection were censored at the end of the

study follow-up period (May 15, 2021). Those who received the Johnson & Johnson vaccine (81 subjects)

without having had a SARS-CoV-2 infection were censored on the day of receipt of the vaccine, and

those whose employment was terminated during the study period before they had SARS-CoV-2 infection

(2245 subjects) were censored on the date of termination of employment. The health system never had a

requirement for asymptomatic employee test screening. Most of the positive tests, therefore, would have

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been tests done to evaluate suspicious symptoms. A small proportion would have been tests done as part

of pre-operative or pre-procedural screening.

Statistical analysis

A Simon-Makuch hazard plot [11] was created to compare the cumulative incidence of SARS-

CoV-2 infection among previously infected subjects who were vaccinated, with those of previously

infected subjects who remained unvaccinated, previously uninfected subjects who were vaccinated, and

previously uninfected subjects who remained unvaccinated. Previous infection was treated as a time-

independent covariate (SARS-CoV-2 infection at least 42 days before Dec 16, 2020), and vaccination (14

days after receipt of the second dose of the vaccine) was treated as a time-dependent covariate (Figure 1).

Curves for the unvaccinated were based on data for those who did not receive the vaccine over the

duration of the study, and for those who did until the date they were considered vaccinated, from which

point onwards their data were recorded into the corresponding vaccinated set. A Cox proportional hazards

regression model was fitted with time to SARS-CoV-2 infection as the outcome variable against

vaccination (as a time-dependent covariate whose value changed on the date a subject was considered

vaccinated)[12]. Previous infection (as a time-independent covariate) and an interaction term for previous

infection and vaccination were included as covariates. The phase of the epidemic was adjusted for by

including the slope of the epidemic curve as a time-dependent covariate whose value changed

continuously with the slope of the epidemic curve. The analysis was performed by NKS and ASN using

the survival package and R version 4.0.5 [12–14].

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RESULTS

Of 52238 employees included in the study, 2579 (5%) were previously infected with SARS-CoV-

2.

Baseline characteristics

Those previously infected with SARS-CoV-2 were significantly younger (mean ± SD age; 39 ±

13 vs. 42 ± 13, p<0.001), and included a significantly higher proportion with patient-facing jobs (65% vs.

51%, p<0.001). Table 1 shows the characteristics of subjects grouped by whether or not they were

previously infected. A significantly lower proportion of those previously infected (47%, 1220 subjects)

were vaccinated by the end of the study compared to 59% (29461) of those not previously infected

(p<0.001). Of those vaccinated, 63% received the Moderna vaccine. Twelve percent of subjects with

previous SARS-CoV-2 infection did not have a symptom onset date, suggesting they may possibly have

been identified on pre-operative or pre-procedural screening, and may not have had symptomatic

infection. When vaccination was begun, the epidemic in Ohio was at the peak of its third wave (Figure 2).

Cumulative incidence of COVID-19

Figure 3 is a Simon-Makuch plot showing that SARS-CoV-2 infections occurred almost

exclusively in subjects who were not previously infected with SARS-CoV-2 and who remained

unvaccinated. The cumulative incidence of SARS-CoV-2 infection among previously infected

unvaccinated subjects did not differ from that of previously infected subjects who were vaccinated, and

that of previously uninfected subjects who were vaccinated. For all three of these groups, the cumulative

incidence of SARS-CoV-2 infection was much lower than that of subjects who were not previously

infected and who remained unvaccinated. Of the 2154 SARS-CoV-2 infections during the study period,

2139 (99.3%) occurred among those not previously infected who remained unvaccinated or were waiting

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to get vaccinated, and15 (0.7%) occurred among those not previously infected who were vaccinated. Not

one of the 2579 previously infected subjects had a SARS-CoV-2 infection, including 1359 who remained

unvaccinated throughout the duration of the study.

Association of vaccination with occurrence of COVID-19

In a Cox proportional hazards regression model, after adjusting for the phase of the epidemic,

vaccination was associated with a significantly lower risk of SARS-CoV-2 infection among those not

previously infected (HR 0.031, 95% CI 0.015 – 0.061) but not among those previously infected (HR

0.313, 95% CI 0 – Infinity). The absence of events among those who were previously infected, whether

they received the vaccine or not, precluded accurate or precise estimates for the latter effect size.

Duration of protection

This study was not specifically designed to determine the duration of protection afforded by

natural infection, but for the previously infected subjects the median duration since prior infection was

143 days (IQR 76 – 179 days), and no one had SARS-CoV-2 infection over the following five months,

suggesting that SARS-CoV-2 infection may provide protection against reinfection for 10 months or

longer.

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DISCUSSION

This study shows that subjects previously infected with SARS-CoV-2 are unlikely to get COVID-

19 reinfection whether or not they receive the vaccine. This finding calls into question the necessity to

vaccinate those who have already had SARS-CoV-2 infection.

It is reasonable to expect that immunity acquired by natural infection provides effective

protection against future infection with SARS-CoV-2. Observational studies have indeed found very low

rates of reinfection over the following months among survivors of COVID-19 [6–8]. Reports of true

reinfections are extremely rare in the absence of emergence of new variants. When such reinfections

occur, it would be purely speculative to suggest that a vaccine might have prevented them. Duration of

protective immunity from natural infection is not known. However, the same also can be said about

duration of protective immunity from vaccination. Uncertainty about the duration of protective immunity

afforded by natural infection is not by itself a valid argument for vaccinating previously infected

individuals. This study provides direct evidence that vaccination with the best available vaccines does not

provide additional protection in previously infected individuals.

A prior study concluded that natural infection cannot be relied on to protect against COVID-19

[9]. That study was based on comparison of PCR-positivity rates during a second COVID-19 surge in

Denmark between those who tested positive and negative during the first COVID-19 surge, and indirectly

calculated that prior infection provided 80.5% protection against repeat infection, and that protection

against those older than 65 years was only 47.1%. The study did not compare vaccinated and

unvaccinated people, and it is therefore an assumption to consider that a vaccine would have provided

better protection in that particular population. Furthermore, there was a gap of only seven weeks between

the end of the first surge and the beginning of the second in that study. It is now well-known that a small

number of people can continue to have positive PCR test results for several weeks to a few months after

infection, one study finding that 5.3% remained positive at 90 days [15]. It is possible that some of the

positives picked up in the early part of the second surge were not necessarily new infections but residual

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virus from the tail end of the first surge. Since the actual number of infections was small, a few such

misclassifications could change the rates substantially. Our study examined rates of SARS-CoV-2

infection in vaccinated and unvaccinated individuals and showed that those previously infected who did

not receive the vaccine did not have higher rates of SARS-CoV-2 infection than those previously infected

who did, thereby providing direct evidence that vaccination does not add protection to those who were

previously infected.

There are several strengths to our study. Its large sample size and follow-up of up to 5 months

provide us with an ample degree of confidence in its findings. A major strength of our study is that we

adjusted the analyses for the phase of the epidemic at all time points. The risk of acquisition of infection

is strongly influenced by the phase of the epidemic at any given time, and it is important to adjust for this

for accurate risk analyses. Given that was this a study among employees of a health system, and that the

health system had policies and procedures in recognition of the critical importance of keeping track of the

pandemic among its employees, we had an accurate accounting of who had COVID-19, when they were

diagnosed with COVID-19, who received a COVID-19 vaccine, and when they received it.

The study has its limitations. Because we did not have a policy of asymptomatic employee

screening, previously infected subjects who remained asymptomatic might have been misclassified as

previously uninfected. Given this limitation, one should be cautious about drawing conclusions about the

protective effect of prior asymptomatic SARS-CoV-2 infection. It should be noted though, that 12% of

the subjects classified as previously infected did not have a symptom onset date recorded, suggesting that

at least some of those classified as previously infected might have been asymptomatic infections. It is

reassuring that none of these possibly asymptomatically infected individuals developed COVID-19 during

the duration of the study. The study follow-up duration was short, being only five months, but this was

longer than published mRNA vaccine efficacy studies [1,2], and longer than the follow-up duration of the

largest published vaccine effectiveness studies to date [3,4]. Median freedom from reinfection (time from

initial infection until end of follow-up) in this study, for those previously infected, of almost 10 months, is

consistent with findings in an earlier study that immunoglobulin G (IgG) to the spike protein remained

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stable over more than six months after an episode of infection [16]. Our study included no children and

few elderly subjects, and the majority would not have been immunosuppressed. Data governance policies

in our institution precluded us from obtaining detailed clinical information on employees. While one

cannot generalize this study’s findings to assume that prior infection would provide adequate immunity in

these groups, there is also no reason to expect a vaccine to provide additional protection in these same

groups. Lastly, it is necessary to emphasize that these findings are based on the prevailing assortment of

virus variants in the community during the study. It is not known how well these results will hold if or

when some of the newer variants of concern become prominent. However, if prior infection does not

afford protection against some of the newer variants of concern, there is little reason to suppose that the

currently available vaccines would either. Vaccine breakthrough infections with variants have indeed

been reported [17].

Our study’s findings have important implications. Worldwide, COVID-19 vaccines are still in

short supply. As of March 9, 2021, dozens of countries had not been able to administer a single dose of

the vaccine [18]. As of May 17, 2021, only 17 countries had been able to reach ten percent or more of

their populations with at least the first dose of vaccine [19]. Given such a scarcity of the vaccine, and the

knowledge that vaccine does not provide additional protection to those previously infected, it would make

most sense to limit vaccine administration to those who have not previously had the infection. In addition

to profession, age, and comorbid conditions, previous infection should be an important consideration in

deciding whom to prioritize to receive the vaccine. A practical and useful message would be to consider

symptomatic COVID-19 to be as good as having received a vaccine, and that people who have had

COVID-19 confirmed by a reliable laboratory test do not need the vaccine.

In conclusion, individuals who have laboratory-confirmed symptomatic SARS-CoV-2 infection

are unlikely to benefit from COVID-19 vaccination, and vaccines can be safely prioritized to those who

have not been infected before.

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TRANSPARENCY DECLARATION

Conflict of Interest

Selection of “no competing interests” reflects that all authors have completed the ICMJE uniform

disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no support from any organization for

the submitted work; no financial relationships with any organizations that might have an interest in the

submitted work in the previous three years; no other relationships or activities that could appear to have

influenced the submitted work.

Funding

None received.

Author contributions

NKS: Conceptualization, Methodology, Validation, Investigation, Data curation, Software, Formal

analysis, Visualization, Writing- Original draft preparation, Writing- Reviewing and Editing, Supervision,

Project administration.

ASN: Methodology, Formal analysis, Visualization, Validation, Writing- Reviewing and Editing.

PCB: Resources, Investigation, Validation, Writing- Reviewing and Editing.

PT: Resources, Writing- Reviewing and Editing.

SMG: Project administration, Resources, Writing- Reviewing and Editing.

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REFERENCES

1. Polack FP, Thomas SJ, Kitchin N, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19

Vaccine. N Engl J Med 2020;383:2603–15.

2. Baden LR, El Sahly HM, Essink B, et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2

Vaccine. N Engl J Med 2021;384:403–16.

3. Dagan N, Barda N, Kepten E, et al. BNT162b2 mRNA Covid-19 Vaccine in a Nationwide Mass

Vaccination Setting. N Engl J Med 2021;384:1412–23.

4. Haas EJ, Angulo FJ, McLaughlin JM, et al. Impact and effectiveness of mRNA BNT162b2

vaccine against SARS-CoV-2 infections and COVID-19 cases, hospitalisations, and deaths

following a nationwide vaccination campaign in Israel: an observational study using national

surveillance data. Lancet 2021;397:1819–29.

5. Beyrer C, Allotey P, Amon JJ, et al. Human rights and fair access to COVID-19 vaccines: the

International AIDS Society–Lancet Commission on Health and Human Rights. Lancet

2021;397:1524–7.

6. Sheehan MM, Reddy AJ, Rothberg MB. Reinfection Rates Among Patients Who Previously

Tested Positive for Coronavirus Disease 2019: A Retrospective Cohort Study. Clin Infect Dis

2021. Available from: https://doi.org/10.1093/cid/ciab234. Accessed May 5, 2021.

7. Pilz S, Chakeri A, Ioannidis JP, et al. SARS-CoV-2 re-infection risk in Austria. Eur J Clin Invest

2021;51:e13520.

8. Lumley SF, O’Donnell D, Stoesser NE, et al. Antibody Status and Incidence of SARS-CoV-2

Infection in Health Care Workers. N Engl J Med 2021;384:533–40.

9. Hansen CH, Michlmayr D, Gubbels SM, Mølbak K, Ethelberg S. Assessment of protection

against reinfection with SARS-CoV-2 among 4 million PCR-tested individuals in Denmark in

2020: a population-level observational study. Lancet 2021;397:1204–12.

10. Centers for Disease Control and Prevention. Frequently Asked Questions about COVID-19

14
Case
medRxiv 1:21-cv-02228
preprint Document 1-1 Filed; this
doi: https://doi.org/10.1101/2021.06.01.21258176 08/17/21 USDC
version posted June 5, Colorado Page
2021. The copyright 23forof
holder this165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

Vaccination. 2021;Available from: https://www.cdc.gov/coronavirus/2019-

ncov/vaccines/faq.html. Accessed April 26, 2021.

11. Simon R, Makuch RW. A non-parametric graphical representation of the relationship between

survival and the occurrence of an event: Application to responder versus non-responder bias. Stat

Med 1984;3:35–44.

12. Therneau TM, Crowson C, Atkinson E. Using Time Dependent Covariates and Time Dependent

Coefficients in the Cox Model. Available from: https://cran.r-

project.org/web/packages/survival/vignettes/timedep.pdf. Accessed May 8, 2021.

13. Therneau TM, Grambsh, PM. Modeling Survival Data: Extending the Cox Model. New York,

NY: Springer International Publishing; 2000.

14. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R

Foundation for Statisical Computing; 2021.

15. Vibholm LK, Nielsen SSF, Pahus MH, et al. SARS-CoV-2 persistence is associated with antigen-

specific CD8 T-cell responses. EBioMedicine 2021;64:103230.

16. Dan JM, Mateus J, Kato Y, et al. Immunological memory to SARS-CoV-2 assessed for up to 8

months after infection. Science 2021;371:eabf4063.https://doi.org/10.1126/science.abf4063.

17. Hacisuleyman E, Hale C, Saito Y, et al. Vaccine Breakthrough Infections with SARS-CoV-2

Variants. N Engl J Med 2021; https://doi.org/10.1056/NEJMoa2105000.

18. The Lancet. Access to COVID-19 vaccines: looking beyond COVAX. Lancet 2021;397:941.

19. Mathieu E, Ritchie H, Ortiz-Ospina E, et al. A global database of COVID-19 vaccinations. Nat

Hum Behav 2021;https://doi.org/10.1038/s41562-021-01122-8.

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TABLES

Table 1. Study Subject Characteristics

Characteristic Previously Infected Not Previously Infected P Value

(N = 2579) (N = 49659)

Age, y, mean ± SD 39±13 42±13 <0.001

Patient-facing job 1676 (65) 25504 (51) <0.001

Job location <0.001

Cleveland Clinic Main Campus 1011 (39) 19595 (40)

Regional hospitals 1096 (43) 16433 (33)

Ambulatory centers 313 (12) 7767 (16)

Administrative centers 138 (5) 4424 (9)

Remote location 21 (<1) 1440 (3)

Job category <0.001

Professional staff 89 (4) 3775 (8)

Residents and fellows 72 (3) 1669 (3)

Advanced practice practitioners 154 (6) 2806 (6)

Nursing 1142 (44) 13623 (27)

Pharmacy 44 (2) 1274 (3)

Research 328 (13) 6776 (14)

Clinical support 111 (4) 3500 (7)

Administration 614 (24) 15050(30)

Administration support 25 (1) 1186 (2)

Data are presented as no. (%) unless otherwise indicated

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FIGURES

Figure 1. Explanation of “previously infected” analyzed as a time-independent covariate and

“vaccinated” treated as a time-dependent covariate.

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Figure 2. COVID-19 epidemic curve before and after vaccine rollout. Points on the scatter plot

represent the proportion of all COVID-19 PCR tests done at Cleveland Clinic that were positive on any

given day. The colored line represents a fitted polynomial curve.

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Figure 3. Simon-Makuch plot showing the cumulative incidence of COVID-19 among subjects

previously infected and not previously infected with COVID-19, who did and did not receive the

vaccine. Curves for the unvaccinated are based on data for those who did not receive the vaccine during

the duration of the study, and for those waiting to receive the vaccine. Day zero was Dec 16, 2020, the

day vaccination was started in our institution. Error bars represent 95% confidence intervals. Seven

subjects who had been vaccinated earlier as participants in clinical trials were considered vaccinated

throughout the duration of the study. Twelve subjects who received their first dose in the first week of the

vaccination campaign managed to get their second dose three weeks later, and were thus considered

vaccinated earlier than 42 days since the start of the vaccination campaign.

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EXHIBIT 3
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ARTICLE
https://doi.org/10.1038/s41467-021-22036-z OPEN

Exposure to SARS-CoV-2 generates T-cell memory

in the absence of a detectable viral infection
Zhongfang Wang1,6, Xiaoyun Yang 1,6, Jiaying Zhong1,6, Yumin Zhou1,6, Zhiqiang Tang2,6, Haibo Zhou3,
Jun He4, Xinyue Mei 1, Yonghong Tang4, Bijia Lin1, Zhenjun Chen 5, James McCluskey 5, Ji Yang1,
Alexandra J. Corbett 5 & Pixin Ran 1 ✉
1234567890():,;

T-cell immunity is important for recovery from COVID-19 and provides heightened immunity
re-infection. However, little is known about the SARS-CoV-2-specific T-cell immunity in
for re-infection
virus-exposed individuals. Here we report virus-specific CD4+ and CD8+ T-cell memory in
recovered COVID-19 patients and close contacts.
contact We also demonstrate the size and quality
of the memory T-cell pool of COVID-19 patients are larger and better than those of close
contacts. However, the proliferation capacity, size and quality of T-cell responses in close
contacts are readily distinguishable from healthy donors, suggesting close contacts are able
to gain T-cell immunity against SARS-CoV-2 despite lacking a detectable infection. Addi-
tionally, asymptomatic and symptomatic COVID-19 patients contain similar levels of SARS-
CoV-2-specific T-cell memory. Overall, this study demonstrates the versatility and potential
of memory T cells from COVID-19 patients and close contacts, which may be important for
host protection
protection.

1 State Key Laboratory of Respiratory Disease & National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, the
First Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China. 2 The Second Peoples Hospital of Changde City,
Hunan, China. 3 The Sixth Affiliated Hospital of Guangzhou Medical University, Guangzhou, China. 4 Affiliated Nanhua Hospital of University of South China,
Hunan, China. 5 The Department of Microbiology and Immunology and The Peter Doherty Institute for Infection and Immunity, University of Melbourne,
Melbourne, Victoria, Australia. 6These authors contributed equally: Zhongfang Wang, Xiaoyun Yang, Jiaying Zhong, Yumin Zhou, Zhiqiang Tang.
✉email: pxran@gzhmu.edu.cn

NATURE COMMUNICATIONS | (2021)12:1724 | https://doi.org/10.1038/s41467-021-22036-z | www.nature.com/naturecommunications 1

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ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-22036-z

S
ince early 2020, SARS-CoV-2 has spread globally, triggering Results
a pandemic that continues to cause devastating damage to Proliferation capacity of memory T cells from recovered
public health and people’s livelihoods. By the middle of COVID-19 patients and close contacts. To assess the SARS-
November, the global COVID-19 cases have reached 50 million CoV-2-specific T-cell memory, human peripheral blood mono-
with the death toll exceeding a grim 1.2 million (John Hopkins nuclear cells (PBMCs) from 90 COVID-19 patients collected
University, USA). Although the mechanisms by which host between 48–86 days after disease onset were stimulated in vitro
immunity combats SARS-CoV-2 infection are far from being for 10 days with peptide pools designed to target the spike gly-
completely understood, significant knowledge in this area has coprotein (S), membrane glycoprotein (M), nucleocapsid (N),
been gained through the investigations of the association of envelope glycoprotein (E) and ORF1ab region of RNA-dependent
COVID-19 clinical features and disease progression with host RNA polymerase (RdRp) of SARS-CoV-2. Our data showed that
immune responses1. For example, our recent study established the memory CD4+ and CD8+ T cells of 94.44% and 83.33%,
that the severity of COVID-19 inversely correlates with T-cell respectively, of the COVID-19 patients successfully underwent
immunity of the host2. In the presence of adequate neutralizing expansion (Fig. 1a–c). These results clearly indicate that most of
antibodies, CD4+ and CD8+ T cells play a major role in the the recovered COVID-19 patients have developed effective T cell
recovery of critical COVID-19 patients2. Other studies showed memory pools against SARS-CoV-2.
that in moderate and severe COVID-19 cases characterized by Although the close contacts in our cohort were all negative in
lymphopenia there was a drastic reduction in the numbers of both nucleic acid test (NAT) and SARS-CoV-2 antibody screen-
both CD4+ and CD8+ T cells3–5. Although the reason for this ing, the possible exposure of these individuals to the virus may
reduction remains unknown, autopsy revealed extensive infiltra- have led to the generation of T cell immunity even in the absence
tion of T cells into the lungs6. Analysis of immune cells from of a successful infection. To test this possibility, we performed a
bronchoalveolar lavage (BAL) fluid of COVID-19 patients 10-day in vitro peptide stimulation assay for 69 close contacts
demonstrated the presence of clonal expansion7. Moreover, virus- from 45 family clusters. The results show that 57.97% (Fig. 1a–c)
specific CD4+ T cell numbers were shown to be associated with and 14.49% (Fig. 1b, c) of close contacts contained virus-specific
the production of IgG that targets the receptor-binding domain memory CD4+ and CD8+ T-cells, respectively. Notably, all close
(RBD) of SARS-CoV-28. Notably, analyses of persistent COVID- contacts developed responses at lower frequencies than 4%, while
19 cases showed that upon activation their T-cells appeared to 64 (71.11%) and 32 (35.56%) of the 90 COVID-19 patients
lose polyfunctionality and cytotoxicity, trending towards an developed marked responses at the frequencies of higher than 4%
exhausted phenotype9,10. for IFNγ+CD4+ T cells (Fig. 1a) and IFNγ+CD8+ T cells
While most acute viral infections result in the development of (Fig. 1b), respectively. In comparison to the COVID-19 patients,
protective immunity, available data suggest that long-term and a significantly lower proportion of close contacts responded
robust-protective memory is not easily acquired for human cor- (p < 0.0001 for CD4+, Fig. 1a; p < 0.0001 for CD8+, Fig. 1b).
onavirus infections11. For example, one year after disease onset In order to investigate whether the observed expanded T cells
following MERS-CoV infection, the viral-specific IgG antibody may have originated from pre-existing cross-reactive T cells
became undetectable for some of the patients with mild specific for common cold coronaviruses from previous infections,
symptoms11–13. The SARS-CoV-1 humoral response was rela- we tested blood samples of 63 healthy donors collected before
tively short-lived and memory B cells disappeared quickly after September of 2019. Following a 10-day in vitro peptide expansion
primary infection14. Recent mathematical modeling suggested a only 3.17% of the healthy donors contained detectable levels of
short duration (likely <2 years) of protective immunity is elicited virus-specific memory CD4+ and CD8+ T cells, respectively
after SARS-CoV-2 infection15. Furthermore, Long et al. have (Fig. 1a–c), suggesting that cross-reactive T cells derived from
reported that the viral-specific IgG levels of SARS-CoV-2-infected exposure to other human coronaviruses do exist but are at a
individuals had an ~70% reduction during the early convalescent significantly lower frequency than those observed in close contacts.
phase and a significant proportion of individuals (40% of The major differences between the proportion of COVID-19
asymptomatic patients and 12.9% of symptomatic patients) patients and healthy donors (p < 0.0001 for CD4+, Fig. 1a;
became IgG seronegative16. In contrast to the short-lived humoral p < 0.0001 for CD8+, Fig. 1b), or between close contacts
response in SARS-CoV-1 survivors, the magnitude and frequency and healthy donors (p < 0.0001 for CD4+, Fig. 1a; p = 0.0157
of specific CD8+ memory T cells, and to a lesser extent CD4+ for CD8+, Fig. 1b) with memory T-cells capable of proliferating
memory T cells, persisted for 6–11 years, suggesting that T cells in response to SARS-CoV2 peptides emphasize that exposure to
may confer long-term immunity15. Although it has been reported SARS-CoV-2 can facilitate the establishment of the T memory
that SARS-CoV-2-specific CD4+ and CD8+ T cells were detected immunity not only in COVID-19 patients, but also in some close
in 100 and 70% of convalescent COVID-19 patients, contacts even in the absence of a successful infection. In addition,
respectively17, to date, it remains largely unclear how well the differences between COVID-19 patients and close contacts were
SARS-CoV-2 T cell memory is established and how the memory observed in the frequency of double-positive (IFNγ+ TNF+)
T cells respond upon re-exposure to viral antigens. Another CD4+ T cells (p < 0.0001 for CD4+, Supplementary Fig. 1a,
important question that remains unresolved is whether close p < 0.0001 for CD8+, Supplementary Fig. 1b), although CD4+, but
contacts, who had been confirmed to be negative in nucleic acid not CD8+ cells producing both cytokines were significantly higher
testing (NAT) and antibody screening, have gained any memory in close contacts than healthy controls (Supplementary Fig. 1a, b).
T cell immunity upon exposure to SARS-CoV-2.
In this study, we examined the proliferation and activation
capability of the SARS-CoV-2 memory T cell pools of a large Ex vivo analyses of SARS-CoV-2-specific memory T cells from
cohort of recovered COVID-19 patients, close contacts, and COVID-19 patients and close contacts. Next, we measured the
unexposed healthy individuals. Our results showed that the sizes of virus-specific memory pools for CD4+ and CD8+ T cells
COVID-19 patients and close contacts developed SARS-CoV-2- from 89 COVID-19 patients (1 COVID-19 sample was used up),
specific T-cell immune memory. In addition, comparable levels of 69 close contacts and 30 healthy donors by using an overnight
SARS-CoV-2-specific memory T cells were detected in the sam- “ex vivo” peptide stimulation assay. Our results demonstrated
ples of asymptomatic and symptomatic COVID-19 patients. that a significant proportion of COVID-19 patients contained

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p<0.0001 COVID-19 Close contact Healthy control

a p<0.0001
p<0.0001
b p<0.0001
c No peptides SARS-CoV-2 No peptides SARS-CoV-2 No peptides SARS-CoV-2

% IFNγ+ CD8 (in vitro)

% IFNγ+ CD4 (in vitro) p=0.016
94.44% p<0.0001 83.33%
30
40
20 CD4
20
57.97%
10

4
14.49%
3
3 3.17%
2
3.17% 2
CD8
1
1

TNF
0 0
C o V ID -1 9 C lo s e c o n ta c ts H C
C o V ID - 1 9 C lo s e c o n t a c t s H C

COVID-19 Close Healthy COVID-19 Close Healthy

contact control contact control IFNγ

p<0.0001 p<0.000
d p=0.007 p<0.0001
e p=0.004 1 p=0.017
f COVID-19 Close contact
No peptides SARS-CoV-2
Healthy control
No peptides SARS-CoV-2
No peptides SARS-CoV-2

% IFNγ+ CD8 (ex vivo)

% IFNγ+ CD4 (ex vivo)

34.83% 15.94% 49.44%

0 .6 1 .5
1 .0 26.09%
0 .4
0 .5 CD4
0 .2 0 .2
3.33% 6.67%
0 .1 0 .1
CD8
0 .0 0 .0

TNF
C o V ID - 1 9
COVID-19 Close
C lo s e c o n ta c ts
Healthy
H C C o V ID - 1 9 C lo s e c o n ta c ts
Close H C
Healthy
contact control COVID-19
contact control IFNγ

Fig. 1 Memory T cells specific to SARS-2 were detected and can proliferate in vitro in COVID-19 patients and in close contacts. Donor PBMCs were
stimulated with 15-mer peptide pools (overlapping by 11 amino acids) encompassing the entire spike (S), nucleocapsid (N), membrane (M), and envelope
(E) proteins for 10 days (in vitro expansion, a–c) or overnight (ex vivo, d–f) in the presence of 10 U/ml rIL-2, IFNγ, and TNF expressing cells were
enumerated by intracellular cytokine staining. Ninety COVID-19 patients (closed circle), their 69 close contacts (open circle), and 63 unexposed healthy
donors (closed triangle) were assayed in vitro. For ex vivo experiments, the samples from the above cohort except for one from the COVID-19 group
because of cells used up, and 30 of the 63 unexposed healthy donors were assayed. Graphs show the frequency of IFNγ expressing cells in (a) CD4+ and
(b) CD8+ T cells after in vitro expansion and overnight stimulation and in (d) CD4+ and (e) CD8+ T cells after overnight stimulation. Dashed line is the cut
off determined by the background staining (no peptide) for the healthy control group. The cut off threshold used for the overnight stimulation experiments
was based on all negative controls (95% CI). The percentages shown are the frequency above this cut off. c, f Representative dot plots showing IFNγ and
TNF expression in T cells after expansion (c) or overnight stimulation (f). a, b, d, e Error bars indicate mean frequencies of IFNγ+ T cells ± SEM; Percentage
shown on top of the plots indicates the frequencies of samples above the cutoff. The student t test was performed with two-sided p values as indicated. No
peptides: no peptide stimulation control. SARS-CoV-2: with stimulation by SARS-CoV-2 overlapping peptide pools.

virus-specific T cells (34.83% for CD4+, Fig. 1d; 49.44% for or CD8+ T cells (Fig. 2e); (iii) SARS-CoV-2 peptides induced
CD8+, Fig. 1e; and cut off = 0.1%) at 48–86 days after disease higher levels of IFNγ production in both CD4+ (Fig. 2b, c) and
onset. In addition, SARS-CoV-2-specific T cells were also detec- CD8+ (Fig. 2d, e) T cells from patients infected with COVID-19
ted in close contacts (15.94% for CD4+, Fig. 1d and 26.09% for compared with close contacts, the MFIs being twice as high in
CD8+, Fig. 1e). Significant differences were seen between the sizes CD4+ T cells from the infected group. Collectively, these results
of T cell memory pools of COVID-19 patients and close contacts indicate that the activation capability of SARS-CoV-2-specific
(p = 0.007 for CD4+, Fig. 1d and p = 0.004 for CD8+, Fig. 1e). In memory T cells from close contacts is lower than that in the
contrast, in the case of the healthy donors, we found that only COVID-19 patients, despite both groups having similar pre-
1/30 (3.33%) and 2/30 (6.67%) of the samples contained cross- existing immunity to CMV.
reactive memory CD4+ and CD8+ T cells, respectively (Fig. 1d,
e), suggesting that the cross-reactive T-cell immunity only exists
in a small number of unexposed healthy donors. Interestingly, Memory T-cell immunity is detectable in both symptomatic
comparing the frequency of double-positive (IFNγ+ TNF+) and asymptomatic patients with COVID-19 infection. PBMCs
CD4+ and CD8+ T cells within individuals, these were higher in from 72 symptomatic and 18 asymptomatic COVID-19 patients
both COVID-19 patients and close contacts than in healthy were used in the overnight ex vivo and 10-day in vitro expansion
controls (Supplementary Fig. 1c, d). assays to evaluate the sizes, qualities and proliferation capacities
of the memory T cell pools. Data in Fig. 3a, d show that following
overnight stimulation by peptide pools, 4/18 (22.22%) and 7/18
IFNγ-producing SARS-CoV-2-specific memory T cells are (38.89%) of the samples from the asymptomatic patients with
detectable in close contacts of infected individuals. To evaluate COVID-19 developed detectable numbers of SARS-CoV-2 spe-
the quality of SARS-CoV-2-specific memory T cells, we measured cific IFNγ-producing CD4+ T cells and CD8+ T cells, respec-
the MFI of IFNγ by intracellular staining in the memory T cells tively. For the symptomatic COVID-19 patients, 27/71 (35.23%)
from COVID-19 patients and close contacts. To increase the and 36/71 (50.70%) of the samples also developed virus-specific
robustness of this experiment, we included an internal control specific CD4+ T cells and CD8+ T cells, respectively (Fig. 3a, d).
where all of the samples were also assessed for the production of There was no significant difference in the sizes of the SARS-CoV-
IFNγ following stimulation with CMV peptide pools spanning the 2-specific memory T-cell pools between the symptomatic and
pp65 protein. From the comparison between the MFI values of the asymptomatic COVID-19 patients (p = 0.58 for CD4+ and p =
different samples, it is clear that; (i) CMV peptides induced similar 0.66 for CD8+, Fig. 3a, d). Meanwhile, the ex vivo analysis
levels of IFNγ production by CD4+ and CD8+ T cells in the showed that the MFI of IFNγ staining of the memory T cells
samples from COVID-19 patients and close contacts (Fig. 2a, c, e), (SARS-CoV-2-specific) from the asymptomatic and symptomatic
(ii) the expression levels of IFNγ in CMV-specific T cells were 2-3 patients were 1536.37 ± 165.28 and 1182.18 ± 219.92 for CD4+
times higher than those of SARS-CoV-2-specific CD4+ (Fig. 2c) (Fig. 3b) and 636.54 ± 56.25 and 578.47 ± 102.37 for CD8+

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No peptide SARS-CoV-2 CMV

a COVID-19 Close contact COVID-19 Close contact COVID-19 Close contact

CD4

CD8
TNF

IFNγ

p = 0 .6 0

b c

MFI IFNγ+ CD4 (X1000)

p < 0 .0 0 0 1 p < 0 .0 0 0 1
MFI IFNγ+ CD4 (X1000)

p < 0 .0 0 0 1 15
6
10

6
4

2
2

0
0 SARS-
S AR S CMV
C M V SARS- CMV
S AR S C M V

COVID-19 Close contact CoV-2 CoV-2

COVID-19 Close contact

d p = 0 .0 3 1
e p = 0 .8 4
MFI IFNγ+ CD8 (X1000)

MFI IFNγ+ CD8 (X1000)

p < 0 .0 0 0 1 p < 0 .0 0 0 1
3
6
5

2 4

1 2

0 0
COVID-19 Close contact SARS-
S A R S CMV
C M V SARS-
S A R S CMV
C M V
CoV-2 CoV-2
COVID-19 Close contact
Fig. 2 Functional analysis of SARS-CoV-2 specific memory T cells in Covid-19 patients and close contacts. Donor PBMCs were stimulated with SARS-
CoV-2 or CMV 15-mer peptide pools overnight in the presence of 10 U/ml rIL-2, IFNγ and TNF expressing cells were enumerated by intracellular cytokine
staining. a Representative FACS plots showing the expression of IFNγ and TNF in CD4+ and CD8+ T cells with or without SARS-CoV-2 or CMV peptide
stimulation overnight, as indicated. b, d Mean Fluorescence Intensity (MFI) of IFNγ staining for (b) CD4+and (d) CD8+ T cells from COVID-19 patients
(close circle, n = 89) and their close contacts (closed square, n = 69) after overnight stimulation with SARS-CoV-2 peptide pool. c, e Paired analyses of
MFI for IFNγ of CD4+ (c) and CD8+ (e) T cells after overnight stimulation with SARS-CoV-2 or CMV peptide pools for COVID-19 patients (n = 79) and
close contacts (n = 69). Each symbol represents a data point from one individual. b–e Error bars represent mean ± SEM. The student t test was performed
with two-sided p values as indicated. No peptides: no peptide stimulation control. SARS-CoV-2: with stimulation by SARS-CoV-2 overlapping peptide
pools. CMV: with stimulation by CMV overlapping peptide pools.

(Fig. 3e), respectively. Thus, there was no significant difference in asymptomatic patients, respectively, proliferated to detectable levels
the qualities of the memory T cells between the asymptomatic (Fig. 3f). For the CD4+ T cells, 97.22% and 83.33% of the samples
and symptomatic patients (p = 0.39 for CD4+ and p = 0.44 for from the symptomatic and asymptomatic patients, respectively,
CD8+, Fig. 3b, e). proliferated to levels above 1% (Fig. 3c). This indicates a slightly
In vitro peptide stimulation and expansion showed that 88.89% reduced proliferation capacity in SARS-CoV-2-specific T-cell
and 72.22% of CD8+ T cells from the symptomatic and immunity of asymptomatic patients (p < 0.0001, Fig. 3c).

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c p<0.0001
a p=0.58
b 9 7 .2 2 %

% IFNγ+ CD4 (in vitro)

6 p=0.39

MFI IFNγ +CD4 (X1000)

40
0 .6
5
% IFNγ+ CD4

4 8 3 .3 3 %
0 .4 20

2
0 .2 6

1 3

0 .0 0 0

Symptomatic Asymptomatic Symptomatic Asymptomatic Symptomatic Asymptomatic

p=0.37
d p=0.66
e f 8 8 .8 9 %

% IFNγ+ CD8 ( in vitro)

MFI IFNγ+ CD8 (X1000)
1 .5
3 p=0.44 30
7 2 .2 2 %
20
% IFN γ + CD8

10
1 .0 2
6

4
0 .5 1

0 .0 0 0

Symptomatic Asymptomatic Symptomatic Asymptomatic Symptomatic Asymptomatic

Fig. 3 Comparisons of the T cell memory and in vitro expansion of SARS-CoV-2 specific T cells between symptomatic and asymptomatic COVID-19
patients. a, d Frequencies of IFNγ expressing cells in CD4+ (a) and CD8+ (d) T cells in recovered symptomatic (n = 71) and asymptomatic (n = 18)
COVID-19 patients ex vivo. b, e MFI of IFNγ staining in CD4+ (b) and CD8+ (e) T cells from symptomatic and asymptomatic COVID-19 patients.
c, f Frequencies of IFNγ expressing (c) CD4+ and (f) CD8+ T cells after in vitro expansion in symptomatic (n = 72) and asymptomatic (n = 18) COVID-19
patients. Percentages shown are the frequencies above the cut off (1%, which was the upper limit observed in no-peptide control stimulations). Error bars
represent mean ± SEM. The student’s t test was performed with two-sided p values indicated.

SARS-CoV-2-specific T cells are stably maintained 48–86 days in Supplementary Table 2. Following in vitro expansion the virus-
after onset of symptoms. We then examined if there was any specific memory CD4+ T cell pool correlated with the titers of
correlation between the magnitude of the T cell responses IgG against the S RBD region (R2 = 0.51, p < 0.0001, Supple-
(measured by an in vitro expansion assay) and the timespan mentary Fig. 3a) and the N protein (R2 = 0.48, p < 0.0001, Sup-
between 48 and 86 days after symptom onset and found no plementary Fig. 3b), whereas no apparent correlation between
relationship between the levels of SARS-CoV-2-specific T cells CD8+ T cells and IgG titers was observed (R2 = 0.28, p < 0.0001,
(CD4+ and CD8+) and the timespan within this period (R2 = anti-S RBD IgG, Supplementary Fig. 3c and R2 = 0.28, p < 0.0001,
0.025, p = 0.14 for CD4+, Supplementary Fig. 2a, and R2 = 0.005, anti-N IgG, Supplementary Fig. 3d). In the ex vivo assay, no
p = 0.52 for CD8+, Supplementary Fig. 2c). Meanwhile, our data correlation was found between either the virus-specific CD4+
also showed that there was no association between the levels of T cells and IgG titres (R2 = 0.01, p = 0.27 anti-S RBD IgG, Sup-
memory T cells measured by an ex vivo assay and the timespan plementary Fig. 3e and R2 = 0.01, p = 0.29, anti-N IgG, Supple-
between 48–86 days after disease onset (R2 = 0.064, p = 0.021 for mentary Fig. 3f) or the virus-specific CD8+ T cells and IgG titres
CD4+, Supplementary Fig. 2b and R2 = 0.066, p = 0.019 for (R2 = 0.03, p = 0.10, anti-S RBD, Supplementary Fig. 3g and
CD8+, Supplementary Fig. 2d). Together, our in vitro and ex vivo R2 = 0.03, p = 0.10, anti-N IgG, Supplementary Fig. 3h), indi-
data suggest that CD4+ T memory and CD8+ T memory may cating that, due to the low numbers of specific T cells that can be
have contracted to a stable plateau by the times these samples detected ex vivo in the memory phase, expansion of T cells
were collected. Furthermore, we also did not see any difference in vitro to increase their numbers may be necessary to observe
between severe COVID-19 and moderate COVID-19 patients in these correlations.
the proportion of SARS-Co-V2-specific IFNγ-producing CD4+
or CD8+ T cells expanded in vitro (p = 0.71 for CD4+, Supple-
mentary Fig. 2e, p = 0.48 for CD8+, Supplementary Fig. 2f). Discussion
COVID-19 patients display a wide range of clinical phenotypes,
Memory CD4+ T-cell responses correlate with IgG titers including severe, moderate, mild, and asymptomatic cases, likely
against N protein and S RBD of SARS-CoV-2. The neutralizing determined by a mix of host genetic factors, and the dose and
antibody response in MERS-CoV-2 infection was previously route of infection. Individuals also exhibit a wide variation in
shown to be dependent on the CD4+ T cell response13. To cellular and humoral immune responses during the primary viral
determine if this is also true for SARS-CoV infection, we per- infection, with some patients displaying balanced viral-specific B
formed correlation analyses between IgG titers (anti N and anti- cell and T cell immunity, whereas others rely either on a higher
RBD, Supplementary Table 1) and magnitude of memory T cells level of activation of neutralizing antibodies or on a stronger T
measured by in vitro and ex vivo assays. The sensitivity and cell response to fight off the virus2. In rare cases, individuals who
accuracy of assays for IgG measurements were verified as shown suffer severe and long-lasting symptoms show highly imbalanced

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cellular and humoral immune responses whereby the levels of correlated to the IgG titers of anti-RBD and anti-N, it is possible
SARS-CoV-2 specific T-cell or antibody immunity are very low2. that the antibody production of asymptomatic individuals is lower
Close contacts, who are SARS-CoV-2-exposed, are often both than that of symptomatic individuals. This observation is con-
NAT negative and antibody negative, indicating that SARS-CoV- sistent with the findings that there is a rapid decay of anti-SARS-
2 failed to establish a successful infection within these individuals, CoV-2 antibodies and IgG antibodies in asymptomatic patients24.
presumably due to their exposure to limited numbers of viral In agreement with recent reports17,25, our data also demon-
particles or a short time of exposure. However, our analysis of the strated the presence of cross-reactive memory CD4+ and CD8+
samples from 69 of these close contacts showed the presence of T cells, which target various surface proteins of SARS-CoV-2, in
SARS-CoV-2 specific memory T-cell immunity. A similar unexposed healthy donors. However, the failure of these cross-
observation was reported during the MERS epidemic where high- reactive memory CD4+ and CD8+ to expand in vitro suggests
risk individuals (e.g., camel workers) who were NAT negative and they have limited potential to function as part of a protective
antibody negative also developed significant levels of MERS-CoV immune response against SARS-CoV-2. It is noteworthy that the
specific memory T cells13. In addition, although in agreement SARS-CoV-2-reactive T cells detected in the unexposed healthy
with Sekine et al.18, we found that some polyfunctional T cells donors in our study were lower than those detected by Grifoni
were detectable in close contacts, cells producing both IFNγ and et al.17 and Braun et al.26, but were consistent with those reported
TNF appear largely specific for infected patients rather than for by Peng et al.27 and Zhou et al.28. Assumably, due to the use of
close contacts and healthy donors, suggesting that for COVID-19 different methodologies in assessing SARS-CoV-2-specific T-cell
patients, the occurrence of stronger antigen stimulation and responses, it is difficult to directly reconcile the cell-number data
greater inflammation during viral infection led to an enhanced between different studies. Thus, a thorough investigation is nee-
polyfunctional T-cell response. ded to determine whether the cross-reactive T memory can
Our ex vivo stimulation analyses demonstrated that the pool provide any protective immunity and exert an influence on the
sizes and quality of the SARS-CoV-2-specific CD4+ and CD8+ T outcomes of COVID-19 disease.
memory cells from close contacts were around half of those from In summary, by examining a substantial number of clinical
COVID-19 patients. Similarly, our in vitro expansion experi- samples, we determined the SARS-CoV-2-specific memory T-cell
ments showed that the SARS-CoV-2-specific CD4+ memory immunity in COVID-19 patients with various clinical symptoms.
T cells of 57.97% and 94.44% of close contacts and COVID-19 Despite some subtle differences, most patients developed mea-
patients, respectively, were able to proliferate. However, a more surable amounts of SARS-CoV-2-specific CD4+ and CD8+
remarkable difference between the CD8+ proliferation fre- memory T cells which were stably maintained between 48–86 days
quencies of the two sample groups was observed, such that the after convalescence. Importantly, our discovery of the presence of
SARS-CoV-2-specific CD8+ memory T cells of 14.49% and significant levels of SARS-CoV-2-specific memory T-cell immu-
83.33% of close contacts and COVID-19 patients, respectively, nity in a group of individuals (close contacts) who were exposed to
underwent proliferation. Theoretically, the initial activation of but not infected by the virus highlights some unique character-
SARS-CoV-2-specific CD8+ and formation of CD8+ T memory istics in the dynamic interactions between SARS-CoV-2 and its
are achieved through the endogenous pathway which processes human host.
host Although cross-reactive memory T cells were present
viral antigens produced within the virus-infected host cells19. in healthy donors who had never been exposed to SARS-CoV-2,
Presumably, without in situ replication of SARS-CoV-2, there are their role in host protection needs to be thoroughly investigated as
insufficient viral antigens within the host cells of close contacts to they were hardly able to proliferate. Together, our analyses add
induce a robust CD8+ response resulting in CD8+ T memory in important information on the landscape of immune responses of a
the majority of individuals. By contrast, the formation of CD4+ T range of individuals in response to the primary SARS-CoV-2
memory does not rely on endogenous viral replication but encounter during the first wave of the pandemic.
involves endocytosis and/or phagocytosis of exogenous viral
antigens, which are mostly derived from non-replicative viral
Methods
particles or soluble viral proteins19. Thus, CD4+ T cell memory COVID-19 patients, close contacts, and healthy donors. For this study, we
may be more easily achieved in uninfected exposed individuals. recruited 90 COVID-19 patients and 69 close contacts. All of the COVID-19
Initially, we observed that SARS-CoV-2-specific memory CD4+ patients (NAT+) had stayed in the hospital and then recovered. The medical data
and CD8+ secreted low levels of IFNγ and only a small proportion collected from the COVID-19 patients included symptoms at disease onset and
records of physical examinations, laboratory tests and imaging. Asymptomatic
of the T cells from COVID-19 patients gained multifunctionality COVID-19 patients were defined using strict criteria: they were negative for any
(IFNγ and TNF dual expression). To vigorously validate this signs of cough, fever, sore throat, runny nose or computed tomography (CT) image
finding, we analysed the CMV-specific memory T cells in the same changes in the lungs. A blood sample was taken from each of the patients in the
PBMC samples. Evidently, the levels of IFNγ and TNF expression period between d48 and d86 after disease onset or returning a NAT+ result.
and the numbers of CMV-specific CD4+ and CD8+ memory Close contacts were identified from family members or friends who had stayed
with a SARS-CoV-2 infected individual(s) at the time from 5 days before their
T cells were all significantly greater than those of the corresponding disease onset to hospitalization. They were classified as a close contact only if they
SARS-CoV-2-specific memory T cells (Fig. 2a), ruling out the also were within a close distance (<1.5 m) of a COVID-19 individual(s) in a
possibility that SARS-CoV-2 infection inhibits the function of confined space for >1 h or were living together with a known case for >24 h. Other
T cells of the host. Recent epidemiological data show that between important criteria were that they were NAT- and negative for SARS-CoV-2-specific
antibodies (IgG and IgM) against S RBD and/or N and virus neutralization tests.
18 and 62% of SARS-CoV-2 infections are asymptomatic20–23. For this study, a blood sample was taken from each of the close contacts at the time
Therefore, determining how well protective immunity is estab- d48 and d86 after exposure to a known COVID-19+ individual.
lished in asymptomatic COVID-19 patients will provide valuable Blood samples of 63 healthy donors were obtained from a local blood donation
information for understanding herd immunity and the design of center in September 2019 (before the start of the COVID-19 pandemic) for
unrelated studies. These donors were considered healthy as they had no known
strategies to combat secondary infections by the virus. To this end, history of any significant systemic diseases. As the blood samples from healthy
we compared the T-memory immunity levels between asympto- donors were frozen for a longer period of time compared to those from patients
matic and symptomatic COVID-19 patients and showed that the and close contacts, we assessed whether prolonged freezing had any effect on assay
sizes and quality of their memory pools are comparable. Only the outcomes by comparing the CMV-specific T-cell responses (which would be
in vitro expansion capacity of memory CD4+ from asymptomatic expected to be the same) of close contacts and healthy donors (HC) in a control
experiment. We found that there is no significant difference in the frequencies of
COVID-19 patients was significantly lower. Since our data showed CMV-specific CD4+ and CD8+ between the two groups of samples (CD4+: p =
the magnitude of in vitro expansion of CD4+ memory T cells is 0.32 and CD8+: p = 0.37).

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This study is approved by the Ethics Commission of the First Affiliated Hospital 2. Wang, Z. et al. COVID-19 severity correlates with weaker T-Cell immunity,
of Guangzhou Medical University (No.2020-51). The signed consent forms from all hypercytokinemia, and lung epithelium injury. Am. J. Respir. Crit. Care Med.
the participants were obtained. 202, 606–610 (2020).
3. Chen, G. et al. Clinical and immunological features of severe and moderate
Peptide pool design and preparation. SARS-CoV-2-specific peptides were coronavirus disease 2019. J. Clin. Invest. 130, 2620–2629 (2020).
designed and synthesized as follows. The protein sequences were derived from the 4. Tong, Z. D. et al. Potential Presymptomatic Transmission of SARS-CoV-2,
SARS-CoV-2 reference (GenBank: MN908947.3). Four hundred and forty-seven 15- Zhejiang Province, China, 2020. Emerg. Infect. Dis. 26, 1052–1054
mer SARS-CoV-2 epitopes (overlapping by 11 amino acids) spanning the entire (2020).
antigen region of spike (S), nucleocapsid (N), membrane (M), and envelope (E) 5. Zheng, M. et al. Functional exhaustion of antiviral lymphocytes in COVID-19
proteins were generated with an online peptide generator (Peptide 2.0), and were patients. Cell Mol. Immunol. 17, 533–535 (2020).
synthesized by GL Biochem Corporation (Shanghai) with a purity of over 80%. One 6. Nienhold, R. et al. Two distinct immunopathological profiles in autopsy lungs
hundred and ten 18-mer peptides (overlapping by 10 amino acids) encompassing of COVID-19. Nat. Commun. 11, 5086 (2020).
the ORF1ab region of RNA-dependent RNA polymerase (RdRP) were synthesized 7. Liao, M. et al. Single-cell landscape of bronchoalveolar immune cells in
by GL Biochem Corporation (Shanghai). Each peptide was dissolved in DMSO, and patients with COVID-19. Nat. Med. 26, 842–844 (2020).
was then pooled, with each at a concentration of 45 μM to form a stock. 8. Ni, L. et al. Detection of SARS-CoV-2-specific humoral and cellular
immunity in COVID-19 convalescent individuals. IImmunity 52, 971–977.
PBMC isolation and ex vivo stimulation. PBMCs were isolated from heparinized e973 (2020).
whole blood by density-gradient sedimentation using Ficoll-Paque according to the 9. Moon, C. Fighting COVID-19 exhausts T cells. Nat. Rev. Immunol. 20, 277
manufacturer’s instructions (GE Healthcare, 17-1440-02). 1 × 106 PBMCs were cultured (2020).
in RPMI 1640 medium (Gibco) supplemented with 10% heat-inactivated FBS (Biolo- 10. Diao, B. et al. Reduction and functional exhaustion of T cells in patients
gical Industries, Israel Beit-Haemek), 100 U/ml penicillin (Gibco) and 0.1 mg/ml with coronavirus disease 2019 (COVID-19). Front. Immunol. 11, 827
streptomycin (Gibco). The PBMCs were treated with the peptide pool containing 447 (2020).
15-mer peptides and 110 18-mer peptides at 125 nM/each peptide in the presence of 11. Choe, P. G. et al. MERS-CoV Antibody Responses 1 Year after Symptom
10 U/ml rIL-2 and 1 μM GolgiPlug (BD Biosciences, San Diego, CA) overnight at 37 °C, Onset, South Korea, 2015. Emerg. Infect. Dis. 23, 1079–1084 (2017).
5% CO2. The approach of using a large peptide pool to stimulate PBMCs was based on 12. Okba, N. M. A. et al. Sensitive and specific detection of low-level antibody
that developed by Chevalier M. F. et al.29 and was validated for CMV peptides. responses in mild Middle East respiratory syndrome coronavirus infections.
Emerg. Infect. Dis. 25, 1868–1877 (2019).
13. Zhao, J. et al. Recovery from the Middle East respiratory syndrome is
PBMC in vitro expansion culture and stimulation. For in vitro culturing and
associated with antibody and T-cell responses. Sci. Immunol. 2, eaan5393
stimulation, 1 × 106 PBMCs were treated with the peptide pool (125 nM/each
(2017).
peptide), and incubated for 10 days. During this culturing, half of the medium was
14. Tang, F. et al. Lack of peripheral memory B cell responses in recovered
changed twice per week with fresh PRMI 1640 supplemented with 10% FBS and
10 U/ml rIL-2. The cells were subcultured when needed. The cells were then re- patients with severe acute respiratory syndrome: a six-year follow-up study. J.
stimulated at day 10 with a medium containing the peptide pool (125 nM/each Immunol. 186, 7264–7268 (2011).
peptide) overnight before being stained for FACS analysis. 15. Ng, O. W. et al. Memory T cell responses targeting the SARS coronavirus
persist up to 11 years post-infection. Vaccine 34, 2008–2014 (2016).
16. Long, Q. X. et al. Clinical and immunological assessment of asymptomatic
Flow cytometry. Cells harvested from the overnight or 10-day stimulation cultures SARS-CoV-2 infections. Nat. Med. 26, 1200–1204. (2020).
were washed and incubated with Live/dead aqua V510 for 15 min on ice. Cells were 17. Grifoni, A. et al. Targets of T cell responses to SARS-CoV-2 coronavirus in
then washed again and surface-stained for 30 min on ice with the following anti- humans with COVID-19 disease and unexposed individuals. Cell 181,
bodies: anti-CD3-FITC (BioLegend, clone UCHT1, 1:200, Cat# 300406), anti-CD4- 1489–1501.e15 (2020).
APC-Cy7 (BD Pharmingen™, clone RPA-T4, 1:200, Cat# 561839), anti-CD8- 18. Sekine, T. et al. Robust T cell immunity in convalescent individuals with
PerCPCy5.5 (BD Bioscience, clone RPA-T8, 1:200, Cat# 560662). After fixation and
asymptomatic or mild COVID-19. Cell 183, 158–168.e114 (2020).
permeabilization with Cytofix and Perm (BD Bioscience, Cat# 554714) on ice for
19. Blum, J. S., Wearsch, P. A. & Cresswell, P. Pathways of antigen processing.
15 min, intracellular staining (ICS) was performed on ice for 30 min with anti-
Annu. Rev. Immunol. 31, 443–473 (2013).
TNF-PE-Cy7 (BD, clone MAb11, 1:200, Cat # 557647) and anti-IFNγ-APC (BD
20. Kimball, A. et al. Asymptomatic and presymptomatic SARS-CoV-2 infections
Pharmingen™, clone B27, 1:200, Cat# 554702). After the final wash, cells were
resuspended in 200 μl FACS buffer. The samples were acquired using an FACSAria in residents of a long-term care skilled nursing facility - King County,
III instrument (BD Bioscience) and analyzed with FlowJo software (Treestar). Washington, March 2020. Morb. Mortal. Wkly. Rep. 69, 377–381 (2020).
21. Ganyani, T. et al. Estimating the generation interval for coronavirus disease
(COVID-19) based on symptom onset data, March 2020. Euro Surveill. 25,
Detection of blood plasma IgG in COVID-19 patients and close contacts. The 2000257 (2020).
SARS-CoV-2-specific IgG in the blood plasma was detected with two ELISA kits 22. Koo, J. R. et al. Interventions to mitigate early spread of SARS-CoV-2 in
targeting N protein and S protein RBD, separately (Guangzhou Darui, China), and Singapore: a modelling study. Lancet Infect. Dis. 20, 678–688 (2020).
one chemiluminescent immunoassay kit targeting N plus S protein (Shenzhen 23. Moriarty, L. F. et al. Public health responses to COVID-19 outbreaks on cruise
YHLO Biotech, China). The IgG levels specific to N plus S protein was also ships - worldwide, February-March 2020. Morb. Mortal Wkly. Rep. 69,
determined by using a lateral flow immunochromatographic assay kit (DIAG- 347–352 (2020).
REAT, Beijing, China). For immunochromatographic assays, the optical signal was 24. Choe, P. G. et al. Waning Antibody Responses in Asymptomatic and
quantified with a time-resolved immunochromatographic analyzer and was cal-
Symptomatic SARS-CoV-2 Infection. Emerging infectious diseases, 27, 327
culated according to established programmed standards. The cut off value for the
(2021).
assignment of positive samples was determined according to the manufacture’s
25. Le Bert, N. et al. SARS-CoV-2-specific T cell immunity in cases of COVID-19
instructions. An individual was considered seropositive if a positive result was
and SARS, and uninfected controls. Nature 584, 457–462 (2020).
generated by all three assays.
26. Braun, J. et al. SARS-CoV-2-reactive T cells in healthy donors and patients
with COVID-19. Nature 587, 270–274 (2020).
Reporting summary. Further information on research design is available in the Nature 27. Peng, Y. et al. Broad and strong memory CD4(+) and CD8(+) T cells induced
Research Reporting Summary linked to this article. by SARS-CoV-2 in UK convalescent individuals following COVID-19. Nat.
Immunol. 21, 1336–1345 (2020).
Data availability 28. Zhou, F. et al. Clinical course and risk factors for mortality of adult inpatients
All relevant data are available from the authors. with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 395,
1054–1062 (2020).
29. Chevalier, M. F. et al. High-throughput monitoring of human tumor-specific
Received: 19 August 2020; Accepted: 24 February 2021; T-cell responses with large peptide pools. Oncoimmunology 4, e1029702
(2015).

Acknowledgements
This work was supported by the National Key Basic Research Project (2019YFC0810900),
References Ministry of Science and Technology of P.R. China, and NSFC 81971485, Guangdong Key
1. Vabret, N. et al. Immunology of COVID-19: current state of the science. Basic Research Project 2019B1515120068, Guangdong Key Research and Development
Immunity 52, 910–941 (2020). Project 2020B1111330001.

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Author contributions Reprints and permission information is available at http://www.nature.com/reprints

Z.W. and P.R. designed the experiments, analysed data, and wrote the paper; X.Y., J.Z.,
and X.M. performed the experiments and analysed data; Y.Z., Z.T., H.Z., J.H., Y.T., and Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
B.L. recruited the cohort and carried out clinical treatments; Z.C., J.C., J.Y., and A.C. published maps and institutional affiliations.
reviewed this work and wrote the paper.

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EXHIBIT 4
DOI: 10.1002/rmv.2260

REVIEW
- -
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Received: 27 April 2021 Revised: 17 May 2021 Accepted: 18 May 2021

Quantifying the risk of SARS
CoV
2 reinfection over time

Eamon O Murchu1,2 | Paula Byrne1 | Paul G. Carty1 | Cillian De Gascun3 |

Mary Keogan4 | Michelle O’Neill1 | Patricia Harrington1 | Máirín Ryan1,5

1
Health Information and Quality Authority,
George’s Court, Dublin, Ireland Summary
2
Trinity College Dublin, Dublin, Ireland Despite over 140 million SARS
CoV
2 infections worldwide since the beginning of the
3
UCD National Virus Reference Laboratory, pandemic, relatively few confirmed cases of SARS
CoV
2 reinfection have been re-
Dublin, Ireland
ported. While immunity from SARS
CoV
2 infection is probable, at least in the short
4
Beaumont Hospital, Dublin, Ireland
5
term, few studies have quantified the reinfection risk. To our knowledge, this is the
Department of Pharmacology & Therapeutics,
Trinity College Dublin, Trinity Health Sciences, first systematic review to synthesise the evidence on the risk of SARS
CoV
2 rein-
Dublin, Ireland fection over time. A standardised protocol was employed, based on Cochrane meth-

Correspondence
odology. Electronic databases and preprint servers were searched from 1 January
Eamon O Murchu, Health Information and 2020 to 19 February 2021. Eleven large cohort studies were identified that estimated
Quality Authority, George’s Court, Dublin 7,
the risk of SARS
CoV
2 reinfection over time, including three that enrolled healthcare
Ireland.
Email: eomurchu@hiqa.ie workers and two that enrolled residents and staff of elderly care homes. Across
studies, the total number o
positive or antibody

of PCR

PCR
positive
positive participants at baseline
antibody
positive
Funding information
Health Research Board, Grant/Award was 615,777, and the maximum duration of follow
up was more than 10 months in
Number: HRB
CICER
2016
1871 three studies. Reinfection was an uncommon event (absolute
absolute rate 0%–1.1%), with no
study reporting an increase in the risk of reinfection over time
time. Only one study esti-
mated the population
level risk of reinfection based on whole genome sequencing in a
subset of patients; the estimated risk was low (0.1% [95% CI: 0.08–0.11%]) with no
evidence of waning immunity for up to 7 months following primary infection. These
data suggest that naturally acquired SARS
CoV
2 immunity does not wane for at least
10 months post
infection. However, the applicability of these studies to new variants
or to vaccine
induced immunity remains uncertain.

KEYWORDS
COVID
19, SARS
CoV
2, reinfection

1 | INTRODUCTION countries worldwide have experienced epidemics of Covid
19. While

much is yet unknown about the immune response following infection
Following the emergence of a novel coronavirus (SARS
CoV
2) in with SARS
CoV
2, evidence is emerging at a fast pace. The Health
China in December 2019 and the declaration by WHO of a public Information and Quality Authority (HIQA) of Ireland has conducted a
health emergency of international concern on 30 January 2020, series of rapid reviews on various public health topics relating to

Abbreviations: Covid
19, coronavirus disease 2019; CI, confidence interval; Ct, cycle threshold; HIQA, Health Information and Quality Authority; IgG, immunoglobulin G; NAAT, nucleic acid
amplification technology; RNA, ribonucleic Acid; RT
PCR, reverse transcription polymerase chain reaction; SARS
CoV
2, severe acute respiratory syndrome coronavirus type 2; WHO, World
Health Organization.

Patricia Harrington and Máirín Ryan are co
senior authors.

Rev Med Virol. 2021;e2260. wileyonlinelibrary.com/journal/rmv © 2021 John Wiley & Sons Ltd. 1 of 11
https://doi.org/10.1002/rmv.2260
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- O MURCHU ET AL.

SARS
CoV
2 infection. These reviews arose directly from questions response through antibody detection (seropositivity) and/or a prior
posed by policy makers and expert clinicians supporting the National SARS
CoV

2 diagnosis by RT
PCR
SARS
CoV
2 RT

PCR or antigen testing followed by
Public Health Emergency Team to inform the national response to recovery (molecular or clinical evidence of viral clearance). No min-
the pandemic in Ireland. imum time interval was defined between primary and secondary in-
Our team at HIQA previously concluded that SARS
CoV
2 fections; however, cases within 90 days of initial infection were
infection produces detectable immune responses in most cases.1 considered suggestive of prolonged viral shedding following the pri-
However, the extent to which previously infected people are immune mary infection.
to reinfection is uncertain. In the short term, protection against All potentially eligible papers, including preprints, were exported
reinfection is probable, as few confirmed SARS
CoV
2 reinfections to Endnote x8.2 and screened for relevance by one reviewer.
have been reported despite over 140 million infections worldwide Following removal of irrelevant citations, two reviewers indepen-
since the beginning of the pandemic.2 dently reviewed the full text of potentially relevant articles. For each
The objective of this systematic review was to evaluate the risk included study, data on study design, participant demographics and
and relative risk of SARS
CoV
2 reinfection over time, comparing relevant clinical and laboratory data were extracted by two re-
previously infected individuals to those without evidence of prior viewers. Quality appraisal was undertaken using the National Heart,
infection. The review informed a range of policy questions relating to Lung and Blood Institute (NIH) quality assessment tool for observa-
the duration of protective immunity (as in, prevention of reinfection) tional cohort studies.6 The findings of the research question were
following SARS
CoV
2 infection. synthesised narratively due to the heterogeneity of study designs
and outcome data.

2 | METHODS
3 | RESULTS
A standardised protocol was employed3 based on Cochrane meth-
odology.4 Electronic databases (PubMed, EMBASE and EuropePMC) The collective database search resulted in 1893 citations, with four
were searched from 1 January 2020 to 19 February 2021 (Data S1). citations retrieved from other sources (grey literature search).
Table 1 outlines the Population, Outcome, Study design (POS) criteria Following removal of duplicates, 1771 citations were screened for
for study selection. relevance. This resulted in 105 studies eligible for full text review
Reinfection was defined as any reverse transcription polymerase (Figure 1), where a further 94 studies were excluded (Table S1).
RT

PCR) or antigen
confirmed
(R
chain reaction (RT
PCR) antigen

confirmed SARS
CoV
2
SARS
CoV

2 infection Eleven studies were identified that met the inclusion criteria.7–17
SARS
CoV

2 infection. Evi-
in an individual with evidence of a prior SARS
CoV
2 Five studies were conducted in the United Kingdom,8,9,11,13,14 of
dence of prior infection included a previously documented immune which three enrolled healthcare workers8,9,11 and two enrolled the

TABLE 1 Population outcome Study design criteria for systematic search

Population Individuals (of any age) with evidence of prior SARS
CoV
2 infection, who subsequently
recovereda

Evidence of prior infection includes diagnosis by RT
PCR or antigen testing, or evidence of

an immune response through antibody detection (seropositivity)

Outcomes 1. Risk of RT
PCR or antigen
confirmed SARS
CoV
2 reinfection over time

2. Relative risk of RT
PCR or antigen
confirmed SARS
CoV
2 reinfection, comparing
populations with evidence of prior infection with populations with no prior evidence
of infection, at specified time points
3. RT
PCR cycle threshold results, if reported
4. Whole genome sequencing results of reinfected cases comparing first and second in-
fections, if reported

Types of studies Include:

Observational cohort studies (prospective or retrospective)
Exclude:
 Cohort studies that enrolled fewer than 100 participants unless the study reported
comparative whole genome sequencing on all reinfection cases
 Studies with durations of follow
up of less than 3 months
 Animal studies

Abbreviation: RT
PCR, reverse transcription polymerase chain reaction.

a
‘Recovered’ refers to molecular or clinical evidence of viral clearance following initial infection; definitions of recovery in primary studies were used.
Common definitions include two consecutive negative respiratory RT
PCR tests 24 h apart and WHO clinical criteria of viral clearance (27 May 2020).5
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O MURCHU ET AL.
- 3 of 11

FIGURE 1 PRISMA diagram of study

selection

staff and residents of elderly care homes.13,14 The remaining six as relative risks, odds ratios, risk ratios and hazard ratios. Due to
16
studies were all general population studies, conducted in Austria, heterogeneity in outcome measures and populations, meta
analysis
Denmark,17 Israel,12 Qatar 7 and the United States.10,15 Six studies of data were not considered appropriate. The following sections
were published as preprints at the time of submission.7,8,10,12,14,15 narratively report the findings of included studies by population
Across studies, the total number of PCR
or antibody
positive par- group (general population, healthcare workers, and residents and
ticipants at baseline was 615,777 (median: 8845; range: 88–378,606). staff of care homes).
The median follow
up of individuals within studies was 131 days
(4.4 months; range of medians: 54–210 days), with a maximum follow

up of ≥300 days (10 months) in three studies.12,14,16 3.1 | General population studies
Studies reported a range of primary endpoints (Table 2 and Ta-
ble S2). Studies either determined evidence of prior infection based 3.1.1 | Austria
on a history of RT
PCR confirmed infection (n = 5 studies), 10,12,15–17

documented antibody detection (n = 4 studies)7,8,11,14 or a combi- In the study by Pilz et al.,16 national SARS
CoV
2 infection data from
nation of both (n = 2 studies). 9,13
Three studies separately reported the Austrian epidemiological reporting system were used to investi-
the relative risks of symptomatic reinfections and ‘all’ reinfections gate potential reinfection events, with a maximum follow
up of
8,11,15
(symptomatic/asymptomatic), one study reported symptomatic 10 months. The primary outcome was the odds of PCR positivity in
reinfections only9 and the remaining studies did not differentiate individuals who recovered from a confirmed SARS
CoV
2 infection
between symptomatic and asymptomatic reinfections.7,10,12–17 In during the first wave (22 February to 30 April 2020) compared with
addition to quantifying the absolute risks of SARS
CoV
2 reinfection, the odds of first infections in the remainder of the general population
the risks compared with PCR
negative or antibody
negative cohorts during the second wave (1 September to 30 November 2020). In
at baseline were expressed by a number of different measures, such total, 40 possible reinfections were recorded out of 14,840
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TABLE 2 Summary of included studies and primary outcome results

First author; country; population Participantsa Follow
up Author reported primary outcomes
7
Abu
Raddad 2021 (preprint); Qatar; General N = 43,044 antibody
positive at Risk of reinfection (confirmed by WGS)b: 0.10% (95% CI:
population baseline 0.08%–0.11%)
Risk over time (any reinfection): Incidence rate of
Median f/u: 114 days (3.8 months)
reinfection by month of follow
up did not show any
Maximum f/u: 242 days (8.1 evidence of waning of immunity over seven months of
months) follow
up

Hall 20218 (preprint); United Kingdom; HCWs N = 6614 antibody
positive at Adjusted odds ratio of reinfection comparing antibody or
baseline PCR
positive group with negative group

Median f/u: 202 days (6.7 • ‘Probable’ reinfectionc: aOR: 0.01 (95% CI 0.00–0.03)
• All ‘possible’ and ‘probable’ reinfections: aOR: 0.17 (95%
months)
CI: 0.13–0.24)
Maximum f/u: 227 days (7.6 • Symptomatic reinfection: aOR: 0.08 (95% CI 0.05–0.13)
months)

Hanrath 20209 United Kingdom; HCWs N = 1038 PCR and/or antibody
Symptomatic reinfection: A positive PCR test was returned
positive at baseline in 0/1038 (0% [95% CI: 0–0.4) of those with previous
infection, compared with 290/10,137 (2.9% [95% CI:
Median f/u: 173 days (5.8 months)
2.6–3.2) of those without (p < 0.0001 2 test)
Maximum f/u: 229 days (7.6
months)

Hansen 202117 Denmark; General population N = 11,068 PCR positive at Main analysis:
baseline aRR (any reinfection): 0.20 (0.16–0.25).
This represents 72 reinfections out of 1,346,920 person

Median f/u: 122 days (4.1 months)
days in PCR
positive group, compared with 16,819 new
Maximum f/u: 295 days (9.8 infections out of 62,151,056 person
days in PCR

months) negative group
Additional cohort analysis (that includes all infection
periods): aRR = 0.21 (0.18–0.25) by age group:
 0–34 years: aRR = 0.17 (0.13–0.23)
 35–49 years: aRR = 0.20 (0.14–0.28)
 50–64 years: aRR = 0.19 (0.13–0.27)
 ≥65 years: aRR = 0.53 (0.37–0.75)

Harvey 202010 (preprint); United States; General N = 378,606 PCR positive at Ratio of positive NAAT results (comparing patients who had
population baseline a positive antibody test at index vs. those without)d: 2.85
(95% CI: 2.73–2.97) at 0
30 days; 0.67 (95% CI: 0.6–
Median f/u: 54 days (1.8 months)
0.74) at 31–60 days; 0.29 (95% CI: 0.24–0.35) at 60–
Maximum f/u: 92 days (3.1 90 days; 0.10 (95% CI: 0.05–0.19) at >90 days; note that
months) NAAT positivity at <90 days is likely due to prolonged
viral shedding

Jeffery
Smith 202113 United Kingdom; N = 88 PCR and/or antibody
Relative risk (any reinfection): 0.04 (95% CI: 0.005–0.27)
Staff &residents at care homes positive at baseline This represents 1 reinfection out of 88 in seropositive
group compared with 22/73 in seronegative group
Mean f/u: 120 days (4 months)

Maximum f/u: Unclear

Krutikov 202114 (preprint); United Kingdom; Staff & N = 634 antibody
positive at Relative adjusted hazard ratios (any reinfection):
residents at care homes baseline Residents of care home: aHR = 0.15 (0.05–0.44)e
Staff of care home: aHR = 0.39 (0.19–0.82)e
Median f/u: 79 days (2.6 months)

Maximum f/u: 300 days (10

months)

Lumley 202111 United Kingdom; HCWs N = 1265 antibody
positive at IRRf(any reinfection): 0.12 (95% CI: 0.03–0.47; p = 0.002);
baseline 2/1265 seropositive (both asymptomatic reinfections)
and N = 223/11,364 seronegative had positive PCR.
Median f/u: 139 days (4.6 months)
Symptomatic reinfection: Incidence was 0.60 per
Maximum f/u: 217 days (7.2 10,000 days at risk in seronegative HCWs; there were
months) no symptomatic infections in seropositive HCWs
Adjusted IRRg: 0.11 (95% CI: 0.03–0.44; p = 0.002) (any
reinfection)
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O MURCHU ET AL.
- 5 of 11

T A B L E 2 (Continued)

First author; country; population Participantsa Follow
up Author reported primary outcomes

Perez 2021 12
(preprint); Israel; General population N = 149,735 PCR positive at Overall reinfection risk: 0.1% (any reinfection between Mar
baseline 2020 and Jan 2021) This represents 154 individuals who
had two positive tests at least 100 days apart out of
Median f/u: 165 days (5.5 months)
149,735 individuals with a record of a prior positive PCR
Maximum f/u: Approx. 325 daysh test
(10.8 months)

Pilz 202116 Austria; General population N = 14,840 PCR positive at Odds ratio: 0.09 (95% CI: 0.07–0.13) (any reinfection)
baseline This represents 40 reinfections out of 14,840 individuals
PCR positive in the first wave (0.27%) compared with
Median f/u: 210 days (7 months)
253,581 infections out of 8,885,640 (2.85%) in the
Maximum f/u: 300 days (10 remaining general population
months)

Sheehan 202115 (preprint); United States; General N = 8845 PCR positive at baseline Protective effectiveness (any reinfection): 78.5% (95% CI:
population 72.0%–83.5%)i
Median f/u: 131 days (4.4 months)
Protective effectiveness against symptomatic infection:
Maximum f/u: 269 days (9 83.1% (95% CI: 75.1%–88.5%)
months)

Note: ‘Any’ reinfection—all reinfections, both symptomatic and asymptomatic. Numbers rounded to two decimal points. No cases were identified on the
basis of antigen testing. The longest duration of follow
up was not stated in all studies or was provided only as an approximate estimate; when not
stated, duration of follow
up was inferred from figures or tables within the study.
Abbreviations: aHR, adjusted hazard ratio; aOR, adjusted odds ratio (adjusted for week group); ARR, adjusted rate ratio; CI, confidence interval; f/u,
follow
up; HCW, healthcare worker; IRR, incidence rate ratio; NAAT, nucleic acid amplification test; WGS, whole genome sequencing.
a
In the baseline antibody and or PCR
positive group (‘seropositive’ or prior positive cohort).
b
Based on cases with WGS confirming the first and second infections were from different viral strains (N = 16).
c
‘Possible’ reinfection was defined as a participant with two PCR
positive samples ≥90 days apart with available genomic data, or an antibody
positive
participant with a new positive PCR at least 4 weeks after the first antibody
positive result. A ‘probable’ case additionally required supportive
quantitative serological data and or supportive viral genomic data from confirmatory samples.
d
NAAT used as proxy; includes all symptomatic reinfections and prolonged viral shedding, comparing patients who had a positive antibody test at index
versus those with a negative antibody.
e
Multivariate analysis of risk of PCR
positive infection by baseline antibody status, stratified by LTCF and adjusted for sex and age.
f
IRR is the relative incidence of subsequent positive SARS
CoV
2 PCR tests and symptomatic infections comparing antibody
positive and antibody

negative groups at baseline.
g
After adjustment for age, gender and month of testing or calendar time as a continuous variable.
h
The midpoint of a range of follow
up dates was taken (300–349 days).
i
Authors report effectiveness with the following calculation: 1−([56/8845]/[4163/141480]).

individuals with a history of prior infection during the first wave 11,068 (2.11%) had tested positive during the first wave. Among
(0.27%), compared with 253,581 infections out of 8,885,640 in- eligible PCR
positive individuals from the first wave, 72 (0.65%,
dividuals of the remaining general population (2.85%). This translated 95% CI: 0.51%–0.82%) tested positive again during the second
into an odds ratio of 0.09 (95% CI: 0.07–0.13). wave compared with 16,819 of 514,271 (3.27%, 95% CI: 3.22%–
3.32%) who tested negative during the first wave. After adjusting
for sex, age group and test frequency, the adjusted RR (aRR) of
3.1.2 | Denmark reinfection was 0.20 (95% CI: 0.16–0.25). Protection against repeat
infection was estimated at 80.5% (95% CI: 75.4–84.5). In an
In the study by Hansen et al.,17 individual
level data were collected alternative analysis, aRR by age category was reported. In in-
on patients who had been tested in Denmark in 2020 from the dividuals aged 65 years or more, the aRR was 0.53 (0.37–0.75),
Danish Microbiology Database, with a maximum follow
up of compared with 0.17, 0.20 and 0.19 in individuals aged 0–34 years,
9.8 months. Infection rates were analysed during the second wave 35–49 years and 50–64 years, respectively.
of the COVID
19 epidemic, from 1 September 2020 to 31
December 2020, comparing PCR
positive individuals with PCR

negative individuals during the first wave (March to May 2020). 3.1.3 | Israel
During the first wave (prior to June 2020), 533,381 people were
tested, of whom 11,727 (2.2%) were PCR positive. Of these, In the study by Perez et al.,12 published as a preprint, preliminary
525,339 were eligible for follow
up in the second wave, of whom reinfection rates within the members of a large healthcare provider
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- O MURCHU ET AL.

(Maccabi Healthcare Services) in Israel were reported, with a estimated at 78.5% (95% CI: 72.0–83.5) and 83.1% (95% CI: 75.1–
maximum follow
up of over 10 months. A total of 149,735 individuals 88.5) against symptomatic reinfection.
had a recorded positive PCR test between March 2020 and January
2021. Among them, 154 members had two positive PCR tests at least
100 days apart and were included in this study. The reinfection rate 3.2 | Healthcare workers
was estimated at approximately 0.1%. In this cohort, 73 individuals
(47.4%) had symptoms at both PCR
positive events. Three UK studies were identified that exclusively enrolled healthcare
workers. In the first study, published as a preprint, 20,787 hospital
staff were followed, of whom 32% (n = 6614) were assigned to the
3.1.4 | Qatar positive cohort (antibody or PCR positive) and 68% (n = 14,173) to the
negative cohort (antibody negative, not previously known to be PCR
In the study by Abu
Raddad et al., published as a preprint, 43,044 or antibody positive) (Hall et al.8). In total, 1,339,078 days of follow
up
anti
SARS
CoV
2 nucleocapsid antibody
positive participants were data were analysed from the baseline positive cohort (maximum
followed for up to 8 months for evidence of reinfection.7 This follow
up of 7.6 months). In total, 44 reinfections (2 probable and 42
retrospective cohort was identified from a database that covers all possible) were detected in the baseline positive cohort (15 of which
serological testing for SARS
CoV
2 conducted in Qatar. were symptomatic), compared with 318 new PCR
positive infections
There was evidence of a decreasing trend in the incidence rate of (249 of which were symptomatic) and 94 antibody seroconversions in
reinfection with each additional month of follow
up from the first the negative cohort. The adjusted odds ratio (aOR) was 0.17 for all
month (incidence rate: 0.97 per 10,000; 52 cases per 167,149 per- reinfections (‘possible’ or ‘probable’; 95% CI: 0.13–0.24). Restricting
son
weeks) to the sixth month (zero cases per 19,148 person
weeks) reinfections to probable reinfections only, participants in the positive
(Mantel
Haenszel trend analysis p
value: <0.001), noting that early cohort had a 99% lower odds of probable reinfection (aOR of 0.01,
reinfection cases (i.e., within 3 months) were likely due to persistent 95% CI: 0.00–0.03). Restricting reinfections to those who were
viral shedding following the primary infection. There was an increase symptomatic, investigators estimated that participants in the positive
at ≥7 months; however, this was based on only one case of rein- cohort had an aOR of 0.08 (95% CI 0.05–0.13).
fection (out of 3094 person
weeks). Applying a confirmation rate In the second study, 1038 healthcare workers with evidence of
obtained through viral genome sequencing in a subset of patients previous infection (PCR and or antibody positive) and 10,137 without
with supporting clinical evidence for reinfection, the risk of docu- (negative antibody and PCR) were followed for a maximum of
mented reinfection was 0.1% (95% CI: 0.08%–0.11%). 7.6 months (Hanrath et al.9). A positive PCR test was returned in 0%
These reinfections were compared to a cohort of 149,923 (0/1038 [95% CI: 0%–0.4%]) of those with previous infection,
antibody
negative individuals followed for a median of 17 weeks compared to 2.9% (290/10,137 [95% CI: 2.6–3.2]) of those without
(range: 0–45.6 weeks). Risk of infection was estimated at 2.15% (95% (p < 0.0001,
2 test).
CI: 2.08%–2.22%). The efficacy of natural infection in protecting In the third study, 12,541 UK healthcare workers were followed
against reinfection was estimated at 95.2% (95% CI: 94.1%–96.0%). for up to 31 weeks to compare the incidence of SARS
CoV
2 infec-
tion in seropositive (N = 1265, including 88 who seroconverted
during follow
up) versus seronegative (N = 11,364) groups at base-
3.1.5 | United States line (Lumley et al.11). A total of 223 anti
spike seronegative health-
care workers had a positive PCR test, 100 during screening while
Two US studies were identified, both published as preprints. In the first, they were asymptomatic and 123 while symptomatic, whereas two
a retrospective database analysis of electronic health records was used anti
spike seropositive healthcare workers had a positive PCR test;
to determine the risk of nucleic acid amplification technology (NAAT) both workers were asymptomatic when tested. Incidence varied by
test positivity, a proxy for reinfection, over a maximum follow
up of calendar time, reflecting the first (March through April) and second
3.1 months (Harvey et al.10). Of 3,257,478 unique patients with an (October and November) waves of the pandemic in the United
index antibody test, 378,606 (11.6%) had a positive antibody result at Kingdom and was consistently higher in seronegative healthcare
baseline. The ratio of positive NAAT test results among patients who workers. After adjustment for age, gender and month of testing or
had a positive antibody test at index versus those with a negative calendar time as a continuous variable, the incidence rate ratio in
antibody test at index declined from 2.85 (95% CI: 2.73–2.97) at 0– seropositive workers was 0.11 (95% CI: 0.03–0.44) compared with
30 days; to 0.67 (95% CI: 0.6–0.74) at 31–60 days; to 0.29 (95% CI: those who were seronegative at baseline.
0.24–0.35) at 60–90 days and to 0.10 (95% CI: 0.05–0.19) at >90 days.
In the second, 150,325 patients were followed for a maximum of
10 months (Sheehan et al.15). In total, 56 reinfections were identified 3.3 | Residents and staff of elderly care homes
from the positive cohort of 8845 individuals, compared with 4163
infections from the negative cohort of 141,480 individuals. The Two studies were identified that enrolled both residents and staff at
protective effectiveness of prior infection against reinfection was UK care homes.13,14
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O MURCHU ET AL.
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In the first study (Jeffery
Smith et al.13), the risk of reinfection reinfection events in their respective populations. A number of studies
according to antibody seropositivity was investigated following out- was downgraded due to lack of controlling for confounders (n = 7
breaks in two London care homes13,18 over 4 months. The median studies). In these studies, potential confounding variables were either
age of residents was 84 and 85 in each care home. not assessed or not measured appropriately, or the statistical analysis
In total, 88 individuals with evidence of prior infection were was not adequately described. As all studies were observational in
investigated for evidence of reinfection (antibody positive N = 87; nature, they cannot be used to demonstrate causality. Therefore, only
PCR positive N = 1). The reinfection rate in this cohort was 1/88 associations between prior infection and reinfection risk can be
(1.1%), and this reinfection event was observed in a staff member. By measured. While estimates of the effectiveness of natural infection to
comparison, infection risk in the seronegative cohort was 30.1% (22/ prevent reinfection were reported in a number of studies, such mea-
73, including four people diagnosed by seroconversion). The RR was sures cannot be reliably estimated on the basis of these data.
estimated at 0.038 (95% CI: 0.005–0.273). The protection against Six studies are currently published as preprints,7,8,10,12,14,15 so
reinfection after four months in seropositive group was estimated at have not yet been formally peer
reviewed, raising additional con-
96.2% (95% CI: 72.7%–99.5%). cerns about overall quality and the potential for results to change
In the second study, published as a preprint, staff and residents prior to formal publication.
in 100 long
term care facilities (LTCFs) in England were followed
between October 2020 and February 2021 (Krutikov et al.14). In
total, 2111 individuals were enrolled (682 residents and 1429 staff). 4 | DISCUSSION
The median age of residents was 86 years (IQR: 79–91) and 47 years
for staff (IQR range: 34–56). Blood sampling was offered to all par- 4.1 | Summary of findings
ticipants at three time points separated by 6–8 weeks intervals in
June, August and October 2020. Samples were tested for IgG anti- Eleven cohort studies estimated the risk or relative risk of SARS

bodies to nucleocapsid and spike protein. PCR testing for SARS
CoV
CoV
2 reinfection in individuals who were either antibody
positive
2 was undertaken weekly in staff and monthly in residents. The or who had a history of PCR
confirmed Covid
19 at baseline,
primary analysis estimated the adjusted hazard ratio (aHR) of a PCR
compared with those who did not, for up to 10 months. Across
positive test by baseline antibody status (Cox regression adjusted for studies, the total number of PCR
or antibody
positive participants at
age and gender, and stratified by LTCF). baseline was 615,777, with a maximum follow
up of over 10 months
IgG antibodies to nucleocapsid were detected at baseline in 226 in three studies. Reinfection was a rare event (median PCR

residents (33%) and 408 staff (29%). Staff and residents contributed w
confirmed reinfection rate: 0.27%, range: 0%–1.1%), with no study
3749 and 1809 months of follow
up time, respectively. There were reporting an increase in the risk of reinfection over time.
93 PCR
positive tests in seronegative residents (0.054 per month at Of the six general population studies, only one estimated the
risk) compared with four in seropositive residents (0.007 per month population
level risk of reinfection based on whole genome
at risk). There were 111 PCR
positive tests in seronegative staff sequencing in a subset of patients with supporting evidence of
(0.042 per month at risk) compared with 10 in seropositive staff reinfection.7 The estimated risk was low (0.1% [95% CI: 0.08%–
(0.009 per month at risk). Controlling for the potential confounding 0.11%]) in this large cohort of 43,044 anti
SARS
CoV
2 nucleocapsid
effect of individual LTCFs, the relative aHRs for PCR
positive infec- antibody
positive participants. Importantly, the incidence rate of
tion were 0.15 (95% CI: 0.05–0.44) and 0.39 (95% CI: 0.19–0.82) reinfection by month did not show any evidence of waning of im-
comparing seropositive versus seronegative residents and staff, munity over the seven months of follow
up. The remaining
respectively. Study authors concluded that the presence of IgG an- population
based studies (conducted in Austria, Denmark, Israel and
tibodies to nucleocapsid was associated with substantially reduced the United States) also reported low absolute and relative risks of
risk of reinfection in staff and residents for up to 10 months after reinfection, and none reported an increased risk over time.
primary infection, assuming that the earliest infections occurred in Only one study reported the relative risk of reinfection by age
March 2020. category, allowing comparisons across groups. In individuals aged
65 years or more, the aRR was 0.53 (0.37–0.75), compared with 0.17,
0.20 and 0.19 in individuals aged 0–34 years, 35–49 years and 50–
3.4 | Quality of included studies 64 years, respectively.17 The lower protection in the over
65s group
may be attributable to immunosenescence; however, little is known
The NIH quality assessment tools was used for appraisal of obser- about this phenomenon in the context of COVID
19.
6
vational cohort studies. Ten studies were considered of ‘good’ or Two UK studies reported lower risks of reinfection in elderly in-
‘fair’ methodological quality (Table S3), with one study10 that used a dividuals. Both studies enrolled residents of care homes (median age
proxy measure for outcomes (NAAT test positivity) considered to be ≥84 years), a group that has been disproportionately affected by the
of poor quality. COVID
19 pandemic, with high rates of infection and deaths among
Each of the 10 studies of ‘good’ (n = 4) or ‘fair’ (n = 6) methodo- frail, elderly residents. In the first study, the relative risk of reinfection
logical quality was considered large enough to adequately capture in staff and residents of two London care homes was very low
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(RR = 0.038; 95% CI: 0.005–0.273), and the protection against rein- demonstrated a substantially lower risk of reinfection in previously
fection after four months in seropositive group was estimated at 96.2% infected individuals without a waning of the protective response over
(95% CI: 72.7%–99.5%).13 This relative risk was based on a single time. However, despite these strengths, there are a number of limi-
reinfection event in a seropositive staff member, indicating the relative tations associated with this review.
risk in the elderly resident cohort is even lower. The second study re- First, as the studies are observational in nature, the prevention
ported higher relative rates of reinfection14 in a sample of staff and of reinfection cannot be causally confirmed, although longitudinal
residents (N = 2111) across 100 LTCFs in England. The study, con- associations can be estimated. Additional concerns relating to
ducted between October 2020 and February 2021, coincided with a observational studies include the greater potential for bias. It is
period of high community prevalence of SARS
CoV
2 in the United possible that antibody test results affected individual behaviour. In-
Kingdom, associated with the rapid emergence of the B.1.1.7 variant.19 dividuals with evidence of prior infection may have believed that they
The estimated aHR for reinfection was 0.15 (95% CI: 0.05–0.44) in possessed immunity to SARS
CoV
2, resulting in a reduction in
residents and 0.39 (95% CI: 0.19–0.82) in staff. The higher relative health
seeking behaviour and testing (outcome ascertainment bias).
rates of infection compared with the earlier UK study raises concerns Conversely, these individuals may have increased their engagement
regarding the impact of new variants on the protective immunity of in social behaviour, placing them at greater risk for infection. The
natural infection. Nonetheless, only four cases of possible reinfection overall direction of bias (whether over
or under
estimating rein-
were identified in residents, and although all cases reported symptoms, fection) cannot be determined.
none required hospital treatment. Taking into consideration that most Second, studies included in this review could not determine
residents were likely first infected during the first wave (up to 6 months whether past seroconversion, or current antibody levels, determine
prior), the risk of reinfection was substantially reduced in residents protection from infection. Furthermore, none could define which
even in the context of high community transmission of the B.1.1.7 characteristics are associated with reinfection. For example, there is
variant. evidence to suggest immune responses are weaker following
Three UK studies estimated the relative risk of reinfection spe- asymptomatic SARS
CoV
2 infections20 and in immunocompromised
8,9,11
cifically among healthcare workers. The first study detected zero patients,21 which may increase susceptibility to repeat infection.
symptomatic infections in 1038 healthcare workers with evidence of Mucosal immunity and neutralising antibodies present in respiratory
a prior infection, compared with 290 in 10,137 without evidence of secretions may be more important for sterilising immunity than
prior infection (p < 0.0001). 9
The second study detected two circulating IgG levels. The role of T
cell immunity was not assessed in
asymptomatic infections (and no symptomatic infections) out of 1265 any study; therefore, it is not possible to determine whether pro-
seropositive individuals, compared with 223 infections (100 during tection from reinfection is conferred through the measured anti-
screening while they were asymptomatic and 123 while symptom- bodies or T
cell immunity. Future longitudinal serological cohorts
11
atic) out of 11,364 seronegative individuals. After adjustment for may be able to determine protective correlates of immunity.
age, gender and month of testing or calendar time, the incidence rate Third, only two studies undertook genomic sequencing of rein-
ratio in seropositive healthcare workers was 0.11 (95% CI: 0.03– fected cases; consequently, the results of nine studies are only based
0.44). The third study reported 44 reinfections in the baseline posi- on potential reinfections. The effect of this, however, is to over-
tive cohort of 6614 individuals (15 of which were symptomatic), estimate the number of reinfections, thereby affirming the conclusion
compared with 318 new PCR
positive infections (249 of which were that reinfection is rare.
symptomatic) and 94 antibody seroconversions in the negative Fourth, due to the nature of a number of retrospective database
8
cohort of 14,173 individuals. The aOR was 0.17 for all reinfections analyses included in this review, many studies could not correlate
(95% CI: 0.13–0.24), and restricting reinfections to those who were symptomatic infections with protection against repeat infection or
symptomatic, the aOR was 0.08 (95% CI 0.05–0.13). This pattern of a evaluate disease progression comparing first and second infections.
lower relative risk of symptomatic reinfections in healthcare workers, This was true for studies that accessed large databases in Austria,16
compared with ‘any’ reinfection (symptomatic and asymptomatic), Denmark17 and the United States.10
was also observed in the study by Sheehan et al. in general pop- Finally, this review included a number of studies that were
ulations.15 This finding suggests that not only is the risk of reinfection published as preprints (n = 6 studies7,8,10,12,14,15). While preprints
following natural infection low, when it does occur, it may represent a have been pivotal to guide policy and practice throughout this
less severe form of disease. pandemic, these studies have not yet been formally peer
reviewed
raising concerns over the quality and accuracy of presented data.

4.2 | Strengths and limitations

4.3 | Generalisability of findings
To our knowledge, this is the first systematic review to quantify the
risk of SARS
CoV
2 reinfection over time. All studies were consid- There are a number of issues relating to the applicability and gen-
ered large enough to adequately capture reinfection events in their eralisability of the presented results. First, all but two studies pre-
respective populations. Results across studies consistently ceded the widespread identification and spread of a number of new
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viral strains of international concern (e.g., variant 202012/01 [also 4.4 | Research in context and policy implications
known as 501Y.V1/B.1.1.7] from the United Kingdom and 501Y.V2
[B.1.351] from South Africa, both identified in December 202022). In This review was expected to inform a range of policy questions
the first study that extended beyond December 2020, reinfection relating to the duration of protective immunity following infection
events between March 2020 and January 2021 in Israel were with SARS
CoV
2, such as:
recorded.12 A higher number of reinfections was recorded in January
2021 compared with previous months. However, genomic sequencing  How long can asymptomatic individuals who have recovered from
was not reported and statistical analysis of the recorded data (e.g., a prior SARS
CoV
2 infection be exempted from restriction of
controlling for confounders and significance testing) was not under- movement policies if they become a close contact of a confirmed
taken. In the second study, elderly care home staff and residents in COVID
19 case?
the United Kingdom were followed between October 2020 and  How long can asymptomatic individuals who have recovered from
February 2021.14 Sequencing data were not available for suspected a prior SARS
CoV
2 infection be exempted from serial testing
reinfections, and study authors did not investigate the potential programmes?
impact of new variants on the risk of reinfection. Nonetheless, the  How long can asymptomatic patients who have recovered from a
risk of reinfection was substantially reduced in elderly residents, prior SARS
CoV
2 infection be exempted from the requirement
most of whom were first infected up to 6 months previously. While for testing prior to scheduled admission to hospital?
these findings are reassuring, further research is needed on the role
of natural immunity in populations that are experiencing the emer- This review identified a large body of evidence that indicates
gence and spread of new variants of concern. the duration of presumptive protective immunity may last for at up
Second, all presented data relate to unvaccinated cohorts as they to 10 months post
infection. However, given the uncertainty that
preceded vaccine roll
out in 10 studies, and in the only study that exists relating to reinfection potential with emerging variants, any
was conducted during vaccine roll
out, all vaccinated individuals policy changes may not be applicable to possible exposure to
14
were excluded once 12 days had passed since their vaccination. emerging immune escape variants of concern. In addition, policies
The applicability of the data to vaccinated populations is therefore should be kept under review and informed by the international
unknown. evidence and national surveillance data. In light of the findings of
23
One preprint study (Lumley et al., 2021 ), identified after our this review, policy was updated in Ireland to extend the period of
database search, reported reinfection rates among healthcare workers presumptive immunity from 3 months to 6 months; therefore, a
according to vaccination status and in relation to the B.1.1.7 variant. person who is an asymptomatic contact of a case and has had a
This study updates the 2020 study included in this review by the same positive test result within the previous 6 months is exempt from
11
authors and presents data up to 28 February 2021. At this time point, restriction of movements and serial testing. A period of 6 months
1456 of 13,109 participating healthcare workers had received two was selected over 10 months due to the ongoing uncertainties
vaccine doses (Pfizer
BioNTech or Oxford
AstraZeneca). Compared to relating to new variants.
unvaccinated seronegative healthcare workers, natural immunity and Increasingly, reinfection cases are being investigated on a
two vaccination doses provided similar protection against symptom- country level and are reported on websites of national public health
atic infection: no healthcare worker who had received two vaccine agencies (e.g., Czechia now report a national reinfection rate of 0.1%,
doses had a symptomatic infection, and incidence was 98% lower in or 1400 cases out of 1,225,000 infections24). Future longitudinal
seropositive healthcare workers (adjusted incidence rate ratio 0.02, studies should focus on the following issues that were not addressed
95% CI: <0.01–0.18). Two vaccine doses or seropositivity reduced the in the aforementioned studies, including:
incidence of any PCR
positive result with or without symptoms by 90%
(0.10, 95% CI: 0.02–0.38) and 85% (0.15 95% CI: 0.08–0.26) respec-  The durability of immunity beyond 10 months
tively. There was no evidence of differences in immunity induced by  Immune correlates of protection
natural infection and vaccination for infections with the B.1.1.7 variant.  Protective immunity in populations with comorbidities and the
These data suggest that both natural infection and vaccination both immunocompromised
provide robust protection against SARS
CoV
2 infection, including  The impact of new variants on protective immunity
against the B.1.1.7 variant. Future studies are expected to expand our
understanding of the differences between natural and vaccine

acquired immunity and the impact of new variants. 5 | CONCLUSIONS
Third, there is much uncertainty in relation to the risk of reinfec-
tion in younger and older age groups. Inconsistent data were identified Eleven large cohort studies were identified that estimated the risk of
relating to elderly populations, with one study reporting higher rates of SARS
CoV
2 reinfection over time, including three that enrolled
reinfection compared with younger age groups17 and two reporting healthcare workers and two that enrolled elderly care home resi-
low rates of reinfection in elderly residents of care homes (although dents. All studies reported low relative SARS
CoV
2 reinfection rates
these two studies did not compare risk across age groups).13,14 in individuals with prior evidence of infection, compared with those
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without, for up to 10 months. The relative risk of reinfection was low 7. Abu
Raddad L, Chemaitelly H, Coyle P, et al. SARS
CoV
2 reinfec-
across studies, although there was some inconsistent evidence of a tion in a cohort of 43,000 antibody
positive individuals followed for
up to 35 weeks. medRxiv; 2021. https://doi.org/10.1101/2021.01.15.
higher risk in older populations compared with younger populations.
21249731
A limitation of this review was the uncertainty regarding the appli- 8. Hall V, Foulkes S, Charlett A, et al. Do antibody positive healthcare
cability of data to new variants of concern and to vaccinated workers have lower SARS
CoV
2 infection rates than antibody
populations. negative healthcare workers? Large multi
centre prospective cohort
study (the SIREN study), England: June to November 2020. medRxiv;
2021. https://doi.org/10.1101/2021.01.13.21249642
ACKNOWLEDGEMENTS 9. Hanrath AT, Payne BAI, Duncan CJA. Prior SARS
CoV
2 infection
The authors would like to thank our executive assistant Debra is associated with protection against symptomatic reinfection. J
Spillane (HIQA) and librarians from the Health Service Executive Infect. 2020;82(4):e29
e30. https://doi.org/10.1016/j.jinf.2020.
12.023
(HSE) for their valued input and support. This research was funded in
10. Harvey R, Rassen J, Kabelac C, et al. Real
world data suggest anti-
part by the Health Research Board under grant no. HRB
CICER
body positivity to SARS
CoV
2 is associated with a decreased risk of
2016
1871. future infection. medRxiv; 2020. https://doi.org/10.1101/2020.12.
18.20248336
CONFLICT OF INTEREST 11. Lumley SF, O'Donnell D, Stoesser NE, et al. Antibody status and
incidence of SARS
CoV
2 infection in health care workers. N. Engl J
No conflict of interest declared.
Med. 2020;384(6):533
540. https://doi.org/10.1056/
nejmoa2034545
AUTHOR CONTRIBUTIONS 12. Perez G, Banon T, Gazit S, et al. A 1 to 1000 SARS
CoV
2 reinfection
Eamon O Murchu: Investigation, formal analysis and writing
original proportion in members of a large healthcare provider in Israel: a
preliminary report. medRxiv; 2021. https://doi.org/10.1101/2021.03.
draft. Paula Byrne: Investigation and writing
original draft. Paul G.
06.21253051
Carty: Investigation and writing
original draft. Cillian De Gascun: 13. Jeffery
Smith A, Iyanger N, Williams SV, et al. Antibodies to SARS

Writing
reviewing and editing. Mary /Keogan: Writing
reviewing and CoV
2 protect against re
infection during outbreaks in care
editing. Michelle O’Neill: Supervision, writing
reviewing and editing. homes, September and October 2020. Euro Surveill.
2021;26(5):2100092. https://doi.org/10.2807/1560
7917.es.2021.
Patricia Harrington: Supervision, writing
reviewing and editing.
26.5.2100092
Máirín Ryan: Supervision, writing
reviewing and editing. All authors 14. Krutikov M, Palmer T, Tut G, et al. Incidence of SARS
CoV
2
attest they meet the ICMJE criteria for authorship. infection according to baseline antibody status in staff and resi-
dents of 100 long term care facilities (VIVALDI study). medRxiv;
2021. https://doi.org/10.1101/2021.03.08.21253110
DATA AVAILABILITY STATEMENT
15. Sheehan M, Reddy A, Rothberg M. Reinfection rates among patients
The data that supports the findings of this study are available in the who previously tested positive for COVID
19: a retrospective
supplementary material of this article. cohort study. medRxiv; 2021. https://doi.org/10.1101/2021.02.14.
21251715
16. Pilz S, Chakeri A, Ioannidis JP, et al. SARS
CoV
2 re
infection risk in
ORCID
Austria. Eur J Clin Invest. 2021;51(4):e13520.
Eamon O Murchu https://orcid.org/0000-0003-3926-0179 17. Hansen CH, Michlmayr D, Gubbels SM, Mølbak K, Ethelberg S.
Assessment of protection against reinfection with SARS
CoV
2
REFERENCES among 4 million PCR
tested individuals in Denmark in 2020: a
1. O Murchu E, Byrne P, Walsh KA, et al. Immune response following population
level observational study. Lancet.
infection with SARS
CoV
2 and other coronaviruses: a rapid re- 2021;397(10280):1204
1212. https://doi.org/10.1016/s0140
6736
view. Rev Med Virol. 2021;31(2):e2162. https://doi.org/10.1002/ (21)00575
4
rmv.2162 18. Ladhani SN, Jeffery
Smith A, Patel M, et al. High prevalence of
2. Johns Hopkins University & Medicine. COVID
19 Dashboard SARS
CoV
2 antibodies in care homes affected by COVID
19: pro-
2020. https://coronavirus.jhu.edu/map.html. Accessed February 6, spective cohort study, England. EClinicalMedicine. 2020;28:100597.
2021. https://doi.org/10.1016/j.eclinm.2020.100597
3. HIQA. Health Information and Quality Authority. Protocol for evi- 19. Public Health England (PHE). Investigation of novel SARS
CoV
2
dence synthesis support—COVID
19 2020. http://www.hiqa.ie/sites/ variant Variant of concern 202012/01 technical briefing 4. https://
default/files/2020
05/Protocol
for
HIQA
COVID
19
evidence
assets.publishing.service.gov.uk/government/uploads/system/
synthesis
support_1
6.pdf uploads/attachment
data/file/952490/Variant
of
Concern
VOC

4. Higgins JPT, Thomas J, Chandler J, Cumpston M, Li T, Page MJ, 20201201
Technical_Briefing_4_England.pdf
Welch VA, eds. Cochrane Handbook for Systematic Reviews of In- 20. Long Q
X, Tang X
J, Shi Q
L, et al. Clinical and immunological
terventions version 6.2 (updated February 2021). Cochrane; 2021. assessment of asymptomatic SARS
CoV
2 infections. Nat Med.
Available from www.training.cochrane.org/handbook 2020;26(8):1200
1204. https://doi.org/10.1038/s41591
020

5. World Health Organization (WHO). Criteria for Releasing COVID19 0965
6
Patients from Isolation: Scientific Brief. 2020. https://www.who.int/ 21. Hunsinger DHP, Kutti Sridharan DG, Rokkam DVRP, Fantry DLE.
news
room/commentaries/detail/criteria
for
releasing
covid
19
COVID
19 reinfection in an immunosuppressed patient without an
patients
from
isolation. Accessed September 22, 2020. antibody response. Am J Med Sci. 2021;S0002

6. National Heart Lung and Blood Institute (NIH). Study quality 9629(0021):00050
00051.
assessment tools. Available at: https://www.nhlbi.nih.gov/health
22. European Centre for Disease Prevention and Control (ECDC). Risk
topics/study
quality
assessment
tools related to the spread of new SARS
CoV
2 variants of concern in the
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 48 of 165
O MURCHU ET AL.
- 11 of 11

EU/EEA—first update. https://www.ecdc.europa.eu/sites/default/ SUPPORTING INFORMATION

files/documents/COVID
19
risk
related
to
spread
of
new
SARS
CoV
Additional supporting information may be found online in the Sup-
2
variants
EU
EEA
first
update.pdf
porting Information section at the end of this article.
23. Lumley SF, Rodger G, Constantinides B, et al. An Observational
Cohort Study on the Incidence of SARS
CoV
2 Infection and B.1.1.7
Variant Infection in Healthcare Workers by Antibody and Vaccina-
tion Status. medRxiv.; 2021. https://doi.org/10.1101/2021.03.09.
21253218 How to cite this article: O Murchu E, Byrne P, Carty PG, et al.
24. State Institute of Public Health (SZU). The number of cases of covid

Quantifying the risk of SARS
CoV
2 reinfection over time. Rev
19 reinfections in the Czech Republic has increased 2021. http://
www.szu.cz/tema/prevence/pocet
pripadu
reinfekci
covid
19
v
cr
Med Virol. 2021;e2260. https://doi.org/10.1002/rmv.2260
vzrostl
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EXHIBIT 5
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Comparison of kinetics of immune responses to SARS-CoV-2 proteins in

individuals with varying severity of infection and following a single dose of the

AZD1222

Deshni Jayathilaka1#, Chandima Jeewandara1#, Laksiri Gomes1, Tibutius Thanesh

Pramanayagam Jayadas1, Achala Kamaladasa1, Dinuka Guruge2, Pradeep Darshana1, Thushali

Ranasinghe1, Inoka Sepali Abyrathna1, Saubhagya Danasekara1, Buddini Gunathilaka1, Heshan

Kuruppu1, Ananda Wijewickrama3, Ruwan Wijayamuni2, Lisa Schimanski4,5, T.K. Tan3,4,

Graham S. Ogg5, Alain Townsend4,5, Gathsaurie Neelika Malavige1,5*

1
Allergy Immunology and Cell Biology Unit, Department of Immunology and Molecular

Medicine, University of Sri Jayewardenepura, Nugegoda, Sri Lanka; 2Colombo Municipal

Council, Colombo, Sri Lanka; 3 National Institute of Infectious Disease, Sri Lanka; 4 MRC

Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of

Oxford, Oxford, United Kingdom; 4 Centre for Translational Immunology, Chinese Academy of

Medical Sciences Oxford Institute, University of Oxford, Oxford, United Kingdom

Correspondence should be addressed to:

Prof. Neelika Malavige DPhil (Oxon), FRCP (Lond), FRCPath (UK)

Allergy Immunology and Cell Biology Unit, Department of Immunology and Molecular

Medicine, Faculty of Medical Sciences, University of Sri Jayawardanapura, Sri Lanka.

Tel +94 (0) 772443193; Fax: +94 (0) 112802026, Email: gathsaurie.malavige@ndm.ox.ac.uk

# Contributed equally

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.
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Abstract

Background: While there have been many studies characterizing the IgG and IgA responses to

different SARS-CoV-2 proteins in individuals with natural infection, the induction of IgG and

IgA to different viral proteins in vaccinees have not been extensively studied. Therefore, we

sought to investigate the antibody responses to SARS-CoV-2 following natural infection and

following a single dose of AZD2221, in Sri Lankan individuals.

Methods: Using Luminex assays, we characterized the IgG and IgA responses in patients with

varying severity of illness and following a single dose of the vaccine at 4 weeks and 12 weeks

since onset of illness or following vaccination. Haemagglutination test (HAT) was used to assess

the antibodies to the receptor binding domain of SARS-CoV-2 wild type (WT), B.1.1.7, B.1.351

and B.1.617.2 (VOCs) and surrogate neutralizing test to measure ACE2 receptor blocking

antibodies.

Results: Those with mild illness and in vaccinees, the IgG responses to S1, S2, RBD and N

protein increased from 4 weeks to 12 weeks, while it remained unchanged in those with

moderate/severe illness. Those who had a febrile illness in 2017 and 2018 (controls) also gave

IgG and IgA high responses to the S2 subunit. In the vaccinees, the most significant rise was

seen for the IgG antibodies to the S2 subunit (p<0.0001). Vaccinees had several fold lower IgA

antibodies to all the SARS-CoV-2 proteins tested than those with mild and moderate/severe

illness at 4 weeks and 12 weeks. At 12 weeks the HAT titres were significantly lower to the

B.1.1.7 in vaccinees and significantly lower in those with mild illness, and in vaccinees to

B.1.351 and for B.1.617.2. No such difference was seen in those with moderate/severe illness.
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Conclusions: Vaccinees
Vaccinees had significantly less IgA to SARS-CoV-2, but comparable IgG

responses to those with natural infection. However, following a single dose, vaccinees had

reduced antibody levels to the variants of concern (VOC), which further declined with time,

compared to natural infection.

infection
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Introduction

The COVID-19 pandemic due to SARS-CoV-2, continues to cause significant mortality and

morbidity, and many countries are currently experiencing a worse situation, than at the beginning

of the pandemic1. Emergence of SARS-CoV-2 variants of concern such as the B.1.1.7 (alpha)

and more recently B.1.617.2 (delta) has led to exponential increase of the number of COVID-19

cases and deaths in many countries1-3. While the higher income countries have vaccinated a large

proportion of their population, resulting in lower hospitalizations and deaths, many lower income

and lower-middle income countries are grappling with the increase in the case loads,

overburdening of health care resources and the inability to secure adequate doses of COVID-19

vaccines4.

Although the duration of protection against re-infection from SARS-CoV-2 in not known, it has

been shown that re-infection does occur, especially among older individuals, probably due to

waning of immunity5. Re-infection has shown to occur particularly with certain variants such as

P.1 (gamma) variant in Brazil despite a very high seroprevalence6, and also with B.1.351 (beta)

due to escape from natural and vaccine induced immunity7. Individuals who had experienced

milder illness have shown to have reduced levels of neutralizing antibodies compared to those

who had severe illness8,9. Apart from the presence of neutralizing antibodies to the receptor

binding domain (RBD), antibodies specific to S2 and N protein of SARS-CoV-2 are also

detected in patients who have recovered from COVID-1910. Although the usefulness of

antibodies directed against S1, S2 and N protein in preventing re-infection are not known,

although IgG and IgA specific to S1, S2 have been detected in breast milk of infected mothers
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and therefore, possibly provide protection to the neonate11. Antibodies against the S2 subunit

have been detected in unexposed individuals and S1, S2 and N protein specific memory B cell

responses have been detected in those who were infected with SARS-CoV-212. Children and

adolescents who were unexposed to SARS-CoV-2 were shown to have a higher frequency of

pre-existing IgG antibodies specific to S2, which were able to cross neutralize SARS-CoV-213.

The presence of high levels of cross-reactive antibodies to the S2 in children and adolescents

have been speculated to reduce disease severity when infected with SARS-CoV-213,14. Although

many studies have investigated the role of SARS-CoV-2 specific IgG responses, virus specific

IgA was detected during early illness and was shown to be able to neutralize the SARS-CoV-2

virus to a greater extent than virus specific IgG15. However, adults with severe illness had higher

levels of SARS-CoV-2 specific IgA levels compared to adults with milder illness and children,

and was shown to enhance neutrophil activation in vitro and thus release of inflammatory

mediators16. Therefore, although virus specific IgA is an important component of mucosal

immunity, its role in protection vs disease pathogenesis is not clear.

Currently there are several vaccines for COVID-19, which have shown to be safe and have high

efficacy rates against the original Wuhan SARS-CoV-2 virus and variable efficacy against

variants of concerm17-19. However, due to non-availability of adequate quantity of vaccines and

also in order to vaccinate as many individuals as fast as possible, some countries have increased

the gap between the two doses of vaccine such as AZD2221 to 12 or 16 weeks20. While there

have been many studies characterizing the IgG and IgA responses to different SARS-CoV-2

proteins in individuals with natural infection, the induction of IgG and IgA to different viral

proteins in vaccinees have not been extensively studied. It was recently shown that the mRNA
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vaccines induce high levels of both IgG and IgA antibodies against the spike protein21. However,

there are limited data characterizing the IgG, IgA, ACE2-receptor blocking antibodies in

individuals with varying severity of natural infection over time, in comparison to those who have

received a single dose of the AZD2221 vaccine. Therefore in this study, we investigated the

antibody responses in those with varying severity of natural infection and in those who received

a single dose of the AZD2221 at 4 weeks and 12 weeks to the S1, S2, RBD and N proteins and

also for SARS-CoV-2 variants of concern in a Sri Lankan population.

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Methods

Patients

Patients confirmed with SARS-CoV2 infection based on the positive RT-PCR who were

admitted to a COVID-19 treatment hospital, from the National Institute of Infectious Diseases

(NIID), Sri Lanka. They were followed throughout their illness while they were in hospital and

the severity grading was based on the worst severity while in hospital. Clinical disease severity

was classified as mild, moderate and severe according to the WHO guidance on COVID-19

disease severity 22 . For this study we recruited two cohorts of patients. Serum samples from the

patient cohort 1 (n=30) was used to determine the IgG and IgA antibody levels at 4 weeks since

onset of illness, the ACE2 receptor blocking antibody levels and the antibodies to RBD by the

HAT assay for the wild type (WT) and SARS-CoV-2 variants. The duration of illness was

defined from the day of onset of symptoms and not the day of PCR positivity or admission to

hospital. Based on the WHO COVID-19 disease classification, 15 patients had mild illness and

15 patients had moderate/severe illness22. As all the patients in the first cohort could not be

traced at 12 weeks, in order to carry out the above assays, we recruited a second cohort of

patients. Based on the WHO COVID-19 disease classification, 14 patients had mild illness and 6

patients had moderate/severe illness22.

In order to compare the antibody responses following infection with one dose of the AZD1222

vaccine, we recruited 20 individuals 4 weeks following vaccination and 73 individuals, 12 weeks

following vaccination. We also included serum samples from individuals who had a febrile

illness in 2017 and early 2018. Ethical approval was received by the Ethics Review Committee
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of Faculty of Medical Sciences, University of Sri Jayewardenepura. Informed written consent

was obtained from patients.

Haemagglutination test (HAT) to detect antibodies to the receptor binding domain (RBD)

23
The HAT was carried out as previously described . The B.1.1.7 (N501Y), B.1.351 (N501Y,

E484K, K417N) and B.1.617.2 versions of the IH4-RBD reagent were produced as described 23,

but included the relevant amino acid changes introduced by site directed mutagenesis. These

variants were titrated in a control HAT with the monoclonal antibody EY-6A (to a conserved

class 4 epitope23,24) and found to titrate identically with the original version so 100ng (50ul of

2ug/ml stock solution) was used for developing the HAT. The assays were carried out and
25
interpreted as previously described . The HAT titration was performed using 11 doubling

dilutions of serum from 1:20 to 1:20,480, to determine presence of RBD-specific antibodies. The

RBD-specific antibody titre for the serum sample was defined by the last well in which the

complete absence of “teardrop” formation was observed.

Surrogate neutralizing antibody test (sVNT) to detect NAbs

The surrogate virus neutralization test (sVNT)26, which measures the percentage of inhibition of

binding of the RBD of the S protein to recombinant ACE226 (Genscript Biotech, USA) was

carried out according the manufacturer’s instructions as previously described by us9. Inhibition

percentage ≥ 25% in a sample was considered as positive for NAbs.

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Results

The kinetics of SARS-CoV-2 specific IgG responses in those with natural infection

IgG responses to the S1, S2, RBD and N protein were measured in individuals with COVID-19

at 4 weeks and at 12 weeks since onset of illness and also in serum samples of 15 individuals

who had a febrile illness in 2017 and early 2018. At 4 weeks since onset of illness, the highest

magnitude of IgG antibody responses was seen for RBD in those with moderate/severe illness,

whereas those with mild disease, had the highest responses to S2 (Figure 1A, table 1). Those

who had a febrile illness in year 2017 and 2018 (controls), also had detectable antibody levels to

S2, but not for other proteins. There was no difference in the antibody levels to S2 in those with

mild illness compared to the controls (p=0.13), although those with milder disease had

significantly higher antibody levels to S1 and RBD (p<0.0001) and N protein (p=0.0004), than

the controls. In those who received a single dose of the AZD1222 vaccine, the IgG responses to

the S1 and S2 components of the spike protein were similar, although the levels for the RBD was

significantly higher (table 1). As expected, the IgG responses to the N protein was very low, but

even lower than for the controls. The antibody levels to S1 (p=0.0002), S2 (p=0.01), RBD

(p=0.002) and N (p<0.0001) proteins were significantly different between the three groups of

individuals at 4 weeks (Figure 1A).

At 12 weeks since onset of illness, those with moderate/severe illness had the highest responses

to N protein, whereas those with mild illness still had the highest responses to S2 (Figure 1B). At

both time points for all proteins, those with moderate/severe disease had significantly higher

antibody levels than those with milder illness (Table 1). The antibody responses to S1 (p=0.03),
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S2 (p=0.04), RBD (p=0.02) and N protein (p=0.0002) were significantly different between the

those with mild illness, moderate/severe disease and the vaccinees (Figure 1B). From 4 to 12

weeks, the S1 specific antibodies significantly rose in those with mild illness (p=0.004), while

there was no significant change in the antibody levels to other proteins at 12 weeks (table 1).

Patients who had moderate/severe illness sustained the same levels of antibodies for all four

proteins from 4 weeks to 12 weeks. In the vaccinees, from 4 weeks to 12 weeks the IgG levels to

S1 (p=0.008), S2 (p<0.0001) and RBD (p=0.003) had significantly increased (table 1).

The kinetics of SARS-CoV-2 specific IgA responses in those with natural infection

IgA responses to the S1, S2, RBD and N protein were measured in the above individuals with

COVID-19 at 4 weeks and at 12 weeks since onset of illness and also in serum samples of 15

individuals who had a febrile illness in 2017 and early 2018. At 4 weeks and 12 weeks of illness,

individuals with both mild and moderate/severe illness, had the highest levels of IgA antibodies

to the RBD (Figure 1C and 1D). However, those with moderate/severe disease had significantly

higher antibody responses to all four proteins when compared to those with mild illness at 4

weeks, but there was no difference at 12 weeks (table 1). Unlike what was observed with SARS-

CoV-2 S2 specific IgG responses, those with mild illness had significantly higher IgA responses

(p=0.02) but not to N protein (p=0.18) (Figure 2A). As expected, vaccinees had low responses to

the N protein, with IgA levels similar to those seen in controls except for IgA to S1, which was

higher in the vaccinees (p=0.003). Significant differences of IgA responses were seen in those

with mild illness, moderate/severe illness and vaccinees for S1 (p=0.001), S2 (p=0.0003), RBD

(p=0.0003) and N protein (p=0.04) (Figure 1C).

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There was no difference in IgA levels to any of the proteins at 4 weeks compared to 12 weeks in

patients with mild illness or with moderate/severe illness (table 1). However, significant

differences were seen between the three groups to S1 (p=0.009), RBD (p=0.003) and N protein

(p=0.02), but not for S2 (p=0.55) (Figure 1D).

ACE2 receptor blocking antibodies following natural infection and one dose of AZD1222

Due the lack of BSL-3 facilities to measure neutralizing antibodies, we used a surrogate test to

measure the inhibition of binding of antibodies in patient sera to the ACE2 receptor. This was

shown to be 100% specific in the Sri Lankan population, with none of the sera of individuals

collected in 2017 and 2018, giving a positive response 9. The ACE2 blocking antibodies were

significantly higher in those with moderate to severe illness, when compared to those with mild

illness at 4 weeks (p=0.03) and at 12 weeks (p=0.03) as reported previously9 (Figure 2). In

addition, the ACE2 receptor blocking antibodies significantly increased from 4 weeks to 12

weeks in those with moderate/severe illness (p=0.02) and in those with mild illness (p=0.03)

(Figure 2). However, in those who received a single dose of the vaccine, the ACE2 blocking

antibodies significantly reduced (p<0.0001) from levels at 4 weeks (median 77.32, IQR 60.05 to

90.77 % of inhibition) to 12 weeks (median 38.17, IQR 28.95 to 57.28 % of inhibition).

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Antibodies to the Receptor Binding Domain of the spike protein, including variants, measured by

the Haemagglutination test (HAT)

We previously evaluated the usefulness of the HAT assay in determining antibody responses to

the RBD of the SARS-CoV-2, wild type (WT) virus, B.1.1.7 variant and the B.1.351 variants at

4 weeks following a single dose of the AZD1222 vaccine and had also evaluated this assay in
27
naturally infected individuals in Sri Lanka . In this study, we proceeded to investigate the

differences in the antibody responses to the RBD in those with natural infection at 4- and 12-

weeks following infection, and after a single dose of the AZD1222 vaccine. The antibody

responses to the WT, B.1.1.7, B.1.351 and B.1.617.2 were measured.

In those with mild illness, at 4 weeks from onset of illness the median antibody titre to the WT

was 160 (IQR 80 to 320), B.1.1.7 was 120 (IQR 70 to 320), B.1.351 was 10 (IQR, 0 to 80) and

for B.1.617.2 it was 40 (IQR 20 to 80). At 12 weeks following the onset of illness, although there

was a slight reduction in the antibody titres to the WT (p=0.91) and B.1.617.2 (0.61), this was

not statistically significant (Figure 3A). In those with moderate/severe illness at 4 weeks from

onset of illness the median antibody titre to the WT was 1280 (IQR 160 to 1280), B.1.1.7 was

640 (IQR 160 to 1280), B.1.351 was 40 (IQR, 0 to 160) and for B.1.617.2 it was 320 (IQR 80 to

1280) (Figure 3B). There was no significant difference between the antibody titres for the WT

compared to B.1.1.7 (p=0.12), but clearly differed for B.1.315 (p<0.0001) and B.1.617.2

(p=0.004). Although the antibody titres for the WT and all the variants reduced from 4 to 12

weeks in those with moderate/severe illness, this was not statistically significant (Figure 3B).
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At 4 weeks following a single dose of the vaccine, the median antibody titre to the WT was 80

(IQR 40 to 280), B.1.1.7 was 40 (IQR 20 to 160), B.1.351 was 20 (IQR, 0 to 70) and for

B.1.617.2 it was 20 (IQR 0 to 70) (Figure 3C). At 12 weeks following a single dose of the

vaccine, the antibody titre for WT was 80 (IQR 20 to 80), for B.1.1.7 it was 20 (IQR 0 to 80), for

B.1.351 it was 20 (0 to 40) and for B.1.617.2 it was 0 (IQR 0 to 20). From 4 to 12 weeks,

although there was no significance difference of the antibody titres of the RBD of the WT

(p=0.05), B.1.351 (p=0.54) and B.617.2 (p=0.07), the antibody titres to B.1.1.7 significantly

reduced (p=0.02) (Figure 3B). As previously described by us at 4 weeks following vaccination,

the HAT titres were significantly lower to the B.1.1.7 (p=0.007), B.1.351 (<0.0001) and for

B.1.617.2 (p<0.0001). However, there was no significance difference in antibody titres between

B.1.351 and B.1.617.2 (p=0.43). At 12 weeks again the HAT titres were significantly lower to

the B.1.1.7 (p<0.0001), B.1.351 (<0.0001) and for B.1.617.2 (p<0.0001) and no difference

between antibody titres to B.1.351 and B.1.617.2.

Antibodies to the RBD were significantly different between those with mild illness,

moderate/severe illness and with those with a single dose of the vaccine at 4 weeks (p=0.004)

and at 12 weeks (p=0.02) (Figure 4A). This difference was also seen for the B.1.1.7 at 4 weeks

between those with mild illness, moderate/severe illness and with those with a single dose of the

vaccine at 4 weeks (p=0.0006) and at 12 weeks (p<0.0001) (Figure 4B) and for B.1.617.2 at 4

weeks (p=0.0002) and at 12 weeks (p=0.0004) (Figure 4C). However, there was no difference

between the antibody titres to the B.1.351 between those with mild, moderate/severe illness and

vaccinees at 4 weeks (p=0.36), but a significant difference was seen at 12 weeks (p=0.02)

(Figure 4D).
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Discussion

In this study we have investigated the kinetics of IgG and IgA responses to S1, S2, RBD and N

protein, ACE2 receptor blocking antibodies and antibodies against SARS-CoV-2 variants, in

individuals at 4 and 12 weeks following natural infection and in those who had a single dose of

the AZD2221. Based on the Luminex assays, IgG and IgA levels to S1, S2, RBD and N, had

increased from 4 weeks to 12 weeks in those with mild illness and in the vaccinees, although the

increase was only significant in the vaccinees. In the vaccinees, the most significant rise was

seen for the S2 subunit, while in those with mild illness the rise was seen for IgG antibodies for

the RBD. In those with moderate/severe illness, while there was no change in the IgG responses

from 4 to 12 weeks, responses to the N protein had increased although this was not significant.

Therefore, the kinetics of antibody responses to S1, S2, RBD and N appears to vary based on the

severity of natural infection and also appeared to be different in vaccinees. Interestingly, blood

samples of those who had a febrile illness in 2017 and 2018 also showed IgG and IgA responses

to the S2 subunit, suggesting the presence of S2 subunit cross-reactive antibodies, in these

donors as previously seen in other studies13,14. Following a single dose of the AZD2221 vaccine,

the antibodies against S2 appears to continue to rise from 4 to 12 weeks, possibly due to

stimulation of pre-existing cross-reactive memory B cell responses to the S2 subunit14.

SARS-CoV-2 specific IgA antibodies have shown to be generated during early illness and have

potent neutralizing ability15. IgA antibodies to the RBD have shown to develop earlier than IgG

and while some studies have shown that serum IgA does not associate with clinical disease
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severity15, in other studies, patients who developed severe disease were shown to have higher

levels of virus specific IgA28. Serum IgA was shown to activate neutrophils, thereby leading to

production of increased levels of inflammatory mediators possibly leading to disease

pathogenesis16. We found that at 4 weeks of illness, those with moderate/severe illness had

significantly higher serum IgA to S1, S2, RBD and N compared to those with mild illness, but

these high levels of IgA declined and there was no differences between these two groups at 12

weeks since onset of illness. Vaccinees had several fold lower IgA antibodies to all the SARS-

CoV-2 proteins tested than those with mild and moderate/severe illness at 4 weeks and 12 weeks.

The importance of serum IgA in preventing re-infection is currently unknown and if those with

lower IgA have reduced protection is currently unknown.

Although the IgG antibodies to S1, S2 and the RBD rose from 4 to 12 weeks in the vaccinees,

the ACE2 receptor blocking antibodies, which were shown to correlate with neutralizing

antibodies significantly decreased26. The HAT assay, which also measures antibodies to the RBD

and has shown to correlate well with the ACE2 receptor blocking assay and with neutralizing

antibodies23,27, also showed that the RBD binding antibodies decreased from 4 to 12 weeks in the

vaccinees. This suggests that although antibodies to RBD, S1 and S2 have increased in vaccinees

from 4 to 12 weeks, they might not be neutralizing antibodies, possibly through targeting other

epitopes in these regions.

Apart from assessing antibodies to the RBD to the wild type, we assessed the antibodies to three

other VOCs, B.1.1.7, B.1.351 and B.1.617.2. At 4 weeks following vaccination, the vaccinees
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had similar levels of antibodies to the RBD of WT as those with mild illness, the levels were

significantly less for B.1.1.7 and for B.1.617.2. The antibody levels among vaccinees were

similar to B.1.351 and B.1.617.2, showing equal reduction compared to antibody binding to the

RBD of the WT. Following vaccination, these levels further declined at 12 weeks to VOCs but

not to the WT, showing that a single dose of the AZD2221 was likely to offer less protection

against VOCs. In fact, it has been shown that one dose of AZD2221 is only 33% effective in

preventing symptomatic disease with B.1.617.2, 3 weeks following the first dose29. The efficacy

of a single dose against B.1.617.2 is likely to decline further by 12 weeks, as the antibodies to

RBD further waned. However, the efficacy of two doses of AZD2221 against hospitalization has

been shown to be 92%, while for Pfizer-BioNTech was 96%30. Therefore, in countries which

have outbreaks due to VOCs, especially B.1.617.2, it would be prudent to encourage second

doses to increase efficacy as recommended. Interestingly, although those with mild or

moderate/severe illness also had a marked reduction in antibodies to the RBD of B.1.351, they

had significantly higher levels of antibodies to the RBD of B.617.2 at 4 weeks compared to

B.1.351. However, by 12 weeks the antibody levels to both B.1.351 and B.1.617.2 were similar.

Therefore, B.1.617.2 had less immune evasion than B.1.351 in those who were naturally

infected, at least during early convalescence.

In summary, we have investigated the kinetics and differences in IgG and IgA antibody

responses to the S1, S2, RBD and N in those with varying severity of infection and vaccinees

who received a single dose of AZD2221, which showed that vaccinees had significantly less IgA

to SARS-CoV-2, but comparable IgG responses those with natural infection. However, following
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a single dose, vaccinees had reduced antibody levels to the VOCs, which further declined with

time.

Acknowledgement

We are grateful to the World Health Organization, UK Medical Research Council and the

Foreign and Commonwealth Office for support. T.K.T. is funded by the Townsend-Jeantet

Charitable Trust (charity number 1011770) and the EPA Cephalosporin Early Career Researcher

Fund. A.T. are funded by the Chinese Academy of Medical Sciences (CAMS) Innovation Fund

for Medical Science (CIFMS), China (grant no. 2018-I2M-2-002).

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References

1 Medicine, J. H. U. a. Coronavrus Resource Centre, <https://coronavirus.jhu.edu/>

(2021).

2 Davies, N. G. et al. Increased mortality in community-tested cases of SARS-CoV-2

lineage B.1.1.7. Nature 593, 270-274, doi:10.1038/s41586-021-03426-1 (2021).

3 England, P. H. SARS-CoV-2 variants of concern and variants under investigation in

England. Report No. 15, 77 (Public Health England, 2021).

4 Ritchie H., O.-O. E., Beltekian D., Mathieu E., Hasell J., Macdonald B., Giattino C.,

Appel C., Lucas Rodés-Guirao, Rose M. (OurWorldInData.org, 2021).

5 Hansen, C. H., Michlmayr, D., Gubbels, S. M., Molbak, K. & Ethelberg, S. Assessment

of protection against reinfection with SARS-CoV-2 among 4 million PCR-tested

individuals in Denmark in 2020: a population-level observational study. Lancet 397,

1204-1212, doi:10.1016/S0140-6736(21)00575-4 (2021).

6 Sabino, E. C. et al. Resurgence of COVID-19 in Manaus, Brazil, despite high

seroprevalence. Lancet 397, 452-455, doi:10.1016/S0140-6736(21)00183-5 (2021).

7 Zhou, D. et al. Evidence of escape of SARS-CoV-2 variant B.1.351 from natural and

vaccine-induced sera. Cell 184, 2348-2361 e2346, doi:10.1016/j.cell.2021.02.037 (2021).

8 Chen, X. et al. Disease severity dictates SARS-CoV-2-specific neutralizing antibody

responses in COVID-19. Signal Transduct Target Ther 5, 180, doi:10.1038/s41392-020-

00301-9 (2020).
Case
medRxiv 1:21-cv-02228
preprint Document 1-1 Filed; this
doi: https://doi.org/10.1101/2021.07.14.21260510 08/17/21 USDC
version posted July 18, Colorado Page
2021. The copyright 68forof
holder this165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

9 Jeewandara, C. et al. SARS-CoV-2 neutralizing antibodies in patients with varying

severity of acute COVID-19 illness. Sci Rep 11, 2062, doi:10.1038/s41598-021-81629-2

(2021).

10 Brochot, E. et al. Anti-spike, Anti-nucleocapsid and Neutralizing Antibodies in SARS-

CoV-2 Inpatients and Asymptomatic Individuals. Front Microbiol 11, 584251,

doi:10.3389/fmicb.2020.584251 (2020).

11 Demers-Mathieu, V. et al. Difference in levels of SARS-CoV-2 S1 and S2 subunits- and

nucleocapsid protein-reactive SIgM/IgM, IgG and SIgA/IgA antibodies in human milk. J

Perinatol 41, 850-859, doi:10.1038/s41372-020-00805-w (2021).

12 Nguyen-Contant, P. et al. S Protein-Reactive IgG and Memory B Cell Production after

Human SARS-CoV-2 Infection Includes Broad Reactivity to the S2 Subunit. mBio 11,

doi:10.1128/mBio.01991-20 (2020).

13 Ng, K. W. et al. Preexisting and de novo humoral immunity to SARS-CoV-2 in humans.

Science 370, 1339-1343, doi:10.1126/science.abe1107 (2020).

14 Fraley, E. et al. Cross-reactive antibody immunity against SARS-CoV-2 in children and

adults. Cellular & molecular immunology, doi:10.1038/s41423-021-00700-0 (2021).

15 Sterlin, D. et al. IgA dominates the early neutralizing antibody response to SARS-CoV-2.

Science translational medicine 13, doi:10.1126/scitranslmed.abd2223 (2021).

16 Bartsch, Y. C. et al. Humoral signatures of protective and pathological SARS-CoV-2

infection in children. Nature medicine 27, 454-462, doi:10.1038/s41591-021-01263-3

(2021).
Case
medRxiv 1:21-cv-02228
preprint Document 1-1 Filed; this
doi: https://doi.org/10.1101/2021.07.14.21260510 08/17/21 USDC
version posted July 18, Colorado Page
2021. The copyright 69forof
holder this165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

17 Abu-Raddad, L. J., Chemaitelly, H., Butt, A. A. & National Study Group for, C.-V.

Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and B.1.351

Variants. The New England journal of medicine, doi:10.1056/NEJMc2104974 (2021).

18 Hall, V. J. et al. COVID-19 vaccine coverage in health-care workers in England and

effectiveness of BNT162b2 mRNA vaccine against infection (SIREN): a prospective,

multicentre, cohort study. Lancet 397, 1725-1735, doi:10.1016/S0140-6736(21)00790-X

(2021).

19 Rubin, R. COVID-19 Vaccines vs Variants-Determining How Much Immunity Is

Enough. JAMA : the journal of the American Medical Association 325, 1241-1243,

doi:10.1001/jama.2021.3370 (2021).

20 Tauh. T, M. M., Meyler, P., Lee, S.M. An updated look at the 16-week window between

doses of vaccines in BC for COVID-19. BC Medical Journal 63, 102-103 (2021).

21 Campillo-Luna, J., Wisnewski, A. V. & Redlich, C. A. Human IgG and IgA responses to

COVID-19 mRNA vaccines. medRxiv, 2021.2003.2023.21254060,

doi:10.1101/2021.03.23.21254060 (2021).

22 WHO. Clinical management of severe acute respiratory infection when novel coronavirus

-
(2019 nCoV) infection is suspected: interim guidance. (WHO, 2020).

23 Townsend, A. et al. A haemagglutination test for rapid detection of antibodies to SARS-

CoV-2. Nature Communications, 2020.2010.2002.20205831,

doi:10.1101/2020.10.02.20205831 (2020).

24 Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat

origin. Nature 579, 270-273, doi:10.1038/s41586-020-2012-7 (2020).

Case
medRxiv 1:21-cv-02228
preprint Document 1-1 Filed; this
doi: https://doi.org/10.1101/2021.07.14.21260510 08/17/21 USDC
version posted July 18, Colorado Page
2021. The copyright 70forof
holder this165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
It is made available under a CC-BY-NC-ND 4.0 International license .

25 Jeewandara, C. et al. Antibody and T cell responses to a single dose of the

AZD1222/Covishield vaccine in previously SARS-CoV-2 infected and naïve health care

workers in Sri Lanka. medRxiv, 2021.2004.2009.21255194,

doi:10.1101/2021.04.09.21255194 (2021).

26 Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-

mediated blockage of ACE2-spike protein-protein interaction. Nature biotechnology 38,

1073-1078, doi:10.1038/s41587-020-0631-z (2020).

27 Kamaladasa, A. et al. Comparison of two assays to detect IgG antibodies to the receptor

binding domain of the SARSCoV2 as a surrogate marker for assessing neutralizing

antibodies in COVID-19 patients. Int J Infect Dis, doi:10.1016/j.ijid.2021.06.031 (2021).

28 Yu, H. Q. et al. Distinct features of SARS-CoV-2-specific IgA response in COVID-19

patients. The European respiratory journal 56, doi:10.1183/13993003.01526-2020

(2020).

29 Iacobucci, G. Covid-19: Single vaccine dose is 33% effective against variant from India,

data show. BMJ (Clinical research ed 373, n1346, doi:10.1136/bmj.n1346 (2021).

30 England, P. H. (Public Health England, 2021).

Case
medRxiv 1:21-cv-02228
preprint Document 1-1 Filed; this
doi: https://doi.org/10.1101/2021.07.14.21260510 08/17/21 USDC
version posted July 18, Colorado Page
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holder this165
preprint
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Tables

4 weeks 12 weeks P value

Median (IQR) Median (IQR)

Mild infection (IgG)

S1 734 (483 to 1071) 1336 (24 to 4714) 0.59

S2 3503 (1656 to 5795) 3579 (106.8 to 9912) 0.68

RBD 539 (840 to 2960) 2952 (38.7 to 7516) 0.59

N 2094 (1554 to 4787) 2694 (51 to 7547) 0.84

Mild infection (IgA)

S1 152 (79 to 490) 192 (19 to 422.1) 0.69

S2 354 (219 to 561.5) 380.2 (165.6 to 869) 0.71

RBD 656.5 (303 to 1616) 770.5 (180.3 to 1520) 0.98

N 207.5 (78 to 468) 276.3 (165.5 to 0.31

496.5)

Moderate/severe infection (IgG)

S1 4776 (1395 to 7833) 5064 (2744 to 6038) 0.96

S2 6869 (2001 to 8931 (7262 to 9607) 0.85

RBD 11,131) 7829 (5083 to 8553) 0.67

N 7486 (2784 to 9538 (8810 to 0.31

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10,218) 10,844)

5831 (3123 to 9383)

Moderate/severe infection (IgA)

S1 1043 (220 to 1784) 391.8 (132.8 to 2021) 0.52

S2 934 (399 to 3679) 1378 (153.9 to 2269) 0.73

RBD 3375 (1192 to 5401) 1837 (506.1 to 4802) 0.38

N 661 (211.5 to 6165) 273 (75.9 to 596.1) 0.18

Vaccinated IgG

S1 2215 (1223 to 3870) 3639 (2190 to 5617) 0.008

S2 1625 (1063 to 4329) 6460 (4143 to 9594) <0.0001

RBD 4393 (2355 to 6131) 6209 (4481 to 8367) 0.003

Vaccinated IgA

S1 76.5 (38.2 to 166.5) 36 (23 to 92) 0.04

S2 203.3 (101.3 to 324.5 (143 to 788) 0.02

RBD 310.9) 221 (116 to 437) 0.11

327.5 (183 to 612.8)

Table 1: Antibody responses to S1, S2, RBD and N protein of the SARS-CoV-2 in those

with varying severity of illness and in those following a single dose of the AZD2221. MFI

indicates the median fluorescence intensity.

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Figure Legends

Figure 1: IgG and IgA antibody levels to S1, S2, RBD and N protein of SARS-CoV-2 in

individuals following natural infection and following a single dose of the AZD1222 vaccine.

Serum IgG antibodies to S1, S2, RBD and N protein were measured by Luminex assays at 4

weeks in those with mild illness (n=15), moderate/severe illness (n=15), vaccinees (n=20) and

controls (n=19) (A) and again at 12 weeks in those with mild illness (n=14), moderate/severe

illness (n=6), vaccinees (n=73) (B). IgA antibodies were also measured in the above groups at 4

weeks (C) and at 12 weeks (D). The Kruskal-Wallis test was used to determine the difference

between the antibody levels between the three different groups (two-tailed). The lines indicate

the median and the interquartile range.

Figure 2: ACE2 receptor blocking antibodies in patients with varying severity of illness and

following a single dose of the AZD1222 vaccine. ACE receptor blocking antibodies were

measured by the surrogate virus neutralizing test following natural infection at 4 weeks in those

with mild illness (n=14) and moderate/severe illness (n=15) and at 12 weeks in those with mild
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(n=14) and moderate/severe illness (n=6). Antibodies were also measured at 4 weeks (n=20) and

12 weeks (n=73) in vaccinees following a single dose of AZD2221. Mann-Whitney test (two

tailed) was used to determine the differences between antibody levels as 4 weeks and 12 weeks.

The lines indicate the median and the interquartile range.

Figure 3: Comparison of antibody titres to RBD of the SARS-CoV-2 using the HAT assay

in those with varying severity of infection and in vaccinees. Antibody titres were measured

individuals with mild illness (n=14) to the WT, B.1.1.7, B.1.351 and B.1.617.2 at 4 weeks and

12 weeks since onset of illness (A), in those with moderate/severe illness at 4 weeks (n=15) and

12 weeks (n=6) since onset of illness (B) and in those who received one dose of AZD1222

vaccine at 4 weeks (n=16) and 12 weeks (n=73) following the vaccine (C). The difference

between antibody titres to WT, B.1.1.7, B.1.351 and B.1.617.2 was determined using the

Wilcoxon paired t test (two tailed) and the differences between antibody titres at 4 weeks and 12

weeks was determined using the Mann-Whitney test (two tailed). The lines indicate the median

and the interquartile range.

Figure 4: Comparison of antibody titres to the RBD of the SARS-CoV-2 using the HAT

assay for the wild type and for variants. Antibody titres were measured in patients with mild

illness (n=14), moderate/severe illness (n=15) from 4 weeks since onset of illness and in those

who received one dose of AZD1222 vaccine at 4 weeks (n=16), and again at 12 weeks in those

who developed mild illness (n=14), moderate/severe illness (n=6) and in those who received 1
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dose of AZD1222 vaccine (n=73), for the WT (A), B.1.1.7 (B), B.1.617.2 (C) and B.1.351 (D).

The Kruskal-Wallis test was used to determine the difference between the antibody levels

between the three different groups (two-tailed). The lines indicate the median and the

interquartile range.
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EXHIBIT 6
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https://doi.org/10.1038/s41586-021-03647-4

Accelerated Article Preview

SARS-CoV-2 infection induces long-lived

bone marrow plasma cells in humans


Received: 20 December 2020 Jackson S. Turner, Wooseob Kim, Elizaveta Kalaidina, Charles W. Goss,, Adriana
A M.
M Rauseo,
Ra


a Pusic,
Aaron J. Schmitz, Lena Hansen, Alem Haile, Michael K. Klebert, Iskra Pusic
c
Accepted: 14 May 2021
Jane A. O’Halloran, Rachel M. Presti & Ali H. Ellebedy
Accelerated Article Preview Published
online 24 May 2021


This is a PDF file of a peer-reviewed paper that has been
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Cite this article as: Turner, J. S. et al.
SARS-CoV-2 infection induces long-lived Although unedited, the content has been subjected ed to pre
prelim
min
preliminary formatting. Nature
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Article

SARS-CoV-2 infection induces long-lived

bone marrow plasma cells in humans

https://doi.org/10.1038/s41586-021-03647-4 Jackson S. Turner1, Wooseob Kim1, Elizaveta Kalaidina2, Charles W. Goss3,
Adriana M. Rauseo4, Aaron J. Schmitz1, Lena Hansen1,5, Alem Haile6, Michael K. Klebert
ber 6,
bert
Received: 20 December 2020 7 4 4,9 1,8,9ಞᅒ
Iskra Pusic , Jane A. O’Halloran , Rachel M. Presti & Ali H. Ellebedy


Accepted: 14 May 2021

Published online: 24 May 2021

Long-lived bone marrow w plasma cells (BMPCs) are a persistent and nd essentia
essential
e sso
source o
of


protective antibodies11–7. Severe acute respiratory syndrome coronavirus
oronaviiruss 2
oronavir
SARS-CoV-2) convalescent individuals have a significantly
(SARS-CoV-2) y low
lowe
lower
wer risk of
einfection8–10. Nonetheless, it has been reported that anti-SARS-CoV-2
reinfection nti-SARS-CoV- serum
nti-SARS-C


antibodies experience rapid decay in the first few months infection, raising
onthss after infe
concerns that long-lived BMPCs may not be generated rated an
and hhumoral immunity against
hum
this virus may be short-lived11–13. Here we demonstrate
o
on ate th
that in patients who
experienced mild infections (n=77), serum m anti-SA Co
anti-SARS-CoV-2 spike (S) antibodies
decline rapidly in the first 4 months afterer in
nfection an
infection and then more gradually over the


following 7 months, remaining detectablectable at le
ctab east 11 months after infection. Anti-S
least
antibody titers correlated with the he frequency
frequ
fre uency of
o S-specific BMPCs obtained from bone
marrow aspirates of 18 SARS-CoV-2CoV-2
V-2 conva
conval e
convalescent patients 7 to 8 months after


infection. S-specific BMPCsCs w
S-CoV-2 inf
S-CoV-
with no history of SARS-CoV-2
were not det
nfecti
detected in aspirates from 11 healthy subjects
infection. We demonstrate that S-bind ng BMPCs are
S-binding

quiescent, indicating
ng
g that
thaatt they
a hey are
a part of a long-lived compartment.
compartment Consistently,
ng memory
circulating resting yBccells directed against the S protein were detected in the


convalescentt ind
individ
iduals. O
idua
individuals. verall, we show that SARS-CoV-2 infection induces a
Overall,
robust antigen-specific,
igen-specific
gen-specifi
n-specific long-lived humoral immune response in hum
n-specific ans
humans.

Reinfections by seasonal coronaviruses occur 6-12 months af aft

after the (49% female, 51% male, median age 49), the majority of whom had expe-


previous infection, indicating that protective immunity immuni y against

immunit again these rienced mild illness (7.8% hospitalized, Extended Data Tables 1 and 2).
viruses may be short-lived14,15. Early reports docu doc
documenting
umenting ra rapidly declin- Follow-up blood samples were collected three times at approximately
ing antibody titers in convalescent SARS-CoV-2 RS-C
RS-CoV-2 2 patients
patien in the first 3-month intervals. Twelve convalescent participants received either
several months after infection suggested este
ted that pro
protective
t immunity the BNT162b2 or the mRNA-1273 SARS-CoV-2 vaccine between the last


against SARS-CoV-2 may be similarly ly

y transi ent11–
transien
transient 11–13
–13
. It
I was also suggested two timepoints; these post-vaccination samples were not included in
that SARS-CoV-2 infection may fail il to
t elicit a functional
fun
un germinal center our analyses. Additionally, bone marrow aspirates were collected from
response, which would interfere erfere with
with the generation of long-lived eighteen of the participants 7 to 8 months after infection and from
plasma cells3–5,7,16. Later reports
ports analyzin
analyzingzing
in samples collected approxi- eleven healthy volunteers with no history of SARS-CoV-2 infection or


mately 4 to 6 months afterfter inf

infe
infection
ection ind
ectio indicate that SARS-CoV-2 antibody vaccination. Follow-up bone marrow aspirates were collected from five
owly8,8,17–21
titers decline more slowly ,17
17–
–21
2
.D
Durable
ura serum antibody titers are main- of the eighteen and one additional convalescent donor approximately
tained by long-lived
ived plasma
plasma cells,cel non-replicating, antigen-specific 11 months after infection. (Fig. 1a, Extended Data Tables 3 and 4). We
plasma cells ththat
at are dete
detected
ecte in bone marrow long after the disap- first performed a longitudinal analysis of circulating anti-SARS-CoV-2


11–7
pearance of the antigen
a . We sought to determine whether they serum antibodies. While anti-SARS-CoV-2 spike (S) IgG antibodies
were detectable
etectable in SASARS-CoV-2
AR convalescent patients approximately were undetectable in blood from controls, 74 of 77 convalescent par-
7 months after
nths af infection.
fter infec ticipants had detectable serum titers approximately 1 month after
onset of symptoms. Between 1- and 4-months post symptom onset,


overall anti-S IgG titers decreased from a mean of 6.3 to 5.7 (mean dif-
Biphasic
Bipha
hasic d
decay of anti-S antibody titers ference 0.59±0.06, P<0.001). However, in the interval between 4- and


Blood samples
sa were collected approximately 1 month after onset of 11-months post symptom onset, the decay rate slowed, and mean titers
symptoms from seventy-seven SARS-CoV-2 convalescent volunteers
sympt declined from 5.7 to 5.3 (mean difference 0.44±0.10, P<0.001, Fig. 1a).

1
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA. 2Division of Allergy and Immunology, Department of Internal Medicine, Washington
University School of Medicine, St. Louis, MO, USA. 3Division of Biostatistics, Washington University School of Medicine, St. Louis, MO, USA. 4Division of Infectious Diseases, Department of
lnternal Medicine, Washington University School of Medicine, St. Louis, MO, USA. 5Influenza Centre, Department of Clinical Science, University of Bergen, 5021, Bergen, Norway. 6Clinical Trials
Unit, Washington University School of Medicine, St. Louis, MO, USA. 7Division of Oncology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
8
The Andrew M. and Jane M. Bursky Center for Human Immunology & Immunotherapy Programs, Washington University School of Medicine, Saint Louis, MO, USA. 9Center for Vaccines and
Immunity to Microbial Pathogens, Washington University School of Medicine, Saint Louis, MO, USA. ᅒe-mail: ellebedy@wustl.edu

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Article
In contrast to the anti-S antibody titers, IgG titers against the 2019/2020 into antibody-secreting plasmablasts. We examined the frequency of
inactivated seasonal influenza virus vaccine were detected in all con- SARS-CoV-2 specific circulating MBCs in convalescent patients as well
trol and SARS-CoV-2 convalescent participants and declined much as in the healthy controls. We stained peripheral blood mononuclear
more gradually, if at all over the course of the study, with mean titers cells with fluorescently labeled S probes and determined the frequency
decreasing from 8.0 to 7.9 (mean difference 0.16±0.06, P=0.042) and of S-binding MBCs among isotype-switched IgDlo CD20+ MBCs by flow
7.9 to 7.8 (mean difference 0.02±0.08, P=0.997) across the 1-to-4- and cytometry. For comparison, we co-stained the cells with fluorescently
4-to-11-month intervals post symptom onset, respectively (Fig. 1b). labeled influenza virus hemagglutinin (HA) probes (Fig. 4a). S-binding
MBCs were identified in convalescent patients in the first sample col- ol-
ol
lected approximately 1 month after onset of symptoms, with compa- ompa-
ompa

Induction of S-binding long-lived BMPCs rable frequencies to influenza HA-binding memory B cells (Fig (Fig. 4b).
g 4b
g. 4b).
).
The relatively rapid early decline in anti-S IgG followed by slower decay S-binding memory B cells were maintained for at least 7m postst sympt
sympto
symptom
tom
m
is consistent with a transition of serum antibodies from being secreted onset and were present at significantly higher frequencies
es compared to o


by short-lived plasmablasts to a smaller but more persistent popula- healthy controls, comparable to frequencies of influenza
uenz
enza HA
HA-binding
A-bindin
tion of long-lived plasma cells generated later in the immune response. memory B cells identified in both groups (Fig. 4c).).
The majority of this latter population resides in bone marrow1–6. To


investigate whether SARS-CoV-2 convalescent patients developed a
virus specific long-lived BMPC compartment, we examined their bone Discussion
marrow aspirates obtained approximately 7 and 11 months after infec- This study sought to determine whether er SARS-CoV-2
SARS--CoV-2 in
SAR infection induces
tion for anti-SARS-CoV-2 S-specific BMPCs. We magnetically enriched antigen-specific long-lived BMPCs in humans. We detected de SARS-CoV-2


BMPCs from the aspirates and then quantified the frequencies of those S-specific BMPCs in aspirates from m 15 o
off 19 cconvalescent
conva patients, and in
secreting IgG and IgA directed against the 2019/2020 influenza virus none from the 11 control participants.
cipants. Frequencies
Freq
F que of anti-S IgG BMPCs
vaccine, tetanus/diphtheria vaccine, and SARS-CoV-2 S protein by ELIS- modestly correlated with serum m IgG titer
titers 7-8 months after infection.
pot (Fig. 2a). Frequencies of influenza and tetanus/diphtheria vaccine Phenotypic analysis by y flow c
cytometry
met demonstrated that S-binding
metry
specific BMPCs were comparable between control and convalescent BMPCs were quiescent, nt, and thei
their frequencies were largely consistent


participants. IgG- and IgA-secreting S-specific BMPCs were detected in five paired aspirates
rates collected
c 7-
7 and 11-months post symptom onset.
in 15 and 9 of the 19 convalescent participants, respectively, but not Importantly, w wee de
detected d no S-binding cells among plasmablasts in
in any of the 11 control participants (Fig. 2b). Importantly, none of the blood samples ples co
coll
collected
lected at the same time as the bone marrow aspirates
convalescent patients had detectable S-specific antibody secreting by ELISpot ott or flow cytometry
cy
cyttom in any of the convalescent or control sam-
cells in blood at the time of bone marrow sampling, indicating that
the detected BMPCs represent bone marrow-resident cells and not  ples. Altogether,
ltog
a long-lived
gether,
ong ved BMPC
ong-liv
ether, thes
B
the
these data indicate mild SARS-CoV-2 infection elicits
response. Additionally, we showed that S-binding

contamination from circulating plasmablasts. Frequencies of anti-S IgG M
MBCs in blood
blood of convalescent patients are present at similar frequen-
BMPCs were stable among the five participants sampled a second time cie
cies
es to tho
those directed against influenza virus HA. Overall, our results are
approximately 4m later, and anti-S IgA BMPC frequencies were stable ble consistent
consis
consististen with SARS-CoV-2 infection eliciting a canonical T-dependent


in four of the five, with one decreasing below the limit of detection ection n B cell rresponse, in which an early transient burst of extrafollicular plas-
(Fig. 2c). Consistent with their stable BMPC frequencies, anti-S IgG G titers mablasts generates a wave of serum antibodies that decline relatively
m
ma
in the five participants remained consistent between 7- and 11-months 1-months
onths quickly. This is followed by more stably maintained serum antibody
post symptom onset. IgG titers measured against the recep rece
receptorr bin
bind
bind-- levels that are supported by long-lived BMPCs.
ing domain (RBD) of S, a primary target of neutralizing lizing antibodies,
antib i
anti While this overall trend captures the serum antibody dynamics of
were detected in four of the five convalescent patients ati nts and we
atie wer
were also the majority of participants, we observed that in three participants,


stable between 7- and 11-months post symptom onset (Fig (F

(Fig. 2d).
g 2d
2d).
). F
Frequen- anti-S serum antibody titers increased between 4- and 7-months post
cies of anti-S IgG BMPCs showed a modest but but significant correlation symptom onset after having initially declined between 1 and 4 months.
with circulating anti-S IgG titers 7-8 months onths post
posst symptom
symp onset in This could be stochastic noise, could represent increased net binding
convalescent participants, consistent nt with
wit long
long-ter
long-term maintenance of affinity as early plasmablast-derived antibodies are replaced by those


antibody levels by these cells. In accordance

ccordance
ordanc ce with previous reports22–24,
th p from affinity-matured BMPCs, or could represent increases in antibody
frequencies of influenza vaccine-specific
ine-
e-specific
pecific Ig
IgG
gG BMPCs and antibody concentration from reencounter with the virus (although none of the
titers exhibited a strong and significan
signific
significant
antt corre
correlation
c (Fig. 2e). Nine of the participants in our cohort tested positive a second time). While anti-S
aspirates from controls and d twelve of the
th eighteen collected 7m post IgG titers in the convalescent cohort were relatively stable in the interval


symptom onset yielded ed a su

suf
sufficient
fficient n
fficie number of BMPCs for additional between 4- and 11-months post symptom onset, they did measurably
analysis by flow cytometry.
ometry. We sta stained these samples intracellularly decrease, in contrast to anti-influenza virus vaccine titers. While this
with fluorescently tly labe
labeled
eled S and influenza virus hemagglutinin (HA) could represent an intrinsically less durable anti-S BMPC response
probes to identify
entify and ch
characterize
hara antigen specific BMPCs. As con- compared to that against influenza virus, the largely stable frequencies


trols, we alsolso intracellularly

intracellula stained PBMC from healthy volunteers of anti-S BMPCs measured in the same individuals 7- and 11-months
1 week after
aftter SARS-CoV-2
SARS-CoV V-2 or seasonal influenza virus vaccination (Fig. 3a, post symptom onset argue against this possibility. It is possible that the
Extended
nded DaData
ata Fig. 1a
Fig. 1a-c). Consistent with the ELISpot data, low frequen- decline reflects a final waning of early plasmablast-derived antibodies.
cies
es of S-binding
S-bindin
S-bind ng BMPCs were detected in ten of the twelve convales- It is also possible that the lack of decline in influenza titers was due to


cent specime
cent specimen
specimens analyzed, but not in any of the nine control specimens boosting through exposure to influenza antigens from infection or
(Fig. 3b).
(Fig. 3b
3bb)). While
Wh both recently generated circulating plasmablasts and vaccination. Our data suggest that SARS-CoV-2 infection induces a


S- and HHA-binding BMPCs expressed Blimp1, BMPCs were differenti- germinal center response in humans because long-lived BMPCs are
ated by
b the lack of expression of Ki-67, indicating a quiescent state, as thought to be predominantly germinal center-derived7. This is consist-
well as higher levels of CD38 (Fig. 3c).
we ent with a report demonstrating increased levels of somatic hypermuta-
tion in MBCs targeting the receptor binding domain of the S protein in
SARS-CoV-2 convalescent patients at 6 months compared to 1 month
Robust S-binding memory B cell response after infection20.
Memory B cells (MBCs) form the second arm of humoral immune mem- To our knowledge, the current study provides the first direct evidence
ory. Upon antigen re-exposure, MBCs rapidly expand and differentiate for induction of antigen specific BMPCs after a viral infection in humans.

2 | Nature | www.nature.com
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 84 of 165

However, we do acknowledge several limitations. Although we detected 5. Halliley, J. L. et al. Long-Lived Plasma Cells Are Contained within the CD19−
CD38hiCD138+ Subset in Human Bone Marrow. Immunity 43, 132–145 (2015).
anti-S IgG antibodies in serum at least 7 months after infection in all 19 6. Mei, H. E. et al. A unique population of IgG-expressing plasma cells lacking CD19 is
of the convalescent donors from whom we obtained bone marrow aspi- enriched in human bone marrow. Blood 125, 1739–1748 (2015).
rates, we failed to detect S-specific BMPCs in four donors. Serum anti-S 7. Nutt, S. L., Hodgkin, P. D., Tarlinton, D. M. & Corcoran, L. M. The generation of
antibody-secreting plasma cells. Nat. Rev. Immunol. 15, 160–171 (2015).
antibody titers in those four donors were low, suggesting that S-specific 8. Hall, V. J. et al. SARS-CoV-2 infection rates of antibody-positive compared with
BMPCs may potentially be present at very low frequencies that are antibody-negative health-care workers in England: a large, multicentre, prospective
below our limit of detection. Another limitation is that we do not know cohort study (SIREN). The Lancet S0140673621006759 (2021) https://doi.org/10.1016/
S0140-6736(21)00675-9.
the fraction of the S-binding BMPCs detected in our study that encodes 9. Houlihan, C. F. et al. Pandemic peak SARS-CoV-2 infection and seroconversion rates in n
neutralizing antibodies. SARS-CoV-2 S protein is the main target of London frontline health-care workers. The Lancet 396, e6–e7 (2020).

neutralizing antibodies17,25–30 and correlation between serum anti-S 10. Lumley, S. F. et al. Antibodies to SARS-CoV-2 are associated with protection against nsst
reinfection. http://medrxiv.org/lookup/doi/10.1101/2020.11.18.20234369 (2020) 20) https
https://
ht s://
//
IgG binding and neutralization titers has been documented17,31. Further doi.org/10.1101/2020.11.18.20234369.
studies will be required to determine the epitopes targeted by BMPCs 11. Long, Q.-X. et al. Clinical and immunological assessment of asymptomatic mat c SARS-CoV-2


and MBCs as well as their clonal relatedness. Finally, while our data infections. Nat. Med. 26, 1200–1204 (2020).
12. Ibarrondo, F. J. et al. Rapid Decay of Anti–SARS-CoV-2 Antibodiess in Persons Perso
Per ns with
wi Mild
document a robust induction of long-lived BMPCs after SARS-CoV-2 Covid-19. N. Engl. J. Med. 383, 1085–1087 (2020).
infection, it is critical to note that our convalescent patients mostly 13. Seow, J. et al. Longitudinal observation and decline of neutralizing tra
tral
alizing antibod
antibody reresponses in


experienced mild infections. Our data are consistent with a report the three months following SARS-CoV-2 infection in humans. mans Na
mans. Nat.
at. Microbiol.
Microbio 5, 1598–1607
Microbiol
(2020).
showing that individuals who recovered rapidly from symptomatic 14. Edridge, A. W. D. et al. Seasonal coronavirus protective ectivee immunity is sh short-lasting.
SARS-CoV-2 infection generated a robust humoral immune response32. Nat. Med. 26, 1691–1693 (2020).
Therefore, it is possible that more severe SARS-CoV-2 infections could 15. Callow, K. A., Parry, H. F., Sergeant, M. & Tyrrell,rell, D. A. The time
ti e course
co of the immune


response to experimental coronavirus infection fect on of man. EpidEpidemiol. Infect. 105, 435–446
lead to a different outcome with respect to long-lived BMPC frequencies (1990).
due to dysregulated humoral immune responses. This, however, has not 16. Kaneko, N. et al. Loss of Bcl-6-Expressingessing T Follicular
Follicul
Fol ar Helper
H Cells and Germinal Centers
been the case in survivors of the 2014 West African Ebola virus outbreak in COVID-19. Cell 183, 143-157.e13 (2020). 20).
17. Wajnberg, A. et al. Robust neutralizing
eutr
eutra antibodies
ntibo
ntibodies to SARS-CoV-2 infection persist for
in whom severe viral infection induced long-lasting antigen-specific months. Science eabd7728 28 (2020)
(2020 https://doi.org/10.1126/science.abd7728.
doi
serum IgG antibodies33. 18. Isho, B. et al. Persistence e of serum and saliva antibody responses to SARS-CoV-2 spike


antigens in COVID-19 19 patients. Sci. Immunol.
patients. Imm 5, (2020).
Long-lived BMPCs provide the host with a persistent source of
19. mm
mmunologi
Dan, J. M. et al. Immunological cal memo
memory to SARS-CoV-2 assessed for greater than six
preformed protective antibodies and are therefore needed to main- infection.
ectio bioRxiv
months after infection. v 2020.11.15.383323
20 (2020) https://doi.
tain durable immune protection. However, longevity of serum org/10.1101/2020.11.15.383323.
1/2020 15.383323
1/2020.11
20. Gaebler, r, C. et al. Evolu
olution o
Evolution of Antibody Immunity to SARS-CoV-2. http://biorxiv.org/
anti-S IgG antibodies is not the only determinant of how durable
lookup/doi/10.1101/2020.11.03.367391
u /doi/10.1101/2020
oi/10.1101/20 (2020) https://doi.org/10.1101/2020.11.03.367391.
immune-mediated protection will be. Indeed, isotype-switched MBCs
can rapidly differentiate into antibody secreting cells upon pathogen
reexposure, offering a second line of defense34. Encouragingly, the
 21.

22.
2.
Rodda,
CO
odda, L.
COVID-19.
COVID
Davis,
Dav C. W.
L B. et al.
et a . Functional
et al
-19. Cell
Func
ll (2020)
Cell
W et al.
SARS-CoV-2-Specific Immune Memory Persists after Mild
(202 https://doi.org/10.1016/j.cell.2020.11.029.
et  l Influenza vaccine–induced human bone marrow plasma cells decline

within
ithin a year
yea after vaccination. Science 370, 237–241 (2020).
frequency of S-binding circulating MBCs 7 months after infection was 23. Turesson,
sson I. Distribution of Immunoglobulin-containing Cells in Human Bone Marrow and
similar compared to those directed against contemporary influenza HA Lym
ymph
Lymphoid Tissues. Acta Med. Scand. 199, 293–304 (2009).


24
24. Pritz
Pritz, T. et al. Plasma cell numbers decrease in bone marrow of old patients.
antigens. Overall, our data provide strong evidence that SARS-CoV-2
-CoV-2-2
E J. Immunol. 45, 738–746 (2014).
Eur.
infection in humans robustly establishes the two arms of humoral umoral 25
25. Shi, R. et al. A human neutralizing antibody targets the receptor binding site of
immune memory: long-lived BMPC and MBCs. These findings ingss pro-
pro SARS-CoV-2. Nature 584, 120–124 (2020).
26. Cao, Y. et al. Potent Neutralizing Antibodies against SARS-CoV-2 Identified by
vide an immunogenicity benchmark for SARS-CoV-2 vaccines vacc s and nd a
High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells. Cell 182,
foundation for assessing the durability of primary humoral iim immune 73-84.e16 (2020).
responses induced after viral infections in humans. ns
ns.


27. Robbiani, D. F. et al. Convergent antibody responses to SARS-CoV-2 in convalescent

individuals. Nature 584, 437–442 (2020).
28. Kreer, C. et al. Longitudinal Isolation of Potent Near-Germline SARS-CoV-2-Neutralizing
Antibodies from COVID-19 Patients. Cell 182, 843-854.e12 (2020).
Online content 29. Alsoussi, W. B. et al. A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2
Infection. J. Immunol. 205, 915–922 (2020).
Any methods, additional references, Natu Nature
ature Res
Resear
Research reporting sum-


30. Wang, C. et al. A human monoclonal antibody blocking SARS-CoV-2 infection. Nat.
maries, source data, extended data, ata, sup
supp
supplementary
ppleme
me information, Commun. 11, 2251 (2020).
acknowledgements, peer review w information;
in ormation;; d
details of author contri- 31. Wang, K. et al. Longitudinal Dynamics of the Neutralizing Antibody Response to Severe
Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection. Clin. Infect. Dis.
butions and competing interests;
ests; and
and stateme
stat
statements
s of data and code avail- ciaa1143 (2020) https://doi.org/10.1093/cid/ciaa1143.
ability are available at https://doi.org/10.1038/s41586-021-03647-4.
ps://doi.org/1
/10
10 32. Chen, Y. et al. Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production.


Cell S0092867420314586 (2020) https://doi.org/10.1016/j.cell.2020.10.051.

33. Davis, C. W. et al. Longitudinal Analysis of the Human B Cell Response to Ebola Virus
1. Benner, R., Meima, F., van der
van der Meulen, G. G M. & van Muiswinkel, W. B. Antibody formation in Infection. Cell 177, 1566-1582.e17 (2019).
mouse bone marrow. rrow. I. Evidence
Evidence for the
t development of plaque-forming cells in situ. 34. Ellebedy, A. H. et al. Defining antigen-specific plasmablast and memory B cell subsets
Immunology 26, 6, 247–255
247 (1974).
(1974). in human blood after viral infection or vaccination. Nat. Immunol. 17, 1226–1234
2. Manz, R. A.,, Thiel,
Thi A. & Radbruch,
Radbruc A. Lifetime of plasma cells in the bone marrow. Nature (2016).


3–134 (1997
388, 133–134 (1997).
3. a, M.
Slifka, M K., Antia, R., W h
Whitmire, J. K. & Ahmed, R. Humoral Immunity Due to Long-Lived Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in
asma Cells.
Plasma C lls. Immunit
Immunity 8, 363–372 (1998). published maps and institutional affiliations.
4. Hammarlund
Hammarlund,, E. et a et al. Duration of antiviral immunity after smallpox vaccination. Nat. Med.
9
9,, 1131–1137 (2
(20
(2003). © The Author(s), under exclusive licence to Springer Nature Limited 2021



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Article



Fig. 1 | SARS-CoV-2 infection elicits durable serum anti-spike antibody lescent donors
collected from five of the eighteen convalescent donor
donorsrs and
an one additional


titers. a, Study design. Seventy-seven SARS-CoV-2 convalescent patients with donor approximately 11 months after infection.
infecction. b,
b, Blood
Bloo IgG titers against S
mild disease (ages 21–69) were enrolled and blood was collected approximately right) measured
right
(left) and influenza virus vaccine (right) me
measured b by ELISA in convalescent
1 month, 4 months, 7 months, and 11 months post onset of symptoms. Bone patients (white circles) at the indicated
ated time
tim post
pos onset of symptoms and
marrow aspirates were collected from eighteen of the participants 7 to controls (black circles). Dotted
ted line indicates
dica
dicates limit of detection. Means and
8 months after infection and from eleven healthy volunteers (ages 23–60) with pairwise differences at each timepoint
ach timep
time were
er estimated using a linear mixed
no history of SARS-CoV-2 infection. Follow-up bone marrow aspirates were model analysis.












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Fig. 2 | SARS-CoV-2 infection elicits S-binding long-lived BMPCs. (n=18 co
(n=18 on
convalescent, 11 control). c, Paired frequencies of BMPC secreting IgG


a, Representative images of ELISpot wells coated with the indicated antigens igens or
igen (left) and
(left) a IgA (right) specific for S from convalescent participants 7m and 11m
anti-Ig and developed in blue and red for IgG and IgA, respectively after ter po symptom onset. d, Paired anti-S (left) and anti-RBD (right) IgG serum
post
incubation of magnetically enriched BMPC from convalescent and control ntrolol antibody titers from convalescent participants 7m and 11m post symptom
participants. b, Frequencies of BMPC secreting IgG (left) or IgA (righ
A (right)
(righ onset. Data in panels (c) and (d, left) are also shown in (b) and Fig. 1b,
antibodies specific for the indicated antigens, indicated as percentages
percentage of total respectively. Each symbol represents one sample (n=5). e, Frequencies of IgG


IgG- or IgA-secreting BMPC in control (black circles) or convalescent

convalescent
conv vales BMPC specific for S (left) and influenza virus vaccine (right) plotted against
participants 7m (white circles) or 11m (grey circles) post symptom
ost symp
sympt onset.
om ons respective IgG titers in paired blood samples from control (black circles) or
Horizontal lines indicate medians. P-values from two-sided
wo-sided Kruskal-Wallis
Krusk l tests convalescent participants 7m post symptom onset (white circles). P- and
with Dunn’s correction for multiple comparisons between
ons b
betwe control and
een contr r-values from two-sided Spearman’s correlations. Each symbol represents one
convalescent participants. Each symbol represents
resents one
pre o sample
sa
amp sample (n=18 convalescent, 11 control).






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Article



Fig. 3 | SARS-CoV-2 BMPCs are quiescent and distinct from circulating PBs.
a, Representative plots of intracellular S staining in CD20lo CD38+ IgDlo CD19+/lo
CD3– live singlet BMPCs (gating in Extended Data Fig. 1a) from control (left) and


convalescent (right) magnetically enriched BMPC 7 months after symptom
onset. b, Frequencies of S-binding BMPCs in total BMPC from control (black
circles) or convalescent participants 7m post symptom onset (white circles).
Horizontal lines indicate medians. P-value from two-sided Mann-Whitney
U-test. Each symbol represents one sample (n=12 convalescent, 9 control).
c, Histographs of Blimp1 (left), Ki-67 (center), and CD38 (right) staining on


S+ (blue) and HA+ (black) BMPC from magnetically enriched BMPC 7m post
symptom onset and S+ plasmablasts (red), and naïve B cells (grey) from healthy
donor PBMC 1 week after SARS-CoV-2 S immunization.











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Fig. 4 | SARS-CoV-2 infection elicits a robust memory B cell response. in (c). c, Frequencies of S- (left) and HA- (right)
ight) binding memory
m
mem
em B cells in PBMC


a, Representative plots of surface influenza virus hemagglutinin (HA) and S alesce
from control (black circles) and convalescent ent (white circles)
cir participants
staining on CD20+ CD38lo/int IgDlo CD19+ CD3– live singlet memory B cells (gating 7 months after symptom onset. The he dotted line
lin
ne in th
the S plot indicates limit of
in Extended Data Fig. 1d) from control (left) and convalescent (right) PBMCs sensitivity, defined as the median + 2× SD o of the controls. Each symbol
7 months after symptom onset. b, Kinetics of S- (top) and HA- (bottom) binding represents one sample (n=18 8c
co scent, 11 control). Horizontal lines
scen
convalescent,
memory B cells in PBMCs from convalescent patients collected at the indicated indicate medians. P-values es from two-sided
ttw ed Mann-Whitney U-tests.
days post onset of symptoms. Data from the 7-month timepoint are also shown












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Article
Methods
ELISA
Sample collection, preparation, and storage Assays were performed in 96-well plates (MaxiSorp; Thermo) coated
All studies were approved by the Institutional Review Board of Wash- with 100 μL of Flucelvax 2019/2020 or recombinant S in PBS, and plates
ington University in St. Louis. Written consent was obtained from all were incubated at 4 °C overnight. Plates were then blocked with 10%
participants. Seventy-seven participants who had recovered from FBS and 0.05% Tween20 in PBS. Serum or plasma were serially diluted
SARS-CoV-2 infection and eleven controls without SARS-CoV-2 infec- in blocking buffer and added to the plates. Plates were incubated for
tion history were enrolled (Extended Data Tables 1 and 3). Blood sam- 90 min at room temperature and then washed 3 times with 0.05% 5%
ples were collected in EDTA tubes and peripheral blood mononuclear Tween-20 in PBS. Goat anti-human IgG-HRP ( Jackson ImmunoRe- noRe-
noRe

cells (PBMCs) were enriched by density gradient centrifugation over search, 1:2,500) was diluted in blocking buffer before adding g to
o wells
Ficoll 1077 (GE) or Lymphopure (BioLegend), remaining red blood and incubating for 60 min at room temperature. Plates were ere washed
was
washhed
ed
cells were lysed with ammonium chloride lysis buffer, and cells were 3 times with 0.05% Tween20 in PBS, and then washed 3 timtimes
mes with PBS S


immediately used or cryopreserved in 10% dimethylsulfoxide in FBS. before the addition of o-Phenylenediamine dihydrochloride
hlorid
oride pperoxidase
eroxidas
Approximately 30 mL bone marrow aspirates were collected in EDTA substrate (Sigma-Aldrich). Reactions were stopped ped by the
he ad
addition
di
tubes from the iliac crest of eighteen convalescent participants and of 1 M HCl. Optical density measurements were re taken
t at 4
490 nm. The


the controls. Bone marrow mononuclear cells were enriched by den- half-maximal binding dilution for each serumum or plasma
pl sma sa
p ssample was
sity gradient centrifugation over Ficoll 1077, remaining red blood calculated using nonlinear regression (Graphpad
Gra
aphpad Prism sm v8). The limit
cells were lysed with ammonium chloride buffer (Lonza) and washed of detection was defined as 1:30.
with PBS supplemented with 2% FBS and 2 mM EDTA. Bone marrow


plasma cells were enriched from bone marrow mononuclear cells Statistics
using CD138 Positive Selection Kit II (Stemcell) and immediately used Spearman’s correlation coefficientsients were
wer estimated
esstim to assess the relation-
for ELISpot or cryopreserved in 10% dimethylsufoxide in FBS for flow ship between 7-month anti-S and d anti-influenza
anti-influ
ant virus vaccine IgG titers
cytometric analysis. and frequencies of BMPCs Cs ssecr
secretingg IgG specific for S and influenza virus
vaccine, respectively.. Means and pairwise differences of antibody titers


Antigens at each timepointt were e estimated using a linear mixed model analysis
Recombinant soluble spike protein (S) and its receptor binding with a first order
err aut
autoregre
autoregressive
essiv covariance structure. Time since symp-
domain (RBD) derived from SARS-CoV-2 was expressed as previously tom onset was tre
trea
treated
ated as a ccategorical fixed effect for the four different
described35. Briefly, mammalian cell codon-optimized nucleotide sample time pace approximately 3 months apart. P-values were
me points spaced
sp
sequences coding for the soluble version of S (GenBank: MN908947.3,
amino acids 1-1213) including a C-terminal thrombin cleavage site,  adjusted
were
ed ffor
ere con
c
or multipl
conducted
nduc
multiple comparisons using Tukey’s method. All analyses
cted
ted u using SAS 9.4 (SAS Institute Inc, Cary, NC, USA) and
and P-values < 0.05 were considered significant.

T4 foldon trimerization domain, and hexahistidine tag cloned into P
Prism 8.4 (G
8 (Graphpad),
Grap
mammalian expression vector pCAGGS. The S protein sequence was
modified to remove the polybasic cleavage site (RRAR to A) and two wo Flow
w cyto
cytometry
c


stabilizing mutations were introduced (K986P and V987P, wild d typee Staining for flow cytometry analysis was performed using cryo-
Staini
numbering). RBD, along with the signal peptide (amino acids cids
ds 1-14) preserved magnetically enriched BMPC and cryo-preserved PBMC.
pr
pre
plus a hexahistidine tag were cloned into mammalian expression pression
ssion For BMPC staining, cells were stained for 30 min on ice with CD45-A532
F
vector pCAGGS. Recombinant proteins were produced ed in Expi293F
pi293F
293 (HI30, Thermo, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD19-PE
cells (ThermoFisher) by transfection with purified ed DNA using
usi the
us th (HIB19, 1:200), CXCR5-PE-Dazzle 594 ( J252D4, 1:50), CD71-PE-Cy7
ExpiFectamine 293 Transfection Kit (ThermoFisher). isher). Supernatants
ishe Superna
Supern (CY1G4, 1:400), CD20-APC-Fire750 (2H7, 1:400), CD3-APC-Fire810


from transfected cells were harvested 3 (forr S) or 4 (for (for RBD)

RB days (SK7, 1:50), and Zombie Aqua (all BioLegend) diluted in Brilliant Staining
post-transfection, and recombinant proteins ote
tein
ns were pu
purified using buffer (BD Horizon). Cells were washed twice with 2% FBS and 2 mM
Ni-NTA agarose (ThermoFisher), then buffer buffe exchanged
ex
xchang into phos- EDTA in PBS (P2), fixed for 1h using the True Nuclear permeabilization kit
phate buffered saline (PBS) and concentrated
ncen
entrated us
using Amicon Ultra- (BioLegend), washed twice with perm/wash buffer, stained for 1h with


cel centrifugal filters (EMD Millipore).

pore).
ore). For
Fo
or flow
ow cytometry
c staining, DyLight 405-conjugated recombinant HA from A/Michigan/45/2015,
recombinant S was labeled with h Al
Alexa
xa Fluor 64
6647- or DyLight 488-NHS DyLight 488- and Alexa 647-conjugated S, Ki-67-BV711 (Ki-67, 1:200,
ester (Thermo Fisher); excess ess Alexa
Alexxa Flu
F
Fluor 6647 and DyLight 488 were BioLegend), and Blimp1-A700 (646702, 1:50, R&D), washed twice with
removed using 7-kDa and 40-kDa Zeb Zeba
ba desalting columns, respec- perm/wash buffer, and resuspended in P2. For memory B cell staining,


tively (Pierce). Recombinant

binant HA
H from
fr A/Michigan/45/2015
A (a.a. 18-529, PBMC were stained for 30 min on ice with biotinylated recombinant
Immune Technology) y) was labeledd with
w DyLight 405-NHS ester (Thermo HAs diluted in P2, washed twice, then stained for 30 min on ice with
Fisher); excess DyLigh
DyLightht 405 was removed using 7-kDa Zeba desalting Alexa 647-conjugated S, IgA-FITC (M24A, Millipore, 1:500), IgG-BV480
columns. Recombinant
coombinant H HA ffrom A/Brisbane/02/2018 (a.a.18–529) (goat polyclonal, Jackson ImmunoResearch, 1:100), IgD-SB702 (IA6-2,


and B/Colorado/06/2017
orado/06/2017 (a.a. 18–546) (both Immune Technology)
orado/0 Thermo, 1:50), CD38-BB700 (HIT2, BD Horizon, 1:500), CD20-Pacific
were biotinylated
iotinylated using
ussin the EZ-Link Micro NHS-PEG4-Biotinylation Blue (2H7, 1:400), CD4-BV570 (OKT4, 1:50), CD24-BV605 (ML5, 1:100),
Kit (Thermo
Thermo Fisher);
Fisher excess biotin was removed using 7-kDa Zeba streptavidin-BV650, CD19-BV750 (HIB19, 1:100), CD71-PE (CY1G4,
desalting
esalting
alting col
colu
columns.
mn 1:400), CXCR5-PE-Dazzle 594 ( J252D4, 1:50), CD27-PE-Cy7 (O323,


1:200), IgM-APC-Fire750 (MHM-88, 1:100), CD3-APC-Fire810 (SK7,

ELISpo
ELISpot
po
ot 1:500), and Zombie NIR (all BioLegend) diluted in Brilliant Staining


Plates
es were
w coated with Flucelvax Quadrivalent 2019/2020 seasonal buffer (BD Horizon), and washed twice with P2. Cells were acquired
influenza virus vaccine (Sequiris), tetanus/diphtheria vaccine (Grifols),
influe on an Aurora using SpectroFlo v2.2 (Cytek). Flow cytometry data were
recombinant S, or anti-human Ig. Direct ex-vivo ELISpot was performed
re analyzed using FlowJo v10 (Treestar). In each experiment, PBMC were
to determine the number of total, vaccine-binding, or recombinant included from convalescent and control participants.
S-binding IgG- and IgA-secreting cells present in BMPC and PBMC sam-
ples using IgG/IgA double-color ELISpot Kits (Cellular Technologies, Reporting summary
Ltd.) according to the manufacturer’s instructions. ELISpot plates were Further information on research design is available in the Nature
analyzed using an ELISpot counter (Cellular Technologies Ltd.). Research Reporting Summary linked to this paper.
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 90 of 165

The WU353, WU367, and 368 studies were reviewed and approved by the Washington
Data availability statement University Institutional Review Board (approval nos. 202003186, 202009100, and 202012081,
respectively).
Relevant data are available from the corresponding author upon rea-
sonable request. Author contributions A.H.E. conceived and designed the study. J.S.T. and A.H.E. designed
experiments and composed the manuscript., A.H., M.K.K., I.P., J.A.O., and R.M.P. wrote and
maintained the IRB protocol, recruited, and phlebotomized participants and coordinated
35. Stadlbauer, D. et al. SARS‐CoV‐2 Seroconversion in Humans: A Detailed Protocol for a
sample collection. J.S.T., W.K., E.K., A.J.S., and L.H. processed specimens. A.J.S. expressed
Serological Assay, Antigen Production, and Test Setup. Curr. Protoc. Microbiol. 57,
S and RBD proteins. J.S.T., W.K., and E.K. performed ELISA and ELISpot. J.S.T. performed flow
(2020).
cytometry. J.S.T., A.M.R., C.W.G. and A.H.E. analyzed data. All authors reviewed the
manuscript.
Acknowledgements We thank the generous participation of the donors for providing precious

specimens. We thank Lisa Kessels, AJ Winingham, the staff of the Infectious Diseases Clinical Competing interests The Ellebedy laboratory received funding under sponsored research sea
arch
Research Unit at Washington University School of Medicine, and the nursing team of the bone agreements that are unrelated to the data presented in the current study from Emergent
merge
mergent
marrow biopsy suite at Washington University School of Medicine and Barnes Jewish Hospital BioSolutions and from AbbVie. J.S.T., A.J.S., and A.H.E. are recipients of a licensing
ing agreement
agreemen
agreem nt
for sample collection and providing care for donors. The SARS-CoV-2 S and RBD protein with Abbvie Inc. unrelated to the data presented in the current study. A.H.E.. is a consultant for


expression plasmids were kindly provided by F. Krammer (Icahn School of Medicine at Mt Mubadala Investment Company and the founder of ImmuneBio Consulting ing
ng L Alll other
LLC. A
Sinai). The Ellebedy laboratory was supported by National Institute of Allergy and Infectious authors declare no competing interests.
Diseases (NIAID) grants U01AI141990 and 1U01AI150747, NIAID Centers of Excellence
for Influenza Research and Surveillance contracts HHSN272201400006C and Additional information


HHSN272201400008C, and NIAID Collaborative Influenza Vaccine Innovation Centers Supplementary information The online version contains supplementary material
materi available at
pplementary materia
pplementa
contract 75N93019C00051. J.S.T. was supported by NIAID 5T32CA009547. L.H. was supported https://doi.org/10.1038/s41586-021-03647-4.
by Norwegian Research Council grant 271160 and National Graduate School in Infection Correspondence and requests for materials should be addressed
e ad
add ressed to A.H.E.
A.H.
Biology and Antimicrobials grant 249062. This study utilized samples obtained from the Peer review information Nature thanks Stanley Perlman, Andreas
lman, An
Andre Radbruch and the other,
eas Radb
Washington University School of Medicine’s COVID-19 biorepository supported by the NIH/ anonymous, reviewer(s) for their contribution to the peer review of
of this
th work. Peer reviewer


National Center for Advancing Translational Sciences grant UL1 TR002345. The content is reports are available.
solely the responsibility of the authors and does not necessarily represent the view of the NIH. vailable at
Reprints and permissions information is available a http://ww
http://www.nature.com/reprints.
http:/











Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 91 of 165

Article
a 4M Lymphocytes 4M 4M 4M

CD3 :: APC-F810
Aqua– 106 106 IgDlo
95.5 92.4 98.5
105

CD19 :: PE
FSC-W
3M 3M 3M 3M 105

SSC-A
SSC-A

SSC-W
CD19
Single CellsF Single CellsS 84.8 104
2M 2M 2M 2M 104
99.3 99.6
1M 1M 1M 1M 0
0
-104
0 0 0 0
4 5 6 4 5 6
0 1M 2M 3M 4M 0 1M 2M 3M 4M 0 1M 2M 3M 4M 0 10 10 10 0 10 10 10 0 104 105 106
FSC-A FSC-H SSC-H Zombie Aqua CD19 :: PE IgD :: PE-Cy5

b c Influenza virus SARS-CoV-2
Control Convalescent vaccinated vaccinated PBMC BMPC
C
HA +
10 6 106 D20lo CD38+
CD20
106

CD38 :: BB700
0.16 0.065 S+ CD20lo CD38+ 24.6
HA :: Dy405

32.2

S :: Dy488
5 0 105 18.5
10 10 5


104
104 104
103
0 0
0


-104 -104 -103
-104 0 104 105 106 -104 0 104 105 106 0 104 105 106
S :: A647 S :: A647 CD20 :: APC-F750
PC-F75
C-F7500

d 4M 4M 4M 4M IgDlo

CD3 :: APC-F810


106 106

750
0
CD19 :: BV750
Lymphocytes NIR- 13.5
105 CD19
CD
D19
SSC-W

SSC-W
5
10
FSC-W

3M 3M 3M 3M
SSC-A

68.9 99.8
Single CellsF 4
7.94
97.2 Single CellsS 104 104
2M 2M 2M 2M
99.8
0 103
1M 1M 1M 1M
0
-104
0 0 0 0 -103


0 1M 2M 3M 4M 0 1M 2M 3M 4M 0 1M 2M 3M 4M 0 104 105 106 0 104 105 106 0 104 105 106
FSC-A FSC-H SSC-H Zombie NIR
R CD19 :: BV750
CD IgD :: SB702

CD20loCD38+

CD38 :: BB700
106
4.94
105

 104

103
CD20+

0 CD38int/lo
-103 94.2
0 104 105
CD20 :: Pacific Blue


Extended Data Fig. 1 | Flow cytometry identification of SARS-CoV-2 2 elicited (HA)

(H staining in BMPC from control (left) and convalescent (right) samples
plasma cells and memory B cells. a, d, Flow cytometry gating strategies
egiess 7 months after symptom onset. c, Representative plots of intracellular
for BMPC in magnetically enriched BMPC and plasmablasts in PBMC BM (a)) and
nd S staining in plasmablasts in PBMC 1 week after seasonal influenza virus or
isotype-switched memory B cells and plasmablasts in PBMC (d). SARS-CoV-2 vaccination.
b, Representative plots of intracellular S and influenza viruss hemagglutinin
hemagglutin
hemagglut






Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 92 of 165

Extended Data Table 1 | SARS-CoV-2 convalescent patient demographics

Bone marrow biopsy

Total N=77
N=19
N (%)
N (%)
Age (median [range]) 49 (21-69) 52 (30-69)
Sex
Female 38 (49.4) 7 (36.8)
Male 39 (50.6) 12 (63.2)

Race
White 70 (90.9) 18 (94.7)


Black 1 (1.3) 0 (0)
Asian 4 (5.2) 0 (0)
Other 2 (2.6) 1 (5.3)


Comorbidities
Asthma 13 (16.9) 3 (15.8)
Lung disease 0 (0) 0 (0)
Heart disease 3 (3.9) 0 (0)


Hypertension 13 (16.9) 6 (31.6)
Diabetes mellitus 3 (3.9) 3 (15.8)
Cancer 10 (13) 3 (15.8)
Autoimmune disease 4 (5.2) 2 (10.5))
Hyperlipidemia 8 (10.4) 2 (10.5)
0.5
5)


Hypothyroidism 5 (6.5) 3 (15.8)
(1
Gastroesophageal reflux disease 5 (6.5) 2 (10.5)
(10.5
(
Other 26 (33.8) 10 (52.6)
0 (52
(52
2.6)
Solid organ transplant 1 (1.3) (5.3)
1 (5
5.3
.3)
Obesity

1 (1.3) 0 (0)
(0








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Article
Extended Data Table 2 | SARS-CoV-2 convalescent patient symptoms

Bone marrow biopsy

Total N=77
N=19
N (%)
N (%)
First symptom
Cough 12 (15.6) 3 (15.8)
Diarrhea 1 (1.3) 0 (0)
Dyspnea 2 (2.6) 1 (5.3)

Fatigue 7 (9.1) 0 (0)
Fever 22 (28.6) 9 (47.4)


Headache 8 (10.4) 2 (10.5)
Loss of taste 3 (3.9) 2 (10.5)
Malaise 4 (5.2) 1 (5.3)


Myalgias 9 (11.7) 0 (0)
Nasal congestion 2 (2.6) 0 (0)
Nausea 1 (1.3) 0 (0)
Night sweats 1 (1.3) 0 (0)


Sore throat 5 (6.5) 1 (5.3)
Symptom present during disease
Fever 65 (84.4) 17 (89.5)
Cough 54 (70.1) 14 (73.7)7)
Dyspnea 31 (40.3) 11 (57.9)
7.9)


Nausea 19 (24.7) 4 (21.1)
(2
Vomiting 9 (11.7) 3 (15.8)
(15.8
(
Diarrhea 39 (50.6) 10 (52.6)
0 (52
(52
2.6)
Headaches 47 (61) (63.2)
12 (663.2
3
Loss of taste
Loss of smell

42 (54.5)
42 (54.5)
11 (5
110
(57.9)
0 (52.6)

Fatigue 38 (49.4) 7 (36.8)
Malaise 6 (7.8) 1 (5.3)


Myalgias or body aches 34 (44.2)

4.2) 8 (42.1)
Sore throat 12 (15.6)
5.6) 1 (5.3)
Chills 25 (32.5)
2.5) 6 (31.6)
Nasal congestion 6 (7.8)
8)) 0 (0)
Other (41.6)
322 (4 7 (36.8)


Duration of symptoms in days 14 (1-43)

4 (1
(1- 13 (6-30)
(median [range])
Days from symptom onset to positive itive 6 (0-36) 6 (1-31)
SARS-CoV-2 PCR test (median n


[range])
Days from symptom onset ett too 1-month
1-montnth 41 (21-84) 34 (22-71)
blood sample collection n (median
(med
(me edian
ian
[range])


Hospitalization 6 (7.8) 1 (5.3)

COVID medications
atio s
ation
Hydroxychloroquine
loroquuine
ui
ine 2 (2.6) 0 (0)
Chloroquine
uine
uin 1 (1.3) 0 (0)


Azithromycin
romycin
romycin 14 (18.2) 6 (31.6)
Lopinavir/ritonavir
opin
navir/ritona 0 (0) 0 (0)
Remdesivir
Remd sivir
Remdes 0 (0) 0 (0)
Convalescent
Convales
Convalesc plasma 0 (0) 0 (0)


None
N 61 (79.2) 12 (63.2)
Other
Ot
Othher 2 (2.6) 1 (5.3)

Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 94 of 165

Extended Data Table 3 | SARS-CoV-2 convalescent patient symptoms and follow up samples (months 4–11)
Month 4 Month 7 Month 11
Bone marrow Bone marrow Bone marrow
Total N= 76 Total N= 76 Total N= 42
biopsy N=19 biopsy N=18 biopsy N=12
N (%) N (%) N (%)
N (%) N (%) N (%)
Days from positive SARS- 125 (102-192) 117 (105-150) 222 (191-275) 213 (200-247) 308 (283-369) 303 (283-325)
CoV-2 PCR test to follow up
visit (median [range])

Days from symptom onset 131 (106-193) 124 (108-155) 227 (194-277) 222 (205-253) 314 (288-373) 309 (297-343)
3433)
343)
to blood sample collection
(median [range])


Any symptom present at 25 (32.9) 8 (42.1) 33 (43) 10 (55.6) 20 (47.6) (50)
6 (50)
follow up visit
Fever 0 (0) 0 (0) 2 (2.6) 0 (0) 1 (2.4) 0 (0)


Cough 1 (1.3) 1 (5.3) 0 (0) 0 (0) 1 (2.4) 0 (0)
(
Dyspnea 7 (9.2) 2 (10.5) 6 (7.9) 3 (16.7) 3)
6 (14.3) (
3 (25)
Nausea 1 (1.3) 0 (0) 1 (1.3) 0 (0) 0 (0) 0 (0)
Vomiting 1 (1.3) 1 (5.3) 0 (0) 0 (0) 0 (0) 0 (0)
Diarrhea 2 (2.6) 1 (5.3) 1 (1.3) 0 (0) 0 (0) 0 (0)


Headaches 1 (1.3) 0 (0) 3 (3.9) 0 (0) 2 (4.8)
(4.8
(4 .8) 0 (0)
Loss or altered taste 8 (10.5) 0 (0) 9 (11.8) 1 (5.6) 5 (1 1 9)
9
(11.9) 1 (8.3)
Loss or altered smell 13 (17.1) 2 (10.5) 12 (15.8) 2 (11.1) (19)
8 (1 2 (16.7)
Fatigue 9 (11.8) 4 (21.1) 13 (17.1) 5 (27.8)) 8 (19) 3 (25)
Forgetfulness/brain fog 8 (10.5) 6 (31.6) 12 (15.8) 6 (33.3)
.3) 10 (23.8) 4 (33.3)


Hair loss 5 (6.6) 1 (5.3) 3 (3.9) 1 (5.6
(5.6)) 2 (4.8) 0 (0)
Other 7 (9.2) 3 (15.8) 12 (15.8) 1 (5.6)
(5.6) 10 (23.8) 1 (8.3)
Joint pain 3 (3.9) 1 (5.3) 7 (9.2) 1 (5.3)
(5. )
(5.3 3 (7.1) 0 (0)










Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 95 of 165

Article
Extended Data Table 4 | Healthy control demographics

Total N= 11
Variable
N (%)
Age (median [range]) 38 (23-53)
Sex
Female 4 (36.4)
Male 7 (63.6)
Race

White 8 (72.7)
Black 1 (9.1)


Asian 1 (9.1)














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EXHIBIT 7
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bioRxiv 1:21-cv-02228
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Immunological memory to SARS-CoV-2 assessed for greater than six

months after infection

Jennifer M. Dan1,3*, Jose Mateus1*, Yu Kato1*, Kathryn M. Hastie1, Caterina E. Faliti1, Sydney I. Ramirez1,3,
April Frazier1, Esther Dawen Yu1, Alba Grifoni1, Stephen A. Rawlings3, Bjoern Peters1,2, Florian Krammer4,
Viviana Simon4,5,6, Erica Ollmann Saphire1,3, Davey M. Smith3, Daniela Weiskopf1^, Alessandro Sette1,3^,
Shane Crotty1,3^

1
Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA
92037, USA
2
Department of Medicine, University of California, San Diego (UCSD), La Jolla, CA 92037, USA
3
Department of Medicine, Division of Infectious Diseases and Global Public Health, University of
California, San Diego (UCSD), La Jolla, CA 92037, USA
4
Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, New York
5
Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount
Sinai, New York, NY 10029, USA
6
The Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai,
New York, NY 10029, USA

* These authors contributed equally

^ Correspondence: shane@lji.org (S.C.), alex@lji.org (A.S.), daniela@lji.org (D.W.)

Case
bioRxiv 1:21-cv-02228
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ABSTRACT
Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and
for assessing the likely future course of the pandemic. We analyzed multiple compartments of circulating
immune memory to SARS-CoV-2 in 185 COVID-19 cases, including 41 cases at > 6 months post-
infection. Spike IgG was relatively stable over 6+ months. Spike-specific memory B cells were more
abundant at 6 months than at 1 month. SARS-CoV-2-specific CD4+ T cells and CD8+ T cells declined with
a half-life of 3-5 months. By studying antibody, memory B cell, CD4+ T cell, and CD8+ T cell memory to
SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune
memory exhibited distinct kinetics.
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
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posted November Page
2020. The copyright 102forof
holder this165
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

INTRODUCTION
Coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2), is a serious disease that has resulted in widespread global morbidity and
mortality. Humans make SARS-CoV-2-specific antibodies, CD4+ T cells, and CD8+ T cells in response to
SARS-CoV-2 infection (1–4). Studies of acute and convalescent COVID-19 patients have observed that T
cell responses are associated with lessened disease (5–7), suggesting that SARS-CoV-2-specific CD4+ T
cell and CD8+ T cell responses may be important for control and resolution of primary SARS-CoV-2
infection. Ineffective innate immunity has been strongly associated with a lack of control of primary SARS-
CoV-2 infection and a high risk of fatal COVID-19 (8–12), accompanied by innate cell immunopathology
(13–18). Neutralizing antibodies have generally not correlated with lessened COVID-19 disease severity
(5, 19, 20), which was also observed for Middle Eastern respiratory syndrome (MERS), caused by
infection with the human coronavirus MERS-CoV (21). Instead, neutralizing antibodies are associated
with protective immunity against secondary (2°) infection with SARS-CoV-2 or SARS-CoV in non-human
primates (3, 22–25). Additionally, human subjects with detectable neutralizing antibodies were
protected from 2° COVID-19 in a ship outbreak (26). Passive transfer of neutralizing antibodies in
advance of infection (mimicking the conditions of 2° infection) effectively limits upper respiratory tract
(URT) infection, lower respiratory tract (lung) infection, and symptomatic disease in animal models (27–
29). Passive transfer of neutralizing antibodies provided after initiation of infection in humans have had
more limited effects on COVID-19 (30, 31), consistent with a substantial role for T cells in control and
clearance of an ongoing SARS-CoV-2 infection. Thus, studying antibody, memory B cell, CD4+ T cell, and
CD8+ T cell memory to SARS-CoV-2 in an integrated manner is likely important for understanding the
durability of protective immunity against COVID-19 generated by primary SARS-CoV-2 infection (1, 19,
32).
While sterilizing immunity against viruses can only be accomplished by high-titer neutralizing
antibodies, successful protection against clinical disease or death can be accomplished by several other
adaptive immune memory scenarios. Possible mechanisms of immunological protection can vary based
on the relative kinetics of the immune memory responses and infection. For example, clinical hepatitis
after hepatitis B virus (HBV) infection is prevented by vaccine-elicited immune memory even in the
absence of circulating antibodies, because of the relatively slow course of HBV disease (33, 34). The
relatively slow course of severe COVID-19 in humans (median 19 days post-symptom onset (PSO) for
fatal cases (35)) suggests that protective immunity against symptomatic or severe 2° COVID-19 may very
well involve memory compartments such as circulating memory T cells and memory B cells (which can
take several days to reactivate and generate recall T cell responses and/or anamnestic antibody
responses) (19, 21, 32).
Immune memory, from either primary infection or immunization, is the source of protective
immunity from a subsequent infection (36–38). Thus, COVID-19 vaccine development is closely tied to
the topic of immunological memory (1, 3). Despite intensive study, the kinetics, duration, and evolution
of immune memory in humans to infection or immunization are not in general predictable based on the
initial effector phase, and immune responses at short time points after resolution of infection are not
very predictive of long-term memory (39–41). Thus, assessing responses over an interval of six months
or more is usually required to ascertain the durability of immune memory.
A thorough understanding of immune memory to SARS-CoV-2 requires evaluation of its various
components, including B cells, CD8+ T cells, and CD4+ T cells, as these different cell types may have
immune memory kinetics relatively independent of each other. Understanding the complexities of
immune memory to SARS-CoV-2 is key to gain insights into the likelihood of durability of protective
immunity against re-infection with SARS-CoV-2 and 2° COVID-19 disease. In the current study, we
assessed immune memory of all three branches of adaptive immunity (CD4+ T cell, CD8+ T cell, and
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humoral immunity) in a cross-sectional study of 185 recovered COVID-19 cases, extending out to greater
than six months post-infection. The findings have implications for immunity against 2o COVID-19, and
thus the potential future course of the pandemic (42, 43).

COVID-19 cohort
185 individuals with COVID-19 were recruited for this study. Subjects (43% male, 57% female)
represented a range of asymptomatic, mild, moderate, and severe COVID-19 cases (Table S1), and were
recruited from multiple sites throughout the United States. The majority of subjects were from California
or New York. The majority of subjects had a mild case of COVID-19, not requiring hospitalization. 92%
of subjects were never hospitalized for COVID-19; 7% of subjects were hospitalized, some of whom
required intensive care unit (ICU) care (Table S1; hospitalization requirement not reported for 1 subject),
consistent with the COVID-19 disease severity distribution in the USA. The majority of subjects (97%)
reported symptomatic disease (Table S1; not reported for 1 subject). The ages of the subjects ranged
from 19 to 81 years old (Table S1). Most subjects provided a blood sample at a single time point,
between 6 days (d) post-symptom onset (PSO) and 240d PSO (Table S1), with 41 samples at > six months
PSO (d178 or longer). Thirty-eight subjects provided longitudinal blood samples over a duration of
several months (2-4 time points. Table S1).

SARS-CoV-2 circulating antibodies over time

The vast majority of SARS-CoV-2 infected individuals seroconvert, at least for a duration of months (1, 2,
4, 20, 44–46). These estimates range from 91-99% in large studies (20, 46). Durability assessments of
circulating antibody titers in Figure 1 were based on data > 20d PSO, using curve fits modeling a
continuous decay, one-phased decay, or two-phased decay, with the best fitting model shown in blue.
Negative and positive controls were used to standardize each assay and normalize across experiments.
SARS-CoV-2 spike IgG endpoint ELISA titers in plasma were measured for all subjects of this cohort (Fig.
1A-B). Spike receptor binding domain (RBD) IgG was also measured (Fig. 1C-D), as RBD is the target of
the vast majority of neutralizing antibodies against SARS-CoV-2 (4, 28, 47, 48). SARS-CoV-2 pseudovirus
(PSV) neutralizing antibody titers were measured in all subjects, as the functional complement of the
antibody binding assays (Fig. 1E-F). Nucleocapsid (N) IgG endpoint ELISA titers were also measured for
all subjects (Fig. 1G-H), as nucleocapsid is a common antigen in commercial SARS-CoV-2 serological
test kits.
SARS-CoV-2 spike IgG titers were nearly stable from d20-d240 PSO, when assessing all COVID-
19 subjects by cross-sectional analysis (half-life t1/2 = 140d, Fig. 1A). Spike IgG titers were heterogenous
among subjects (range 5 to 73,071; 575 median), as has been widely observed (20, 48). This gave a wide
confidence interval for the spike IgG t1/2 (95% CI: 89 to 329d). While the antibody responses likely have
underlying bi-phasic decay kinetics, the best fit curve was a linear decay, probably related to
heterogeneity between individuals. SARS-CoV-2 nucleocapsid IgG kinetics were similar to spike IgG
over 8 months (t1/2 67d, 95% CI: 49-105d. Fig. 1G). As a complementary approach, using paired samples
from the subset of subjects who donated at two or more time points, the calculated spike IgG titer
average t1/2 was 100d, (95% CI: 64-220d, Fig. 1B) and the nucleocapsid IgG titer average t1/2 was 67d,
(95% CI: 54-88d, Fig. 1H). The percentage of subjects seropositive for spike IgG at 1 month PSO (d20-
50) was 98% (54/55). The percentage of subjects seropositive for spike IgG at 6 to 8 months PSO (d
>178) was 90% (36/40).
Cross-sectional analysis SARS-CoV-2 RBD IgG titers from d20-d240 PSO gave an estimated t1/2
of 83d, 95% CI 62-127d (Fig. 1C). As a complementary approach, we again used paired samples, which
gave an average t1/2 of 68d, 95% CI: 57-85d (Fig. 1D). The percentage of subjects seropositive for RBD
IgG at 6 to 8 months PSO was 88% (35/40). Thus, the RBD IgG titer maintenance largely matched that of
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spike IgG. SARS-CoV-2 PSV neutralization titers in the full cohort largely matched the results of SARS-
CoV-2 RBD IgG ELISA binding titers (Fig.1E-F). A one-phase decay model was the best fit (P=0.015, F
test. Initial decay t1/2 27d, followed by an extended plateau phase. Fig. 1E), while a linear decay gave an
estimated t1/2 of 114d. Paired timepoints analysis of the PSV neutralization titers gave an estimated t1/2
87d, (95% CI: 68-123d, Fig. 1F). The percentage of subjects seropositive for SARS-CoV-2 neutralizing
antibodies (titer > 20) at 6 to 8 months PSO was 90% (36/40). Notably, even low levels of circulating
neutralizing antibody titers (> 1:20) were associated with a substantial degree of protection against
COVID-19 in non-human primates (24, 49). Thus, modest levels of circulating SARS-CoV-2 neutralizing
antibodies are of biological interest in humans.
SARS-CoV-2 spike IgA (Fig. 1I-J) and RBD IgA (Fig.1K-L) titers were also assessed. Paired
timepoints analysis of spike IgA titers yielded an estimated t1/2 of 214d, 95% CI 126-703d (Fig. 1J).
Cross-sectional analysis of spike IgA fit a shohrt one-phase decay model with an extended plateau phase
(initial t1/2 of 11d, Fig. 1I). Circulating RBD IgA had an estimated t1/2 of 27d, 95% CI 15-58d, decaying by
~90d in a majority of COVID-19 cases to levels indistinguishable from uninfected controls (Fig. 1K),
consistent with observations 3 months PSO (46, 50). By paired sample analysis, long-lasting RBD IgA was
made in some subjects, but often near the limit of sensitivity (LOS) (Fig. 1L).

SARS-CoV-2 memory B cells

To identify SARS-CoV-2-specific memory B cells, fluorescently labeled multimerized probes were used
to detect B cells specific to spike, RBD, and nucleocapsid (Fig 2A, Fig. S1). Antigen-binding memory B
cells (defined as IgD– and/or CD27+) were further distinguished according to surface immunoglobulin
(Ig) isotypes: IgM, IgG or IgA (Fig. 2B, Fig. S1).
Spike-specific memory B cells in SARS-CoV-2 unexposed donors were rare (median 0.0078%.
Fig 2A, 2C.). Cross-sectional analysis revealed that frequencies of SARS-CoV-2 spike-specific memory B
cells increased over the first ~150d PSO and then plateaued (Pseudo-first order model for best fit curve.
R2 = 0.14. Better fit than second order polynomial model by Akaike’s Information Criterion. Fig 2C, Fig.
S2A). Spike-specific memory B cell frequencies increased from the first time-point (d36-d163) to the
second time-point (d111-d240) in paired samples from 24 of 36 longitudinally tracked donors (Fig 2D).
RBD-specific memory B cells displayed similar kinetics to spike-specific memory B cells. As
expected, RBD-specific memory B cells were undetectable in SARS-CoV-2 unexposed subjects (Fig. 2E.
Fig. S2C). RBD-specific memory B cells appeared as early as 16d PSO, and the frequency steadily
increased in the following 4-5 months (Fig. 2E. Fig. S2B-C). 29 of 36 longitudinally tracked individuals
had higher frequencies of RBD-specific memory B cells at the later time point (Fig. 2F), again showing
an increase in SARS-CoV-2 specific memory B cells several months post-infection. ~10-30% of spike-
specific memory B cells from SARS-CoV-2 convalescent donors were specific for the RBD domain (Fig.
2A, Fig. S2B).
SARS-CoV-2 nucleocapsid-specific memory B cells were also detected after SARS-CoV-2
infection (Fig. 2A). Similar to spike- and RBD-specific memory B cells, nucleocapsid-specific memory B
cell frequency steadily increased during the first ~5 months PSO (Fig. 2G, 2H, Fig. S2D). Antibody
affinity maturation could potentially explain the increased frequencies of SARS-CoV-2-specific memory
B cells detected by the antigen probes. However, geometric mean fluorescent intensity (MFI) of probe
binding was stable over time (Fig. S2I-J), not supporting an affinity maturation explanation for the
increased memory B cell frequencies.
Representation of Ig isotypes among the SARS-CoV-2 spike-specific memory B cell population
shifted with time (Fig. 2I-2O). During the earliest phase of memory (20-60d PSO), IgM+ and IgG+
isotypes were similarly represented (Fig. 2O), but the IgM+ memory B cells gradually disappeared (Fig.
2M, 2N, 2O), and IgG+ spike-specific memory B cells then dominated by 6 months PSO (Fig. 2O). IgA+
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spike-specific memory B cells were detected as a small fraction of the total spike-specific memory B cells
(~5%, Fig. 2O). IgG+ spike-specific memory B cell frequency increased while IgA+ was low and stable
over the 8 month period (Fig. 2I-2L). Similar patterns of increasing IgG+ memory, short-lived IgM+
memory, and stable IgA+ memory were observed for RBD- and nucleocapsid-specific memory B cells
over the 8 month period (Fig. 2O-2Q, Fig. S2E-S2H).
There is limited knowledge of memory B cell kinetics following primary acute viral infection in
humans. We are not aware of other cross-sectional or longitudinal analyses of antigen-specific memory
B cells covering a 6+ month window after an acute infection by flow cytometry, except for four individuals
with Ebola (51) and two individuals studied after yellow fever virus immunization (52), and also excepting
influenza vaccines, for which people have repeated exposures and complex immune history. In the
yellow fever study, short-lived IgM+ memory and longer-lasting isotype-switched memory B cells were
observed in the two individuals. Overall, based on the observations here, development of B cell memory
to SARS-CoV-2 appeared to be robust and likely long-lasting.

SARS-CoV-2 memory CD8+ T cells

SARS-CoV-2 memory CD8+ T cells in 155 subjects were identified using a series of 23 peptide pools
covering the entirety of the SARS-CoV-2 ORFeome (2, 5). The most commonly recognized ORFs were
spike (S), membrane (M), nucleocapsid (N), and ORF3a (CD69+ CD137+, Fig. 3A and Fig. S3A-B),
consistent with our previous study (2). The percentage of subjects with detectable circulating SARS-CoV-
2 memory CD8+ T cells at 1 month PSO (d20-50) was 61% (30/49, Fig. 3B). The proportion of subjects
positive for SARS-CoV-2 memory CD8+ T cells at > 6 months PSO was 50% (9/18). SARS-CoV-2 memory
CD8+ T cells declined with an apparent t1/2 of 166d in the full cohort (Fig. 3B) and t1/2 139d among 24
paired samples (Fig. 3C). Spike-specific memory CD8+ T cells exhibited similar kinetics to the overall
SARS-CoV-2-specific memory CD8+ T cells (t1/2 271d for the full cohort and 164d among paired samples,
Fig. 3D-E, respectively). Phenotypic markers indicated that the majority of SARS-CoV-2-specific memory
CD8+ T cells were TEMRA (53), with small populations of TCM and TEM (Fig. 3F). In the context of influenza,
CD8+ TEMRA were associated with protection against severe disease in humans (54). The memory CD8+
T cell half-lives observed herein were comparable to the 123d t1/2 observed for memory CD8+ T cells
within 1-2 years after yellow fever immunization (55). Overall, the decay of circulating SARS-CoV-2-
specific CD8+ T cell is consistent with what has been reported for another acute virus.

SARS-CoV-2 memory CD4+ T cells

SARS-CoV-2 memory CD4+ T cells in 155 subjects were identified using the same series of 23 peptide
pools covering the SARS-CoV-2 ORFeome (2, 5). The most commonly recognized ORFs were spike, M,
N, ORF3a, and nsp3 (CD137+ OX40+, Fig. 4A and Fig. S4A-B), consistent with our previous study (2).
Circulating SARS-CoV-2 memory CD4+ T cell responses were quite robust (Fig. 4B). Approximately one
third (35%, 17/49) of COVID-19 cases at 1 month PSO had > 1.0% SARS-CoV-2-specific CD4+ T cells.
SARS-CoV-2 memory CD4+ T cells declined over the 6 month time frame of this study with an apparent
t1/2 of 96d in the full cohort (Fig. 4B) and t1/2 64d among paired samples (Fig. 4C). The percentage of
subjects with detectable circulating SARS-CoV-2 memory CD4+ T cells at 1 month PSO (d20-50) was 94%
(46/49, Fig. 4B). The proportion of subjects positive for SARS-CoV-2 memory CD4+ T cells at > 6 months
PSO was 89% (16/18). Spike-specific and M-specific memory CD4+ T cells exhibited similar kinetics to
the overall SARS-CoV-2-specific memory CD4+ T cells (whole cohort t1/2 150d and 174d, respectively.
Fig. 4D-E, and Fig. S4D). A plurality of the SARS-CoV-2 memory CD4+ T cells present at > 6 months PSO
were TCM (Fig. 4F).
T follicular helpers (TFH) are the specialized subset of CD4+ T cells required for B cell help (56),
and, therefore, critical for the generation of neutralizing antibodies and long-lived humoral immunity in
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most contexts. Thus, we examined circulating TFH (cTFH) memory CD4+ T cells, with particular interest in
spike-specific memory cTFH cells due to the importance of antibody responses against spike. Memory
cTFH cells specific for predicted epitopes across the remainder of the SARS-CoV-2 genome were also
measured, using the MP_R megapool (2). Memory cTFH cells specific for SARS-CoV-2 spike and MP_R
were detected in the majority of COVID-19 cases at early time points (16/17 & 17/17. Fig. 4G-H, and
Fig. S5A-C). cTFH memory appeared to be stable, with 100% of subjects positive for spike cTFH and 92%
positive for MP_R cTFH memory at 6 months PSO (Fig. 4G-H).
Recently activated cTFH cells are PD-1hi (56). Consistent with conversion to resting memory cTFH
cells, the percentage of PD-1hi SARS-CoV-2-specific memory cTFH dropped over time (Fig. 4I). CCR6+
SARS-CoV-2-specific cTFH cells have been associated with reduced COVID-19 disease severity (5) and
have been reported to be a major fraction of spike-specific cTFH cells (5, 57). Here we confirmed that a
significant fraction of both spike-specific and MP_R cTFH were CCR6+. We also observed significant
increases in the fraction of CCR6+ cTFH memory over time (P < 0.001 and P < 0.01 compared to bulk cTFH
at > 6 months PSO. Fig. 4J). Overall, substantial cTFH memory was observed after SARS-CoV-2 infection,
with durability > 6 months PSO.

Immune memory relationships

Additional features of immune memory to SARS-CoV-2 were considered, including relationships
between the compartments of immune memory. Immune memory was examined for associations
between magnitude of memory and disease severity. Circulating antibody titers of severe COVID-19
cases trended higher, consistent with other studies (Fig. S6A). No distinction was observed in B and T
cell memory between hospitalized and non-hospitalized COVID-19 cases (Fig. S6B-F), though
interpretations are limited by the relatively low number of severe cases in this cohort. The influence of
gender on immune memory was also assessed. Overall, males had higher spike IgG (ANCOVA
p=0.00019, Fig. 5A) and nucleocapsid and RBD IgG (Fig. S7A-D). Higher spike IgG in males was also
observed in another convalescent cohort (48). In contrast, no differences were observed in SARS-CoV-2
memory B cell frequencies or T cells between males and females (Fig. S7E-I). In sum, the heterogeneity
in immune memory to SARS-CoV-2 was not primarily attributable to gender or COVID-19 disease
severity.
Very few published data sets compare antigen-specific antibody, B cell, CD8+ T cell, and CD4+
T cell memory to an acute viral infection in the same individuals. To our knowledge, this is the largest
study of its kind, for any acute infection. We examined relationships between immune memory
compartments to gain insights into the interrelationships between immune memory types and better
interpret the totality of immune memory to SARS-CoV-2. We focused on RBD IgG, RBD IgA, RBD memory
B cells, total SARS-CoV-2-specific CD8+ T cells, and total SARS-CoV-2-specific CD4+ T cells, due to their
putative potential roles in protective immunity. The majority (59%) of COVID-19 cases were positive for
all five of these immune memory compartments at 1-2 months PSO (Fig. 5B), with the incomplete
responses largely reflecting individuals with no detectable CD8+ T cell memory and/or poor RBD IgA
responses (Fig. 5C). By 5+ months after COVID-19, the proportion of individuals positive for all five of
these immune memory compartments had dropped to 40%; nevertheless, 96% of individuals were still
positive for at least three out of five SARS-CoV-2 immune memory responses (Fig. 5B). Immune memory
at 5+ months PSO represented different contributions by immune memory compartments in different
individuals (Fig. 5C), again demonstrating heterogeneity of immune memory, with increasing
heterogeneity in the population over time.
Interrelationships between the components of memory were examined by assessing ratios over
time. The ratio of SARS-CoV-2 CD4+ and CD8+ T cell memory was largely stable over time (Fig. 5D, Fig.
S8A). Given that serological measurements are the simplest measurements of immune memory at a
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population scale, we examined how well such measurements may serve as surrogate markers of other
components of SARS-CoV-2 immune memory over time. The relationship between circulating RBD IgG
and RBD-specific memory B cells changed ~20-fold over the time range studied (R=0.60, Fig. 5D, Fig.
S8B). The changing relationship between circulating RBD IgA and RBD-specific memory B cells was even
larger, with a 40-fold shift (R=0.62, Fig. 5D, Fig. S8C). The relationship between RBD IgG and SARS-
CoV-2 CD4+ T cell memory was relatively flat over the time range studied (Fig. 5D); however, variation
spanned a ~1000-fold range and thus predictive power of circulating RBD IgG for assessing T cell
memory was poor due to heterogeneity between individuals (R=0.02, Fig. S8D-E). In aggregate, while
heterogeneity of immune responses is a defining feature of COVID-19, immune memory to SARS-CoV-
2 develops in almost all subjects, with complex relationships between the individual immune memory
compartments.

Conclusion
In this study, we aimed to fill a gap in our basic understanding of immune memory after COVID-19. This
required simultaneous measurement of circulating antibodies, memory B cells, CD8+ T cells, and CD4+
T cells specific for SARS-CoV-2, in a group of subjects with a full range of disease and distributed from
short time points PSO out to > 8 months PSO. To our knowledge, this is the first study of its kind,
incorporating antigen-specific antibody, memory B cell, CD8+ T cell, and CD4+ T cell measurements, out
past 6 months post-infection. By studying these multiple compartments of adaptive immunity in an
integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited
distinct kinetics.
The spike IgG titers were durable, with modest declines in titers at 6 to 8 months PSO at the
population level. RBD IgG and SARS-CoV-2 PSV neutralizing antibody titers were potentially similarly
stable, consistent with the RBD domain of spike being the dominant neutralizing antibody target.
However, due to the nature of only having data at two time points, the paired sample longitudinal data
set could not distinguish between models of a continuous log-linear decay and a bi-phasic decay with a
slower half-life later. It is well recognized that the magnitude of the antibody response against SARS-
CoV-2 is highly heterogenous between individuals. We observed that heterogenous initial antibody
responses did not collapse into a homogeneous circulating antibody memory. That heterogeneity is
thus a central feature of immune memory to this virus. For antibodies, the responses spanned a ~200-
fold range. Additionally, the heterogeneity showed that long-term longitudinal studies will be required
to precisely define antibody kinetics to SARS-CoV-2. Nevertheless, at 5+ months PSO, almost all
individuals were positive for SARS-CoV-2 spike and RBD IgG.
Notably, memory B cells specific for spike or RBD were detected in almost all COVID-19 cases,
with no apparent half-life at 5+ months post-infection. B cell memory to some other infections has been
observed to be long-lived, including 60+ years after smallpox vaccination (58), or 90+ years after
infection with influenza (59), another respiratory virus like SARS-CoV-2. The memory T cell half-lives
observed over 6+ months PSO in this cohort (~166-271d for CD8+ and ~96-174d for CD4+ T cells) were
comparable to the 123d t1/2 observed for memory CD8+ T cells soon after yellow fever immunization
(55). Notably, the durability of a fraction of the yellow fever virus-specific memory CD8+ T cells possessed
an estimated t1/2 of 485d by deuterium labeling (55). Using different approaches, the long-term
durability of memory CD4+ T cells to smallpox, over a period of many years, was an estimated t1/2 of ~10
years (58, 60), which is also consistent with recent detection of SARS-CoV T cells 17 years after the initial
infection (61). These data suggest that T cell memory might reach a more stable plateau, or slower decay
phase, later than the first 6 months post-infection.
While immune memory is the source of long-term protective immunity, direct conclusions about
protective immunity cannot be made on the basis of quantifying SARS-CoV-2 circulating antibodies,
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memory B cells, CD8+ T cells, and CD4+ T cells, because mechanisms of protective immunity against
SARS-CoV-2 or COVID-19 are not defined in humans. Nevertheless, some reasonable interpretations
can be made. Antibodies are the only component of immune memory that can provide truly sterilizing
immunity. Immunization studies in non-human primates have indicated that circulating neutralization
titers of ~200 may provide sterilizing immunity against a relatively high dose URT challenge (62), and
neutralizing titers of ~3,400 may provide sterilizing immunity against a very high dose URT challenge
(63), although direct comparisons are not possible because the neutralizing antibody assays have not
been standardized (3). Conclusions are also constrained by the limited overall amount of data on the
topic of protective immunity to SARS-CoV-2, though progress in this field has been exceptionally rapid
by any standard.
Beyond sterilizing immunity, confining SARS-CoV-2 to the URT and oral cavity would minimize
COVID-19 disease severity to ‘common cold’ or asymptomatic disease. This outcome is the primary goal
of current COVID-19 vaccine clinical trials (3, 64). Such an outcome could potentially be mediated by a
mixture of memory CD4+ T cells, memory CD8+ T cells, and memory B cells specific for RBD producing
anamnestic neutralizing antibodies, based on mechanisms of action demonstrated in mouse models of
other viral infections (65–67). In human COVID-19 infections, SARS-CoV-2-specific CD4+ T cells and CD8+
T cells are associated with lessened COVID-19 disease severity of an ongoing SARS-CoV-2 infection (5),
and rapid seroconversion was associated with significantly reduced viral loads in acute disease over 14
days (30). Both of those associations are consistent with the hypothesis that SARS-CoV-2 memory T cells
and B cells would be capable of substantially limiting SARS-CoV-2 dissemination and/or cumulative viral
load, resulting in substantially reduced COVID-19 disease severity
severity. The likelihood of such outcomes is
also closely tied to the kinetics of the infection, as memory B and T cell responses can take 3-5 days to
successfully respond to an infection. As noted above, given the relatively slow course of severe COVID-
19 in humans, a large window of time is available for resting immune memory compartments to
potentially contribute in meaningful ways to protective immunity against pneumonia or severe or fatal
2° COVID-19. The presence of sub-sterilizing neutralizing antibody titers at the time of SARS-CoV-2
exposure would blunt the size of the initial infection, and may provide an added contribution to limiting
COVID-19 severity, based on observations of protective immunity for other human respiratory viral
infections (38, 68–70) and observations of SARS-CoV-2 vaccines in non-human primates (49, 63, 71).
This study has limitations. Longitudinal data for each subject, with at least 3 time points per
subject, would be required to distinguish between linear, one-phase with plateau, and two-phase decay
best fit models for more precise understanding of long-term kinetics of SARS-CoV-2 antibodies.
Nevertheless, the current cross-sectional data describe well the dynamics of SARS-CoV-2 memory B
cells, CD8+ T cell, and CD4+ T cell over 6 months PSO. Additionally, circulating memory was assessed
here; it is possible that local URT immune memory is a minimal, moderate, or large component of
immune memory after a primary infection with SARS-CoV-2. This remains to be determined.
When considering potential connections between immune memory and protective immunity, it
is key to consider the available epidemiological data. Individual case reports demonstrate that
reinfections with SARS-CoV-2 are occurring (72, 73). What is currently lacking is an epidemiological
framework for quantifying how rare or common such reinfection events are. Thus, interpretations of
current events are very constrained. There is a high degree of heterogeneity in the magnitude of
adaptive immune responses to this novel coronavirus. That heterogeneity was observed in this study to
be carried on into the immune memory phase to SARS-CoV-2. As a result of the immune response
heterogeneity, as observed in the cohort here, it may be expected that at least a fraction of the SARS-
CoV-2-infected population with particularly low immune memory would be susceptible to re-infection
relatively quickly. The source of heterogeneity in immune memory to SARS-CoV-2 is unknown and worth
further examination. It is possible that some of that heterogeneity is a result of low cumulative viral load
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or initial inoculum, essentially resulting in a very minor or transient infection that barely triggered an
adaptive immune response in some individuals. Nevertheless, immune memory consisting of at least
three immunological compartments was measurable in ~90% 90% of subjects > 5 months PSO, indicating
that durable immunity against 2o COVID-19 disease is a possibility in most individuals.
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METHODS

Human Subjects
The Institutional Review Boards of the University of California, San Diego (UCSD; 200236X) and the La
Jolla Institute for Immunology (LJI; VD-214) approved the protocols used for blood collection for
subjects with COVID-19 who donated at all sites other than Mt. Sinai. The Icahn School of Medicine at
Mt. Sinai IRB approved the samples collected at this institution in New York City (IRB-16-00791). All
human subjects were assessed for medical decision-making capacity using a standardized, approved
assessment, and voluntarily gave informed consent prior to being enrolled in the study. Study inclusion
criteria included a diagnosis of COVID-19 or suspected COVID-19, age of 18 years or greater,
willingness and ability to provide informed consent. Although not a strict inclusion criterion, evidence of
positive PCR-based testing for SARS-CoV-2 was requested from subjects prior to participation. 143 cases
were confirmed SARS-CoV-2 positive by PCR-based testing (Table S1). Two subjects tested negative by
SARS-CoV-2 PCR (Table S1). The remainder were not tested or did not have test results available for
review (Table S1). Subjects who had a medical history and/or symptoms consistent with COVID-19, but
lacked positive PCR-based testing for SARS-CoV-2 and subsequently had negative laboratory-based
serologic testing for SARS-CoV-2 were then excluded; i.e., all COVID-19 cases in this study were
confirmed cases by SARS-CoV-2 PCR or SARS-CoV-2 serodiagnostics, or both. Adults of all races,
ethnicities, ages, and genders were eligible to participate. Study exclusion criteria included lack of
willingness to participate, lack of ability to provide informed consent, or a medical contraindication to
blood donation (e.g. severe anemia). Subject samples at LJI were obtained from individuals in California
and at least seven other states.
Blood collection and processing methods at LJI were performed as previously described (5).
Briefly, whole blood was collected via phlebotomy in acid citrate dextrose (ACD) serum separator tubes
(SST), or ethylenediaminetetraacetic acid (EDTA) tubes and processed for peripheral blood
mononuclear cells (PBMC), serum, and plasma isolation. Most donors were screened for symptoms prior
to scheduling blood draws, and had to be symptom-free and approximately 3-4 weeks out from
symptom onset at the time of the initial blood draw at UCSD or LJI, respectively. Samples were coded,
and then de-identified prior to analysis. Other efforts to maintain the confidentiality of participants
included the labeling samples with coded identification numbers. An overview of the characteristics of
subjects with COVID-19 is provided in Table S1.
COVID-19 disease severity was scored from 0 to 10 using a numerical scoring system based on
the NIH ordinal scale (5, 74). A categorical descriptor was applied based on this scoring system:
“asymptomatic” for a score of 1, “mild” for a score of 2-3, “moderate” for a score of 4-5, and “severe” for
a score of 6 or more. Subjects with a numerical score of 4 or higher required hospitalization (including
admission for observation) for management of COVID-19. The days PSO was determined based on the
difference between the date of the blood collection and the date of first reported symptoms consistent
with COVID-19. For asymptomatic subjects, the day from first positive SARS-CoV-2 PCR-based testing
was used in place of the date of first reported COVID-19 symptoms.

SARS-CoV-2 ELISAs
SARS-CoV-2 ELISAs were performed as previously described (2, 5, 75). Briefly, Corning 96-well half area
plates (ThermoFisher 3690) were coated with 1μg/mL of antigen overnight at 4°C. Antigens included
recombinant SARS-CoV-2 RBD protein, recombinant spike protein (5), and recombinant nucleocapsid
protein (GenScript Z03488). The following day, plates were blocked with 3% milk in phosphate buffered
saline (PBS) containing 0.05% Tween-20 for 1.5 hours at room temperature. Plasma was heat inactivated
at 56°C for 30-60 minutes. Plasma was diluted in 1% milk containing 0.05% Tween-20 in PBS starting at
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a 1:3 dilution followed by serial dilutions by 3 and incubated for 1.5 hours at room temperature. Plates
were washed 5 times with 0.05% PBS-Tween-20. Secondary antibodies were diluted in 1% milk
containing 0.05% Tween-20 in PBS. For IgG, anti-human IgG peroxidase antibody produced in goat
(Sigma A6029) was used at a 1:5,000 dilution. For IgA, anti-human IgA horseradish peroxidase antibody
(Hybridoma Reagent Laboratory HP6123-HRP) was used at a 1:1,000 dilution. The HP6123 monoclonal
anti-IgA was used because of its CDC and WHO validated specificity for human IgA1 and IgA2 and lack
of crossreactivity with non-IgA isotypes (76).
Endpoint titers were plotted for each sample, using background subtracted data. A positive
control standard was created by pooling plasma from 6 convalescent COVID-19 donors to normalize
between experiments. The limit of detection (LOD) was defined as 1:3 for IgG, 1:10 for IgA. Limit of
sensitivity (LOS) for SARS-CoV-2 infected individuals was established based on uninfected subjects,
using plasma from normal healthy donors never exposed to SARS-CoV-2. For cross-sectional analyses,
modeling for the best fit curve (e.g., one phase decay versus simple linear regression) was performed
using GraphPad Prism 8.0. Best curve fit was defined by an extra sum-of-squares F Test, selecting the
simpler model unless P < 0.05 (77). To calculate the t1/2, log2 transformed data was utilized. Using the
best fit curve, either a one phase decay non-linear fit or a simple linear regression (-1/slope) was utilized.
Pearson R was calculated for correlation. For longitudinal samples, a simple linear regression was
performed, with t1/2 calculated from log2 transformed data for each pair. For gender analyses, modeling
and t1/2 was performed similar to cross-sectional analyses; ANCOVA (VassarStats or GraphPad Prism 8.4)
was then performed between male and female data sets.

Neutralizing antibody assays

The pseudovirus neutralizing antibody assay was performed as previously described (5). Briefly, Vero
cells were seeded in 96 well plates to produce a monolayer at the time of infection. Pre-titrated amounts
of rVSV-SARS-Cov-2 (phCMV3-SARS-CoV-2 spike SARS-CoV-2-pseduotyped VSV-ΔG-GFP were
generated by transfecting 293T cells) were incubated with serially diluted human plasma at 37°C for 1
hour before addition to confluent Vero monolayers in 96-well plates. Cells were incubated for 12-16
hours at 37°C in 5% CO2. Cells were then fixed in 4% paraformaldehyde, stained with 1ug/mL Hoechst,
and imaged using a CellInsight CX5 imager to quantify total number of cells expressing GFP. Infection
was normalized to the average number of cells infected with rVSV-SARS-CoV-2 incubated with normal
human plasma. The limit of detection (LOD) was established as < 1:20 based on plasma samples from a
series of unexposed control subjects. Data are presented as the relative infection for each concentration
of sera. Neutralization IC50 titers were calculated using One-Site Fit LogIC50 regression in GraphPad
Prism 8.0.

Detection of antigen-specific memory B cells

To detect SARS-CoV-2 specific B cells, biotinylated protein antigens were individually multimerized with
fluorescently labeled streptavidin at 4°C for one hour. Full-length SARS-CoV-2 spike (2P-stabilized,
double Strep-tagged) and RBD were generated in-house. Biotinylation was performed using biotin-
protein ligase standard reaction kit (Avidity, Cat# Bir500A) following the manufacturers standard
protocol and dialyzed over-night against PBS. Biotinylated spike was mixed with streptavidin BV421
(BioLegend, Cat# 405225) and streptavidin Alexa Fluor 647 (Thermo Fisher Scientific, Cat# S21374) at
20:1 ratio (~6:1 molar ratio). Biotinylated RBD was mixed with streptavidin PECy7 (BioLegend, Cat#
405206) at 2.2:1 ratio (~4:1 molar ratio). Biotinylated SARS-CoV-2 full length nucleocapsid (Avi- and His-
tagged; Sino Biological, Cat# 40588-V27B-B) was multimerized using streptavidin PE (BioLegend, Cat#
405204) and streptavidin BV711 (BioLegend, Cat# 405241) at 5.5:1 ratio (~6:1 molar ratio). Streptavidin
PECy5.5 (Thermo Fisher Scientific, Cat# SA1018) was used as a decoy probe to gate out SARS-CoV-2
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non-specific streptavidin-binding B cells. The antigen probes prepared individually as above were then
mixed in Brilliant Buffer (BD Bioscience, Cat# 566349) containing 5μM free d-biotin (Avidity, Cat#
Bir500A). Free d-biotin ensured minimal cross-reactivity of antigen probes. ~107 previously frozen PBMC
samples were prepared in U-bottom 96-well plates and stained with 50μL antigen probe cocktail
containing 100ng spike per probe (total 200ng), 27.5ng RBD, 40ng nucleocapsid per probe (total 80ng)
and 20ng streptavidin PECy5.5 at 4°C for one hour to ensure maximal staining quality before surface
staining with antibodies as listed in Table S2 was performed in Brilliant Buffer at 4°C for 30min. Dead
cells were stained using LIVE/DEAD Fixable Blue Stain Kit (Thermo Fisher Scientific, Cat# L34962) in
DPBS at 4°C for 30min. ~80% of antigen-specific memory (IgD– and/or CD27+) B cells detected using
this method were IgM+, IgG+, or IgM– IgG– IgA+, which were comparable to non-specific memory B cells.
Based on these observations, we concluded that the antigen probes did not significantly impact the
quality of surface immunoglobulin staining. Stained PBMC samples were acquired on Cytek Aurora and
analyzed using FlowJo10.7.1 (BD Bioscience). Frequency of antigen-specific memory B cells were
expressed as a percentage of total B cells (CD19+ CD20+ CD38int/–, CD3–, CD14–, CD16–, CD56–,
LIVE/DEAD–, lymphocytes), or as numbers per 106 PBMC (LIVE/DEAD– cells). LOD was set based on
median + 2×SD of [1 / (number of total B cells recorded)] or median + 2×SD of [106 / (number of PBMC
recorded)]. LOS was set as the median + 2×SD of the results in unexposed donors. Phenotype analysis
of antigen-specific B cells was performed only in subjects with at least 10 cells detected in the respective
antigen-specific memory B cell gate. In each experiment, PBMC from a known positive control (COVID-
19 convalescent subject) and unexposed subjects were included to ensure consistent sensitivity and
specificity of the assay. For each data set, second order polynomial, simple linear regression, and
pseudo-first order kinetic models were considered. The model with a lower Akaike’s Information
Criterion value was determined to be better-fit and visualized.

Activation induced markers (AIM) T cell assay

Antigen-specific CD4+ T cells were measured as a percentage of AIM+ (OX40+CD137+) CD4+ T and
(CD69+CD137+) CD8+ T cells after stimulation of PBMCs with overlapping peptide pools spanning the
entire ORFeome, as previously described (2). Cells were cultured for 24 hours in the presence of SARS-
CoV-2 specific MPs [1 μg/mL] or 5 μg/mL phytohemagglutinin (PHA, Roche) in 96-wells U-bottom plates
at 1x106 PBMCs per well. A stimulation with an equimolar amount of DMSO was performed as negative
control, PHA, and stimulation with a combined CD4 and CD8 cytomegalovirus MP (CMV, 1 μg/mL) were
included as positive controls. Any sample with low PHA signal was excluded as a quality control. Antigen-
specific CD4+ and CD8+ T cells were measured as background (DMSO) subtracted data, with a minimal
DMSO level set to 0.005%. All positive ORFs (> 0.02% for CD4s, > 0.05% for CD8s) were then
aggregated into a combined sum of SARS-CoV-2-specific CD4+ or CD8+ T cells. The threshold for
positivity for antigen-specific CD4+ T cell responses (0.03%) and antigen-specific CD8+ T cell responses
(0.12%) has been calculated using the median two-fold standard deviation of all negative controls
measured (>150). The antibody panel utilized in the (OX40+CD137+) CD4+ T and (CD69+CD137+) CD8+
T cells AIM staining is shown in Table S2.
For surface CD40L+ OX40+ CD4+ T cell AIM assays, experiments were performed as previously
described (5), with the following modifications. Cells were cultured in complete RPMI containing 5%
Human AB Serum (Gemini Bioproducts), 2Me, PenStrep, NaPy, and NE-AA. Prior to addition of peptide
MPs, cells were blocked at 37C for 15 minutes with 0.5ug/mL anti-CD40 mAb (Miltenyi Biotec).

ACKNOWLEDGEMENTS
We would like to thank the LJI Clinical Core, specifically Gina Levi, RN and Brittany Schwan for healthy
donor enrollment and blood sample procurement. We are also grateful to the Mt. Sinai Personalized
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Virology Initiative for sharing banked samples from study participants with COVID-19. We are grateful
to Dr. A. Wajnberg for study participant referrals and to the Personalized Virology Initiative (Dr. G.
Kleiner, Dr. LCF Mulder, Dr. M. Saksena, K. Srivastava, C. Gleason, C. M. Bermúdez-González, K. Beach,
K. Russo, L. Sominsky, E. Ferreri, R. Chernet, L. Eaker, A. Salimbangon, D. Jurczyszak, H. Alshammary, W.
Mendez, A. Amoako, S. Fabre, S. Suthakaran, M. Awawda, E. Hirsch, A. Shin) for sharing banked samples
from study participants with COVID-19. This work was funded by the NIH NIAID under awards AI142742
(Cooperative Centers for Human Immunology) (A.S., S.C.), NIH contract Nr. 75N9301900065 (D.W.,
A.S.), U01 AI141995-03 (A.S., P.B.), and U01 CA260541-01 (D.W). This work was additionally supported
in part by the John and Mary Tu Foundation (D.S.), the NIAID under K08 award AI135078 (J.D.), UCSD
T32s AI007036 and AI007384 Infectious Diseases Division (S.Ram., S.Raw.), and the Bill and Melinda
Gates Foundation INV-006133 from the Therapeutics Accelerator, Mastercard, Wellcome, private
philanthropic contributions (K.H., E.O.S., S.C.), and a FastGrant from Emergent Ventures in aid of COVID-
19 research. This work was partially supported by the NIAID Centers of Excellence for Influenza Research
and Surveillance (CEIRS) contract HHSN272201400008C (F.K., for reagent generation), the
Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051 and the
generous support of the JPB foundation (F.K., V.S.), the Cohen Foundation (VS, FK), the Open
Philanthropy Project (#2020-215611; F.K., V.S.), as well as other philanthropic donations. We would also
like to thank all of the COVID-19 and healthy human subjects who made this research possible through
their generous blood donations.

COMPETING INTERESTS
A.S. is a consultant for Gritstone, Flow Pharma, Merck, Epitogenesis, Gilead and Avalia. S.C. is a
consultant for Avalia. LJI has filed for patent protection for various aspects of T cell epitope and
vaccine design work. Mount Sinai has licensed serological assays to commercial entities and has filed
for patent protection for serological assays. D.S., F.A., V.S. and F.K. are listed as inventors on the
pending patent application (F.K., V.S.), and Newcastle disease virus (NDV)-based SARS-CoV-2 vaccines
that name F.K. as inventor. All other authors declare no conflict of interest.
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FIGURE LEGENDS

Figure 1. SARS-CoV-2 circulating antibodies over time. (A) Cross-sectional spike IgG from COVID-
19 subject plasma samples (n=228). Linear decay preferred model for best fit curve, t1/2 = 140d, 95% CI:
89-329 days. R = -0.20, p=0.003. (B) Longitudinal spike IgG (n=50), average t1/2 100d, 95% CI: 64-220d
(C) Cross-sectional RBD IgG. Linear decay preferred model for best fit curve, t1/2 = 83d, 95% CI: 62 to
127d. R = -0.34, p<0.0001. (D) Longitudinal RBD IgG, average t1/2 of 68d, 95% CI: 57-86d (E) Cross-
sectional PSV neutralizing titers. One-phase decay (blue line) preferred model for best fit curve, t1/2 =
27d, 95%: CI: 11 to 153d. R = -0.27, p <0.0001. Linear fit shown as black line. (F) Longitudinal PSV
neutralizing titers of SARS-CoV-2 infected subjects, average t1/2 87d, 95% CI: 68-123d (G) Cross-
sectional nucleocapsid IgG. Linear decay preferred model for best fit curve, t1/2 = 67d, 95% CI: 49-105d.
R = -0.32, p<0.0001. (H) Longitudinal nucleocapsid IgG, average t1/2 was 67d, 95% CI: 54-88d. (I) Cross-
sectional Spike IgA titers. One-phase decay (blue line) preferred model for best fit curve, t1/2 = 11d, 95%:
CI: 5 to 25d. R = -0.14, p=0.04. Linear fit shown as black line. (J) Longitudinal Spike IgA, t1/2 = 214d, 95%
CI 126-703d. (K) Cross-sectional RBD IgA. One phase decay (blue line) preferred model for best fit
curve, t1/2 = 27d, 95% CI: 15 to 58d. R = -0.40, p<0.0001. Linear fit shown in black. (L) Longitudinal RBD
IgA, average t1/2 was 72d, 95% CI: 55-104d. For cross-sectional analyses, SARS-CoV-2 infected subjects
(white circles, n=238) and unexposed subjects (gray circles, n=51). For longitudinal samples, SARS-CoV-
2 subjects (n=50). The dotted black line indicates limit of detection (LOD). The dotted green line
indicates limit of sensitivity (LOS) above uninfected controls. Unexposed = gray, COVID subjects = white.
Thick blue line represents best fit curve. When two fit curves are shown, the thin black line represents
the alternative fit curve.

Figure 2. Kinetics of SARS-CoV-2 memory B cell responses. (A) Example plots showing staining
patterns of SARS-CoV-2 antigen probes on memory B cells (See Fig S1 for gating). One unexposed
donor and three convalescent COVID-19 subjects are shown. Numbers indicate percentages. (B) Gating
strategies to define IgM+, IgG+, or IgA+ SARS-CoV-2 spike-specific memory B cells. The same gating
strategies were used for RBD- or nucleocapsid-specific B cells. (C) Cross-sectional analysis of frequency
(% of CD19+ CD20+ B cells) of SARS-CoV-2 S-specific total (IgG+, IgM+, or IgA+) memory B cells. Pseudo-
first order kinetic model for best fit curve (R2 = 0.14). (D) Longitudinal analysis of SARS-CoV-2 spike-
specific memory B cells. (E) Cross-sectional analysis of SARS-CoV-2 RBD-specific total (IgG+, IgM+, or
IgA+) memory B cells. Second order polynomial model for best fit curve (R2 = 0.21). (F) Longitudinal
analysis of SARS-CoV-2 RBD-specific memory B cells. (G) Cross-sectional analysis of SARS-CoV-2
nucleocapsid-specific total (IgG+, IgM+, or IgA+) memory B cells. Pseudo-first order kinetic model for
best fit curve (R2 = 0.19). (H) Longitudinal analysis of IgG+ SARS-CoV-2 spike-specific memory B cells. (I)
Cross-sectional analysis of SARS-CoV-2 spike-specific IgG+ memory B cells. Pseudo-first order kinetic
model for best fit curve (R2 = 0.24). (J) Longitudinal analysis of SARS-CoV-2 spike-specific IgG+ memory
B cells. (K) Cross-sectional analysis of SARS-CoV-2 spike-specific IgA+ memory B cells. Second order
polynomial model for best fit curve (R2 = 0.10). (L) Longitudinal analysis of SARS-CoV-2 spike-specific
IgA+ memory B cells. (M) Cross-sectional analysis of SARS-CoV-2 spike-specific IgM+ memory B cells.
Second order polynomial model for best fit curve (R2 = 0.17). (N) Longitudinal analysis of SARS-CoV-2
spike-specific IgM+ memory B cells. (O) Fraction of SARS-CoV-2 antigen-specific memory B cells that
belong to indicated Ig isotypes at 1-8 months PSO. (P) Cross-sectional analysis of SARS-CoV-2 RBD-
specific IgG+ memory B cells. Second order polynomial model for best fit curve (R2 = 0.27). (Q) Cross-
sectional analysis of SARS-CoV-2 nucleocapsid-specific IgG+ memory B cells. Second order polynomial
model for best fit curve (R2 = 0.26). n = 20 unexposed subjects (gray circles) and n = 180 COVID-19
subjects (n = 217 data points, white circles) for cross-sectional analysis. n = 36 COVID-19 subjects (n =
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73 data points, white circles) for longitudinal analysis. The dotted black line indicates limit of detection
(LOD). The dotted green line indicates limit of sensitivity (LOS).

Figure 3. SARS-CoV-2 memory CD8+ T cells. (A) Representative flow cytometry plots of SARS-CoV-2-
specific CD8+ T cells (CD69+ CD137+, See Fig S3 for gating) after overnight stimulation with S, N, M,
ORF3a, or nsp3 peptide pools, compared to negative control (DMSO). (B) Cross-sectional analysis of
frequency (% of CD8+ T cells) of total SARS-CoV-2-specific CD8+ T cells. (C) Longitudinal analysis of total
SARS-CoV-2-specific CD8+ T cells in paired samples from the same subjects. (D) Cross-sectional analysis
of spike-specific CD8+ T cells. (E) Longitudinal analysis of spike-specific CD8+ T cells in paired samples
from the same subjects. (F) Distribution of TCM, TEM, and TEMRA among total SARS-CoV-2-specific CD8+ T
cells. n = 155 COVID-19 subject samples (white circles) for cross-sectional analysis. n = 30 COVID-19
subjects (white circles) for longitudinal analysis. The dotted black line indicates limit of detection (LOD).

Figure 4. SARS-CoV-2 memory CD4+ T cells. (A) Representative flow cytometry plots of SARS-CoV-2-
specific CD4+ T cells (CD137+ OX40+, See Fig S4 for gating) after overnight stimulation with S, N, M,
ORF3a, or nsp3 peptide pools, compared to negative control (DMSO). (B) Cross-sectional analysis of
frequency (% of CD4+ T cells) of total SARS-CoV-2-specific CD4+ T cells. (C) Longitudinal analysis of total
SARS-CoV-2-specific CD4+ T cells in paired samples from the same subjects. (D) Cross-sectional analysis
of spike-specific CD4+ T cells. (E) Longitudinal analysis of spike-specific CD4+ T cells in paired samples
from the same subjects. (F) Distribution of TCM, TEM, and TEMRA among total SARS-CoV-2-specific CD4+ T
cells. (G, H) Quantitation of SARS-CoV-2-specific TFH cells (surface CD40L+ OX40+, as % of CD4+ T cells.
See Fig S5 for gating) after overnight stimulation with (G) spike (S) or (H) MP_R peptide pools. (I) PD-1hi
SARS-CoV-2-specific TFH at 1-2 months (mo) and 6 mo PSO. (J) CCR6+ SARS-CoV-2-specific TFH in
comparison to bulk cTFH cells in blood.
For A-F, n = 155 COVID-19 subject samples (white circles) for cross-sectional analysis. n = 30 COVID-19
subjects (white circles) for longitudinal analysis. The dotted black line indicates limit of detection (LOD).
For G-J, n = 34 COVID-19 subject samples (white circles), n = 21 COVID-19 subjects at 1-2 mo, n = 13
COVID-19 subjects at 6 mo. The dotted black line indicates limit of detection (LOD).* p<0.05, **p<0.01,
*** p<0.001, **** p<0.0001.

Figure 5. Immune memory relationships. (A) Relationship between gender and spike IgG titers over
time. Males: One phase decay preferred model, t1/2 = 23d, 95% CI: 7-224d, R = -0.26, p=0.0057.
Females: linear decay preferred model, t1/2 = 159d, 95% CI 88-847d, R = -0.18, p=0.05. (B) Immune
memory to SARS-CoV-2 during the early phase (1-2 mo, black line), medium phase (3-4 mo, red line), or
late phase (5+ mo, blue line). For each individual, a score of 1 was assigned for each response above
LOS in terms of RBD-specific IgG, RBD-specific IgA, RBD-specific memory B cells, SARS-CoV-2 specific
CD4+ T cells, and SARS-CoV-2-specific CD8+ T cells, giving a maximum total of 5 components of SARS-
CoV-2 immune memory. Only COVID-19 convalescent subjects with all five immunological parameters
tested were included in the analysis. n = 83 (1-2 mo), n = 53 (3-4 mo), n = 28 (5+ mo). (C) Percentage
dot plots showing frequencies (normalized to 100%) of subjects with indicated immune memory
components as described in (B) during the early (1-2 mo) or late (5+ mo) phase. “G”, RBD-specific IgG.
“B”, RBD-specific memory B cells. “4”, SARS-CoV-2 specific CD4+ T cells. “8”, SARS-CoV-2 specific CD8+
T cells. “A”, RBD-specific IgA. (D) Relationships between immune memory compartments in COVID-19
subjects over time, as ratios (full curves and data shown in Fig. S8). AU = arbitrary units, scaled from Fig.
S8. “B:IgA”, RBD-specific memory B cell ratio to RBD IgA antibodies. “B:IgG”, RBD-specific memory B
cell ratio to RBD IgG antibodies. “B:CD4”, RBD-specific memory B cell ratio to SARS-CoV-2-specific CD4+
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T cells. “CD4:CD8”, SARS-CoV-2-specific CD4+ T cells ratio to SARS-CoV-2-specific CD8+ T cells.

“CD4:IgG”, SARS-CoV-2-specific CD4+ T cells ratio to RBD IgG antibodies.

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SUPPLEMENTARY MATERIALS

Table S1. Participant characteristics

COVID-19 (n = 185)

Age (years) 19-81 [Median = 40, IQR = 19.5]

Gender
Male (%) 43% (79/185)
Female (%) 57% (106/185)
Race
African American 3% (5/185)
or Black (%)
Alaskan Native or 1% (1/185)
American Indian
(%)
Asian (%) 8% (14/185)
Native Hawaiian or 0% (0/185)
Pacific Islander (%)
Multiracial (%) 1% (2/185)
Other (%) 1% (1/185)
Unknown (%) 10% (19/185)
White (%) 77% (143/185)
Ethnicity
Hispanic or Latino (%) 15% (27/185)
Non-Hispanic (%) 80% (148/185)
Unknown (%) 5% (10/185)
Hospitalization status
Never hospitalized (%) 92% (171/185)
Hospitalized (%) 7% (13/185)
Unknown if hospitalized 1% (1/185)
(%)
Sample Collection Dates March-October 2020

SARS-CoV-2 PCR Positivity

Positive 77% (143/185)
Negative 1% (2/185)
Not performed 20% (37/185)
Unknown 2% (3/185)
Peak Disease Severity
Asymptomatic (score 1) 2% (4/185)
Mild (non-hospitalized. Score 2-3) 90% (167/185)
Moderate (hospitalized. Score 4-5) 3% (6/185)
Severe (hospitalized. Score 6+) 4% (7/185)
Unknown 1% (1/185)
Days Post Symptom Onset at Collection; n = 233 6-240 (Median 90.5, IQR 99)

Blood Collection Frequency

Multiple Time Point 21% (38/185)
Donors (2-4 times)
Single Time Point Donors 79% (147/185)


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Table S2. Memory B cell flow cytometry panel.


Reagents SOURCE IDENTIFIER
Mouse anti-human CD62L BV615 (clone SK11) BD Bioscience Cat# 565219
Mouse anti-human CD19 BUV563 (clone SJ25C1) BD Bioscience Cat# 612916
Mouse anti-human FCRL5 (CD307e) BUV615 (clone 509F6) BD Bioscience Cat# 751131
Mouse anti-human CD95 BUV737 (clone DX2) BD Bioscience Cat# 612790
Mouse anti-human CCR6 BUV805 (clone 11A9) BD Biosicnece Cat# 749361
Mouse anti-human CD138 BV480 (clone MI15) BD Bioscience Cat# 566140
Mouse anti-human IgD BV510 (clone IA6-2) BioLegend Cat# 348220
Mouse anti-human IgM BV570 (clone MHM-88) BioLegend Cat# 314517
Mouse anti-human CD24 BV605 (clone ML5) BioLegend Cat# 311124
Mouse anti-human CD20 BV650 (clone 2H7) BioLegend Cat# 302336
Rat anti-human CXCR5 BV750 (clone RF8B2) BD Bioscience Cat# 747111
Mouse anti-human CD71 BV786 (clone M-A712) BD Bioscience Cat# 563768
Mouse anti-human CD27 BB515 (clone M-T271) BD Bioscience Cat# 564642
Mouse anti-human IgA Vio Bright FITC (clone IS11-8E10) Miltenyi Biotec Cat# 130-113-480
Mouse anti-human CD3 PerCP (clone SK7) BioLegend Cat# 344814
Mouse anti-human CD14 PerCP (clone 63D3) BioLegend Cat# 367152
Mouse anti-human CD16 PerCP (clone 3G8) BioLegend Cat# 302030
Mouse anti-human CD56 PerCP (clone HCD56) BioLegend Cat# 318342
Rat anti-human IgG PerCP/Cyanine5.5 (clone M1310G05) BioLegend Cat# 410710
Mouse anti-human CD85j PE/Dazzle 594 BioLegend Cat# 333716
Mouse anti-human CD11c PE/Cyanine5 (clone 3.9) BioLegend Cat# 301610
Mouse anti-human CD21 Alexa Fluor 700 (clone Bu32) BioLegend Cat# 354918


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Table S3. Antibodies utilized in the CD8+ and CD4+ T cell activation induced markers (AIM) assays

Membrane Antibody Fluorochrome Clone/vendor/catalog Dilution

CD45RA BV421 HI100/BioLegend/304130 1:50
CD14 BUV563 M5E2/BD/741360 1:100
CD19 BUV805 HIB19/BD/742007 1:100
Live/Dead ef506/Aqua Thermo Fisher/65-0866-18 1:200
CD8 BV650 RPA-T8/BioLegend/301042 1:50
CD4 BV605 RPA-T4/BD/562658 1:25
CCR7 FITC G043H7/BioLegend/353216 1:50
CD69 PE FN50/BD/555531 1:10
OX40 PE-Cy7 Ber-ACT35/BioLegend/350012 1:50
CD137 APC 4B4-1/BioLegend/309810 1:25
CD3 AF700 UCHT1/Thermo Fisher/56-0038-42 1:25

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SUPPLEMENTARY FIGURE LEGENDS

Figure S1. SARS-CoV-2 memory B cells. (A) Gating strategies to define spike-, RBD-, or nucleocapsid-
specific memory B cells.

Figure S2. Kinetics of memory B cell responses. (A) Cross-sectional analysis showing SARS-CoV-2
Spike-specific memory B cell numbers per 106 PBMC. Second order polynomial model for best fit curve
(R2 = 0.14). (B) Percentage of Spike-specific B cells that are specific to RBD. Simple linear regression (R2
= 0.024) (C) Cross-sectional analysis showing SARS-CoV-2 RBD-specific memory B cell numbers per 106
PBMC. Second order polynomial model for best fit curve (R2 = 0.15). (D) Cross-sectional analysis showing
SARS-CoV-2 nucleocapsid-specific memory B cell numbers per 106 PBMC. Second order polynomial
model for best fit curve (R2 = 0.14). (E) Cross-sectional analysis of frequency (% of CD19+ CD20+ B cells)
of SARS-CoV-2 RBD-specific IgA+ memory B cells. Second order polynomial model for best fit curve (R2
= 0.036). (F) Cross-sectional analysis of frequency (% of CD19+ CD20+ B cells) of SARS-CoV-2 RBD-
specific IgM+ memory B cells. Second order polynomial model for best fit curve (R2 = 0.034). (G) Cross-
sectional analysis of frequency (% of CD19+ CD20+ B cells) of SARS-CoV-2 nucleocapsid-specific IgA+
memory B cells. Second order polynomial model for best fit curve (R2 = 0.0031). (H) Cross-sectional
analysis of frequency (% of CD19+ CD20+ B cells) of SARS-CoV-2 nucleocapsid-specific IgM+ memory B
cells. Second order polynomial model for best fit curve (R2 = 0.029). (I) Cross-sectional analysis of
geometric mean fluorescence intensity of spike, RBD and nucleocapsid probes on S-, RBD- and
nucleocapsid-specific memory B cells, respectively. Data shown are simple linear-regression lines for
individual probes. (J) Cross-sectional analysis of geometric mean fluorescence intensity of spike, RBD
and nucleocapsid probes on S-, RBD- and nucleocapsid-specific memory B cells, respectively,
normalized to a positive control sample. Data shown are simple linear-regression lines for individual
antigen.

Figure S3. SARS-CoV-2 circulating memory CD8+ T cells. (A) Gating strategies to define SARS-CoV-
2-specific CD8+ T cells by AIM assay, using individual SARS-CoV-2 ORF peptide pools. (B)
Representative examples of flow cytometry plots of SARS-CoV-2-specific CD8+ T cells (CD69+ CD137+,
after overnight stimulation with S, M, N, ORF3a, or nsp3 peptide pools, compared to negative control
(DMSO) from three COVID-19 subjects and one uninfected control. (C) Cross-sectional analysis of total
SARS-CoV-2-specific CD4+ T cells, as per Figure 3, but graphing stimulation index (SI). n = 155 COVID-
19 subject samples (clear circles) for cross-sectional analysis. n = 30 COVID-19 subjects (white circles)
for longitudinal analysis.

Figure S4. SARS-CoV-2 circulating memory CD4+ T cells. (A) Gating strategies to define SARS-CoV-
2-specific CD4+ T cells by AIM assay, using individual SARS-CoV-2 ORF peptide pools. (B)
Representative examples of flow cytometry plots of SARS-CoV-2-specific CD4+ T cells (OX40+ CD137+,
after overnight stimulation with S, M, N, ORF3a, or nsp3 peptide pools, compared to negative control
(DMSO). From three COVID-19 subjects and one uninfected control. (C) Cross-sectional analysis of total
SARS-CoV-2-specific CD4+ T cells, as per Figure 4, but graphing stimulation index (SI). (D) Cross-
sectional analysis of M-specific CD4+ T cells. (E) Longitudinal analysis of M-specific CD4+ T cells in paired
samples from the same subjects. n = 155 COVID-19 subject samples (white circles) for cross-sectional
analysis. n = 30 COVID-19 subjects (white circles) for longitudinal analysis.

Figure S5. SARS-CoV-2 memory TFH cells. (A) Gating strategies to define SARS-CoV-2-specific CD4+ T
cells by AIM assay, using S and MP_R peptide pools. (B) Representative examples of flow cytometry plots
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of SARS-CoV-2-specific CD4+ T cells. Surface CD40L+ OX40+, after overnight stimulation with S and
MP_R peptide pools, compared to negative control (DMSO) from a representative COVID-19 subject
and an uninfected control. (C, D) SARS-CoV-2-specific CD4+ T cells based on surface CD40L+ OX40+,
gated as in A, after overnight stimulation with S or MP_R peptide pools. n = 34 COVID-19 subject
samples (white circles), n = 21 at 1-2 mo, n = 13 at 6 mo. The dotted black line indicates LOD.* p<0.05,
**p<0.01, *** p<0.001, **** p<0.0001.

Figure S6. Immune memory and disease severity. (A) Spike IgG, as per Figure 1. Symbol colors
represent disease severity (white: asymptomatic, gray: mild, black: moderate, red: severe). (B) Cross-
sectional analysis of SARS-CoV-2 spike-specific total (IgG+, IgA+, or IgA+) memory B cells, as per Figure
2C, color coded based on subject COVID-19 disease severity. (C) Cross-sectional analysis of SARS-CoV-
2 RBD-specific total (IgG+, IgA+, or IgA+) memory B cells, as per Figure 2E, color coded based on subject
COVID-19 disease severity. (D) Cross-sectional analysis of SARS-CoV-2 nucleocapsid-specific total (IgG+,
IgA+, or IgA+) memory B cells, as per Figure 2F, color coded based on subject COVID-19 disease
severity. (E) Cross-sectional analysis of SARS-CoV-2-specific CD8+ T cells, as per Figure 3B, color coded
based on subject COVID-19 disease severity. (F) Cross-sectional analysis of SARS-CoV-2-specific CD4+
T cells, as per Figure 4B, color coded based on subject COVID-19 disease severity.

Figure S7. Immune memory and gender. Cross-sectional analyses of SARS-CoV-2 serologies by male
and female gender. (A) Nucleocapsid IgG titers. Males: Linear decay preferred model, t1/2 = 69d, 95%
CI: 42-209d, R = -0.28, p=0.0035. Females: linear decay preferred model, t1/2 = 64d, 95% CI: 47-104d,
R = -0.41, p<0.0001. (B) RBD IgG titers. Males: One phase decay preferred model, t1/2 = 24d, 95% CI
10-122d, R = -0.38, p<0.0001. Females: linear decay preferred model, t1/2 = 94d,95% CI: 64-179d R = -
0.34, p=0.0.0002. (C) RBD IgA titers. Males: One phase decay preferred model, t1/2 = 15d,95% CI 8-30d,
R = -0.45, p<0.0001. Females: linear decay preferred model, t1/2 = 92d, 95% CI: 60-195d, R = -0.32,
p=0.0004. (D) Pseudovirus neutralizing titers. Males: One phase decay preferred model, t1/2 = 16d, 95%
CI: 7-49d, R = -0.35, p=0.0022. Females: linear decay preferred model, t1/2 = 169d, 95% CI: 96-710d, R
= -0.25, p=0.0069. (E) Spike IgA titers. Males: One phase decay preferred model, t1/2 = 8d, 95% CI 4-
13d, R = -0.22, p=0.019. Females: linear decay preferred model, t1/2 = 337d, 95% CI 116-370d: R = -
0.056, p=0.54. (F) Cross-sectional analysis of frequency (% of CD19+ CD20+ B cells) of SARS-CoV-2
spike-specific memory B cells (IgG+, IgA+, or IgM+), as per Figure 2C, color coded based on subject
gender Pseudo-first order kinetic model for best fit curves. R2 = 0.27 (females), R2 = 0.057 (males). No
significant difference between males and females. p = 0.10 by One-way ANCOVA. (G) Cross-sectional
analysis of SARS-CoV-2 RBD-specific total (IgG+, IgA+, or IgA+) memory B cells, as per Figure 2E, color
coded based on subject gender. Second order polynomial model for best fit curves. R2 = 0.37 (females)
and R2 = 0.12 (males). No significant difference between males and females. p = 0.24 by one-way
ANCOVA. (H) Cross-sectional analysis of SARS-CoV-2 nucleocapsid-specific total (IgG+, IgA+, or IgA+)
memory B cells, as per Figure 2F, color coded based on subject gender. Second order polynomial
model for best fit curves. R2 = 0.28 (females), R2 = 0.16 (males). No significant difference between males
and females. p = 0.45 by one-way ANCOVA. (I). No significant difference between males and females.
p = 0.16 by one-way ANCOVA. (J) No significant difference between males and females. p = 0.24 by
one-way ANCOVA.

Figure S8. Immune memory relationships. (A) The ratio of SARS-CoV-2 specific CD4+ T cell frequency
relative to SARS-CoV-2 specific CD8+ T cell frequency (best-fit simple linear regression line, R2 =
0.02932). Two data points are outside the axis limits. (B) The ratio of RBD-specific memory B cell
frequency (percentage) relative to RBD-specific IgG (pseudo-first order kinetic model, R2 = 0.3659).
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Three data points are outside the axis limits (C) The ratio of RBD-specific memory B cell frequency
(percentage) relative to RBD IgA antibodies (pseudo-first order kinetic model, R2 = 0.3804). Two data
points are outside the axis limits. (D) The ratio of SARS-CoV-2 specific CD4+ T cell frequency relative to
RBD IgG antibodies (best-fit simple linear regression line, R2 = 0.0003891). Two data points are outside
the axis limits. (E) The ratio of RBD-specific memory B cell frequency (percentage) relative to total SARS-
CoV-2 specific CD4+ T cell frequency (best-fit simple linear regression line, R2 = 0.2351). One data point
is outside the axis limits. For Figure 5: The ratio of RBD-specific memory B cell frequency (percentage)
relative to RBD IgA antibodies (orange curve; best-fit second order polynomial curve transformed by
×105), RBD IgG antibodies (magenta; best-fit simple linear regression line transformed by ×105) and
total SARS-CoV-2 specific CD4+ T cell frequency (blue; best-fit simple linear regression line transformed
by ×102), or the ratio of SARS-CoV-2 specific CD4+ T cell frequency relative to SARS-CoV-2 specific CD8+
T cell frequency (till; best-fit simple linear regression line) and RBD IgG antibodies (black; best-fit simple
linear regression line transformed by ×103).

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REFERENCES

1. D. S. Stephens, M. J. McElrath, COVID-19 and the Path to Immunity. Jama. 324 (2020),
doi:10.1001/jama.2020.16656.
2. A. Grifoni, D. Weiskopf, S. I. Ramirez, J. Mateus, J. M. Dan, C. R. Moderbacher, S. A. Rawlings, A.
Sutherland, L. Premkumar, R. S. Jadi, D. Marrama, A. M. de Silva, A. Frazier, A. Carlin, J. A. Greenbaum,
B. Peters, F. Krammer, D. M. Smith, S. Crotty, A. Sette, Targets of T cell responses to SARS-CoV-2
coronavirus in humans with COVID-19 disease and unexposed individuals. Cell. 181, 1489-1501.e15
(2020).
3. F. Krammer, SARS-CoV-2 vaccines in development. Nature, 1–16 (2020).
4. M. S. Suthar, M. G. Zimmerman, R. C. Kauffman, G. Mantus, S. L. Linderman, W. H. Hudson, A.
Vanderheiden, L. Nyhoff, C. W. Davis, S. Adekunle, M. Affer, M. Sherman, S. Reynolds, H. P. Verkerke,
D. N. Alter, J. Guarner, J. Bryksin, M. Horwath, C. M. Arthur, N. Saakadze, G. H. Smith, S. Edupuganti, E.
M. Scherer, K. Hellmeister, A. Cheng, J. A. Morales, A. S. Neish, S. R. Stowell, F. Frank, E. Ortlund, E.
Anderson, V. D. Menachery, N. Rouphael, A. Mehta, D. S. Stephens, R. Ahmed, J. D. Roback, J.
Wrammert, Rapid generation of neutralizing antibody responses in COVID-19 patients. Cell Reports
Medicine. 1, 100040 (2020).
5. C. R. Moderbacher, S. I. Ramirez, J. M. Dan, A. Grifoni, K. M. Hastie, D. Weiskopf, S. Belanger, R. K.
Abbott, C. Kim, J. Choi, Y. Kato, E. G. Crotty, C. Kim, S. A. Rawlings, J. Mateus, L. P. V. Tse, A. Frazier, R.
Baric, B. Peters, J. Greenbaum, E. O. Saphire, D. M. Smith, A. Sette, S. Crotty, Antigen-specific adaptive
immunity to SARS-CoV-2 in acute COVID-19 and associations with age and disease severity. Cell
(2020), doi:10.1016/j.cell.2020.09.038.
6. R. Zhou, K. K.-W. To, Y.-C. Wong, L. Liu, B. Zhou, X. Li, H. Huang, Y. Mo, T.-Y. Luk, T. T.-K. Lau, P.
Yeung, W.-M. Chan, A. K.-L. Wu, K.-C. Lung, O. T.-Y. Tsang, W.-S. Leung, I. F.-N. Hung, K.-Y. Yuen, Z.
Chen, Acute SARS-CoV-2 infection impairs dendritic cell and T cell responses. Immunity (2020),
doi:10.1016/j.immuni.2020.07.026.
7. M. Liao, Y. Liu, J. Yuan, Y. Wen, G. Xu, J. Zhao, L. Cheng, J. Li, X. Wang, F. Wang, L. Liu, I. Amit, S.
Zhang, Z. Zhang, Single-cell landscape of bronchoalveolar immune cells in patients with COVID-19.
Nat Med. 26, 842–844 (2020).
8. A. G. Laing, A. Lorenc, I. del M. del Barrio, A. Das, M. Fish, L. Monin, M. Muñoz-Ruiz, D. R. McKenzie,
T. S. Hayday, I. Francos-Quijorna, S. Kamdar, M. Joseph, D. Davies, R. Davis, A. Jennings, I. Zlatareva, P.
Vantourout, Y. Wu, V. Sofra, F. Cano, M. Greco, E. Theodoridis, J. Freedman, S. Gee, J. N. E. Chan, S.
Ryan, E. Bugallo-Blanco, P. Peterson, K. Kisand, L. Haljasmägi, L. Chadli, P. Moingeon, L. Martinez, B.
Merrick, K. Bisnauthsing, K. Brooks, M. A. A. Ibrahim, J. Mason, F. L. Gomez, K. Babalola, S. Abdul-
Jawad, J. Cason, C. Mant, J. Seow, C. Graham, K. J. Doores, F. D. Rosa, J. Edgeworth, M. Shankar-Hari,
A. C. Hayday, A dynamic COVID-19 immune signature includes associations with poor prognosis. Nat
Med, 1–13 (2020).
9. D. Blanco-Melo, B. E. Nilsson-Payant, W.-C. Liu, S. Uhl, D. Hoagland, R. Møller, T. X. Jordan, K. Oishi,
M. Panis, D. Sachs, T. T. Wang, R. E. Schwartz, J. K. Lim, R. A. Albrecht, B. R. tenOever, Imbalanced Host
Response to SARS-CoV-2 Drives Development of COVID-19. Cell. 181, 1036-1045.e9 (2020).
10. P. S. Arunachalam, F. Wimmers, C. K. P. Mok, R. A. P. M. Perera, M. Scott, T. Hagan, N. Sigal, Y.
Feng, L. Bristow, O. T.-Y. Tsang, D. Wagh, J. Coller, K. L. Pellegrini, D. Kazmin, G. Alaaeddine, W. S.
Leung, J. M. C. Chan, T. S. H. Chik, C. Y. C. Choi, C. Huerta, M. P. McCullough, H. Lv, E. Anderson, S.
Edupuganti, A. A. Upadhyay, S. E. Bosinger, H. T. Maecker, P. Khatri, N. Rouphael, M. Peiris, B.
Pulendran, Systems biological assessment of immunity to mild versus severe COVID-19 infection in
humans. Science, eabc6261 (2020).
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 124forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

11. P. Bastard, L. B. Rosen, Q. Zhang, E. Michailidis, H.-H. Hoffmann, Y. Zhang, K. Dorgham, Q.

Philippot, J. Rosain, V. Béziat, J. Manry, E. Shaw, L. Haljasmägi, P. Peterson, L. Lorenzo, L. Bizien, S.
Trouillet-Assant, K. Dobbs, A. A. de Jesus, A. Belot, A. Kallaste, E. Catherinot, Y. Tandjaoui-Lambiotte,
J. L. Pen, G. Kerner, B. Bigio, Y. Seeleuthner, R. Yang, A. Bolze, A. N. Spaan, O. M. Delmonte, M. S.
Abers, A. Aiuti, G. Casari, V. Lampasona, L. Piemonti, F. Ciceri, K. Bilguvar, R. P. Lifton, M. Vasse, D. M.
Smadja, M. Migaud, J. Hadjadj, B. Terrier, D. Duffy, L. Quintana-Murci, D. van de Beek, L. Roussel, D. C.
Vinh, S. G. Tangye, F. Haerynck, D. Dalmau, J. Martinez-Picado, P. Brodin, M. C. Nussenzweig, S.
Boisson-Dupuis, C. Rodríguez-Gallego, G. Vogt, T. H. Mogensen, A. J. Oler, J. Gu, P. D. Burbelo, J.
Cohen, A. Biondi, L. R. Bettini, M. D’Angio, P. Bonfanti, P. Rossignol, J. Mayaux, F. Rieux-Laucat, E. S.
Husebye, F. Fusco, M. V. Ursini, L. Imberti, A. Sottini, S. Paghera, E. Quiros-Roldan, C. Rossi, R.
Castagnoli, D. Montagna, A. Licari, G. L. Marseglia, X. Duval, J. Ghosn, H. Lab§, N.-U. I. R. to C. Group§,
C. Clinicians§, C.-S. Clinicians§, I. C. Group§, F. C. C. S. Group§, T. M. I. Consortium§, C.-C. Cohort§, A.
U. C.-19 Biobank§, C. H. G. Effort§, J. S. Tsang, R. Goldbach-Mansky, K. Kisand, M. S. Lionakis, A. Puel,
S.-Y. Zhang, S. M. Holland, G. Gorochov, E. Jouanguy, C. M. Rice, A. Cobat, L. D. Notarangelo, L. Abel,
H. C. Su, J.-L. Casanova, Auto-antibodies against type I IFNs in patients with life-threatening COVID-19.
Science, eabd4585 (2020).
12. Q. Zhang, P. Bastard, Z. Liu, J. L. Pen, M. Moncada-Velez, J. Chen, M. Ogishi, I. K. D. Sabli, S.
Hodeib, C. Korol, J. Rosain, K. Bilguvar, J. Ye, A. Bolze, B. Bigio, R. Yang, A. A. Arias, Q. Zhou, Y. Zhang,
F. Onodi, S. Korniotis, L. Karpf, Q. Philippot, M. Chbihi, L. Bonnet-Madin, K. Dorgham, N. Smith, W. M.
Schneider, B. S. Razooky, H.-H. Hoffmann, E. Michailidis, L. Moens, J. E. Han, L. Lorenzo, L. Bizien, P.
Meade, A.-L. Neehus, A. C. Ugurbil, A. Corneau, G. Kerner, P. Zhang, F. Rapaport, Y. Seeleuthner, J.
Manry, C. Masson, Y. Schmitt, A. Schlüter, T. L. Voyer, T. Khan, J. Li, J. Fellay, L. Roussel, M. Shahrooei,
M. F. Alosaimi, D. Mansouri, H. Al-Saud, F. Al-Mulla, F. Almourfi, S. Z. Al-Muhsen, F. Alsohime, S. A.
Turki, R. Hasanato, D. van de Beek, A. Biondi, L. R. Bettini, M. D’Angio, P. Bonfanti, L. Imberti, A. Sottini,
S. Paghera, E. Quiros-Roldan, C. Rossi, A. J. Oler, M. F. Tompkins, C. Alba, I. Vandernoot, J.-C. Goffard,
G. Smits, I. Migeotte, F. Haerynck, P. Soler-Palacin, A. Martin-Nalda, R. Colobran, P.-E. Morange, S.
Keles, F. Çölkesen, T. Ozcelik, K. K. Yasar, S. Senoglu, Ş. N. Karabela, C. R. Gallego, G. Novelli, S.
Hraiech, Y. Tandjaoui-Lambiotte, X. Duval, C. Laouénan, C.-S. Clinicians†, C. Clinicians†, I. C. Group†,
F. C. C. S. Group†, C.-C. Cohort†, A. U. Covid-19, Biobank†, C. H. G. Effort†, NIAID-USUHS, T. C. I.
Group†, A. L. Snow, C. L. Dalgard, J. Milner, D. C. Vinh, T. H. Mogensen, N. Marr, A. N. Spaan, B.
Boisson, S. Boisson-Dupuis, J. Bustamante, A. Puel, M. Ciancanelli, I. Meyts, T. Maniatis, V. Soumelis, A.
Amara, M. Nussenzweig, A. García-Sastre, F. Krammer, A. Pujol, D. Duffy, R. Lifton, S.-Y. Zhang, G.
Gorochov, V. Béziat, E. Jouanguy, V. Sancho-Shimizu, C. M. Rice, L. Abel, L. D. Notarangelo, A. Cobat,
H. C. Su, J.-L. Casanova, Inborn errors of type I IFN immunity in patients with life-threatening COVID-
19. Science, eabd4570 (2020).
13. D. M. D. Valle, S. Kim-Schulze, H.-H. Huang, N. D. Beckmann, S. Nirenberg, B. Wang, Y. Lavin, T. H.
Swartz, D. Madduri, A. Stock, T. U. Marron, H. Xie, M. Patel, K. Tuballes, O. V. Oekelen, A. Rahman, P.
Kovatch, J. A. Aberg, E. Schadt, S. Jagannath, M. Mazumdar, A. W. Charney, A. Firpo-Betancourt, D. R.
Mendu, J. Jhang, D. Reich, K. Sigel, C. Cordon-Cardo, M. Feldmann, S. Parekh, M. Merad, S. Gnjatic,
An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat Med, 1–8 (2020).
14. L. Kuri-Cervantes, M. B. Pampena, W. Meng, A. M. Rosenfeld, C. A. G. Ittner, A. R. Weisman, R. S.
Agyekum, D. Mathew, A. E. Baxter, L. A. Vella, O. Kuthuru, S. A. Apostolidis, L. Bershaw, J. Dougherty,
A. R. Greenplate, A. Pattekar, J. Kim, N. Han, S. Gouma, M. E. Weirick, C. P. Arevalo, M. J. Bolton, E. C.
Goodwin, E. M. Anderson, S. E. Hensley, T. K. Jones, N. S. Mangalmurti, E. T. L. Prak, E. J. Wherry, N. J.
Meyer, M. R. Betts, Comprehensive mapping of immune perturbations associated with severe COVID-
19. Sci Immunol. 5, eabd7114 (2020).
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 125forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

15. S. Li, L. Jiang, X. Li, F. Lin, Y. Wang, B. Li, T. Jiang, W. An, S. Liu, H. Liu, P. Xu, L. Zhao, L. Zhang, J.
Mu, H. Wang, J. Kang, Y. Li, L. Huang, C. Zhu, S. Zhao, J. Lu, J. Ji, J. Zhao, Clinical and pathological
investigation of severe COVID-19 patients. Jci Insight. 5 (2020), doi:10.1172/jci.insight.138070.
16. C. Radermecker, N. Detrembleur, J. Guiot, E. Cavalier, M. Henket, C. d’Emal, C. Vanwinge, D.
Cataldo, C. Oury, P. Delvenne, T. Marichal, Neutrophil extracellular traps infiltrate the lung airway,
interstitial, and vascular compartments in severe COVID-19. J Exp Med. 217 (2020),
doi:10.1084/jem.20201012.
17. B. Schurink, E. Roos, T. Radonic, E. Barbe, C. S. C. Bouman, H. H. de Boer, G. J. de Bree, E. B. Bulle,
E. M. Aronica, S. Florquin, J. Fronczek, L. M. A. Heunks, M. D. de Jong, L. Guo, R. du Long, R. Lutter, P.
C. G. Molenaar, E. A. Neefjes-Borst, H. W. M. Niessen, C. J. M. van Noesel, J. J. T. H. Roelofs, E. J.
Snijder, E. C. Soer, J. Verheij, A. P. J. Vlaar, W. Vos, N. N. van der Wel, A. C. van der Wal, P. van der
Valk, M. Bugiani, Viral presence and immunopathology in patients with lethal COVID-19: a prospective
autopsy cohort study. Lancet Microbe (2020), doi:10.1016/s2666-5247(20)30144-0.
18. M. Aid, K. Busman-Sahay, S. J. Vidal, Z. Maliga, S. Bondoc, C. Starke, M. Terry, C. A. Jacobson, L.
Wrijil, S. Ducat, O. R. Brook, A. D. Miller, M. Porto, K. L. Pellegrini, M. Pino, T. N. Hoang, A.
Chandrashekar, S. Patel, K. Stephenson, S. E. Bosinger, H. Andersen, M. G. Lewis, J. L. Hecht, P. K.
Sorger, A. J. Martinot, J. D. Estes, D. H. Barouch, Vascular Disease and Thrombosis in SARS-CoV-2
Infected Rhesus Macaques. Cell (2020), doi:10.1016/j.cell.2020.10.005.
19. N. Baumgarth, J. Nikolich-Žugich, F. E.-H. Lee, D. Bhattacharya, Antibody Responses to SARS-CoV-
2: Let’s Stick to Known Knowns. J Immunol, ji2000839 (2020).
20. A. Wajnberg, F. Amanat, A. Firpo, D. Altman, M. Bailey, M. Mansour, M. McMahon, P. Meade, D. R.
Mendu, K. Muellers, D. Stadlbauer, K. Stone, S. Strohmeier, J. Aberg, D. Reich, F. Krammer, C. Cordon-
Cardo, SARS-CoV-2 infection induces robust, neutralizing antibody responses that are stable for at
least three months (n.d.), doi:10.1101/2020.07.14.20151126.
21. A. Sariol, S. Perlman, Lessons for COVID-19 immunity from other coronavirus infections. Immunity.
53, 248–263 (2020).
22. K. Subbarao, SARS-CoV-2: A New Song Recalls an Old Melody. Cell Host Microbe. 27, 692–694
(2020).
23. W. Deng, L. Bao, J. Liu, C. Xiao, J. Liu, J. Xue, Q. Lv, F. Qi, H. Gao, P. Yu, Y. Xu, Y. Qu, F. Li, Z. Xiang,
H. Yu, S. Gong, M. Liu, G. Wang, S. Wang, Z. Song, Y. Liu, W. Zhao, Y. Han, L. Zhao, X. Liu, Q. Wei, C.
Qin, Primary exposure to SARS-CoV-2 protects against reinfection in rhesus macaques. Science,
eabc5343 (2020).
24. Q. Gao, L. Bao, H. Mao, L. Wang, K. Xu, M. Yang, Y. Li, L. Zhu, N. Wang, Z. Lv, H. Gao, X. Ge, B. Kan,
Y. Hu, J. Liu, F. Cai, D. Jiang, Y. Yin, C. Qin, J. Li, X. Gong, X. Lou, W. Shi, D. Wu, H. Zhang, L. Zhu, W.
Deng, Y. Li, J. Lu, C. Li, X. Wang, W. Yin, Y. Zhang, C. Qin, Rapid development of an inactivated vaccine
candidate for SARS-CoV-2. Science, eabc1932 (2020).
25. A. Chandrashekar, J. Liu, A. J. Martinot, K. McMahan, N. B. Mercado, L. Peter, L. H. Tostanoski, J.
Yu, Z. Maliga, M. Nekorchuk, K. Busman-Sahay, M. Terry, L. M. Wrijil, S. Ducat, D. R. Martinez, C. Atyeo,
S. Fischinger, J. S. Burke, M. D. Slein, L. Pessaint, A. V. Ry, J. Greenhouse, T. Taylor, K. Blade, A. Cook,
B. Finneyfrock, R. Brown, E. Teow, J. Velasco, R. Zahn, F. Wegmann, P. Abbink, E. A. Bondzie, G.
Dagotto, M. S. Gebre, X. He, C. Jacob-Dolan, N. Kordana, Z. Li, M. A. Lifton, S. H. Mahrokhian, L. F.
Maxfield, R. Nityanandam, J. P. Nkolola, A. G. Schmidt, A. D. Miller, R. S. Baric, G. Alter, P. K. Sorger, J.
D. Estes, H. Andersen, M. G. Lewis, D. H. Barouch, SARS-CoV-2 infection protects against rechallenge
in rhesus macaques. Science, eabc4776 (2020).
26. A. Addetia, K. H. D. Crawford, A. Dingens, H. Zhu, P. Roychoudhury, M.-L. Huang, K. R. Jerome, J.
D. Bloom, A. L. Greninger, Neutralizing antibodies correlate with protection from SARS-CoV-2 in
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 126forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

humans during a fishery vessel outbreak with high attack rate. J Clin Microbiol (2020),
doi:10.1128/jcm.02107-20.
27. S. J. Zost, P. Gilchuk, J. B. Case, E. Binshtein, R. E. Chen, J. P. Nkolola, A. Schäfer, J. X. Reidy, A.
Trivette, R. S. Nargi, R. E. Sutton, N. Suryadevara, D. R. Martinez, L. E. Williamson, E. C. Chen, T. Jones,
S. Day, L. Myers, A. O. Hassan, N. M. Kafai, E. S. Winkler, J. M. Fox, S. Shrihari, B. K. Mueller, J. Meiler,
A. Chandrashekar, N. B. Mercado, J. J. Steinhardt, K. Ren, Y.-M. Loo, N. L. Kallewaard, B. T. McCune, S.
P. Keeler, M. J. Holtzman, D. H. Barouch, L. E. Gralinski, R. S. Baric, L. B. Thackray, M. S. Diamond, R. H.
Carnahan, J. E. Crowe, Potently neutralizing and protective human antibodies against SARS-CoV-2.
Nature, 1–10 (2020).
28. T. F. Rogers, F. Zhao, D. Huang, N. Beutler, A. Burns, W. He, O. Limbo, C. Smith, G. Song, J. Woehl,
L. Yang, R. K. Abbott, S. Callaghan, E. Garcia, J. Hurtado, M. Parren, L. Peng, S. Ramirez, J. Ricketts, M.
J. Ricciardi, S. A. Rawlings, N. C. Wu, M. Yuan, D. M. Smith, D. Nemazee, J. R. Teijaro, J. E. Voss, I. A.
Wilson, R. Andrabi, B. Briney, E. Landais, D. Sok, J. G. Jardine, D. R. Burton, Isolation of potent SARS-
CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science, eabc7520
(2020).
29. A. Baum, R. Copin, D. Ajithdoss, A. Zhou, K. Lanza, N. Negron, M. Ni, Y. Wei, G. S. Atwal, A.
Oyejide, Y. Goez-Gazi, J. Dutton, E. Clemmons, H. M. Staples, C. Bartley, B. Klaffke, K. Alfson, M. Gazi,
O. Gonzales, E. Dick, R. Carrion, L. Pessaint, M. Porto, A. Cook, R. Brown, V. Ali, J. Greenhouse, T.
Taylor, H. Andersen, M. G. Lewis, N. Stahl, A. J. Murphy, G. D. Yancopoulos, C. A. Kyratsous, Biorxiv, in
press, doi:10.1101/2020.08.02.233320.
30. Regeneron, REGN-COV2 ANTIBODY COCKTAIL PROGRAM UPDATE (n.d.), (available at
https://newsroom.regeneron.com/static-files/a596a85e-e72d-4529-8eb5-d52d87a99070).
31. Lilly announces proof of concept data for neutralizing antibody LY-CoV555 in the COVID-19
outpatient setting | Eli Lilly and Company (n.d.), (available at https://investor.lilly.com/news-
releases/news-release-details/lilly-announces-proof-concept-data-neutralizing-antibody-ly).
32. D. M. Altmann, R. J. Boyton, SARS-CoV-2 T cell immunity: Specificity, function, durability, and role
in protection. Sci Immunol. 5, eabd6160 (2020).
33. P. V. Damme, K. V. Herck, A review of the long-term protection after hepatitis A and B vaccination.
Travel Med Infect Di. 5, 79–84 (2007).
34. M. M. Rosado, M. Scarsella, E. Pandolfi, S. Cascioli, E. Giorda, P. Chionne, E. Madonne, F.
Gesualdo, M. Romano, C. M. Ausiello, M. Rapicetta, A. R. Zanetti, A. Tozzi, R. Carsetti, Switched
memory B cells maintain specific memory independently of serum antibodies: The hepatitis B
example. Eur J Immunol. 41, 1800–1808 (2011).
35. F. Zhou, T. Yu, R. Du, G. Fan, Y. Liu, Z. Liu, J. Xiang, Y. Wang, B. Song, X. Gu, L. Guan, Y. Wei, H. Li,
X. Wu, J. Xu, S. Tu, Y. Zhang, H. Chen, B. Cao, Clinical course and risk factors for mortality of adult
inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 395, 1054–1062
(2020).
36. W. A. Orenstein, R. Ahmed, Simply put: Vaccination saves lives. Proc National Acad Sci. 114, 4031–
4033 (2017).
37. P. Piot, H. J. Larson, K. L. O’Brien, J. N’kengasong, E. Ng, S. Sow, B. Kampmann, Immunization: vital
progress, unfinished agenda. Nature. 575, 119–129 (2019).
38. S. Plotkin, W. Orenstein, P. Offit, Plotkin’s vaccines, 7th edition (Elsevier, 2018), Elsevier.
39. F. Sallusto, A. Lanzavecchia, K. Araki, R. Ahmed, From vaccines to memory and back. Immunity. 33,
451–463 (2010).
40. S. Crotty, R. Ahmed, Immunological memory in humans. Seminars in Immunology. 16, 197–203
(2004).
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 127forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

41. F. Weisel, M. Shlomchik, Memory B Cells of Mice and Humans. Annual review of immunology. 35,
255–284 (2017).
42. S. M. Kissler, C. Tedijanto, E. Goldstein, Y. H. Grad, M. Lipsitch, Projecting the transmission
dynamics of SARS-CoV-2 through the postpandemic period. Science. 368, 860–868 (2020).
43. C. M. Saad-Roy, C. E. Wagner, R. E. Baker, S. E. Morris, J. Farrar, A. L. Graham, S. A. Levin, M. J.
Mina, C. J. E. Metcalf, B. T. Grenfell, Immune life history, vaccination, and the dynamics of SARS-CoV-2
over the next 5 years. Science, eabd7343 (2020).
44. L. B. Rodda, J. Netland, L. Shehata, K. B. Pruner, P. M. Morawski, C. Thouvenel, K. K. Takehara, J.
Eggenberger, E. A. Hemann, H. R. Waterman, M. L. Fahning, Y. Chen, J. Rathe, C. Stokes, S. Wrenn, B.
Fiala, L. P. Carter, J. A. Hamerman, N. P. King, M. Gale, D. J. Campbell, D. Rawlings, M. Pepper,
Functional SARS-CoV-2-specific immune memory persists after mild COVID-19. Medrxiv Prepr Serv
Heal Sci (2020), doi:10.1101/2020.08.11.20171843.
45. Q.-X. Long, X.-J. Tang, Q.-L. Shi, Q. Li, H.-J. Deng, J. Yuan, J.-L. Hu, W. Xu, Y. Zhang, F.-J. Lv, K. Su,
F. Zhang, J. Gong, B. Wu, X.-M. Liu, J.-J. Li, J.-F. Qiu, J. Chen, A.-L. Huang, Clinical and immunological
assessment of asymptomatic SARS-CoV-2 infections. Nat Med. 26, 1200–1204 (2020).
46. D. F. Gudbjartsson, G. L. Norddahl, P. Melsted, K. Gunnarsdottir, H. Holm, E. Eythorsson, A. O.
Arnthorsson, D. Helgason, K. Bjarnadottir, R. F. Ingvarsson, B. Thorsteinsdottir, S. Kristjansdottir, K.
Birgisdottir, A. M. Kristinsdottir, M. I. Sigurdsson, G. A. Arnadottir, E. V. Ivarsdottir, M. Andresdottir, F.
Jonsson, A. B. Agustsdottir, J. Berglund, B. Eiriksdottir, R. Fridriksdottir, E. E. Gardarsdottir, M.
Gottfredsson, O. S. Gretarsdottir, S. Gudmundsdottir, K. R. Gudmundsson, T. R. Gunnarsdottir, A.
Gylfason, A. Helgason, B. O. Jensson, A. Jonasdottir, H. Jonsson, T. Kristjansson, K. G. Kristinsson, D.
N. Magnusdottir, O. T. Magnusson, L. B. Olafsdottir, S. Rognvaldsson, L. le Roux, G. Sigmundsdottir, A.
Sigurdsson, G. Sveinbjornsson, K. E. Sveinsdottir, M. Sveinsdottir, E. A. Thorarensen, B. Thorbjornsson,
M. Thordardottir, J. Saemundsdottir, S. H. Kristjansson, K. S. Josefsdottir, G. Masson, G. Georgsson, M.
Kristjansson, A. Moller, R. Palsson, T. Gudnason, U. Thorsteinsdottir, I. Jonsdottir, P. Sulem, K.
Stefansson, Humoral Immune Response to SARS-CoV-2 in Iceland. New Engl J Med (2020),
doi:10.1056/nejmoa2026116.
47. L. Piccoli, Y.-J. Park, M. A. Tortorici, N. Czudnochowski, A. C. Walls, M. Beltramello, C. Silacci-
Fregni, D. Pinto, L. E. Rosen, J. E. Bowen, O. J. Acton, S. Jaconi, B. Guarino, A. Minola, F. Zatta, N.
Sprugasci, J. Bassi, A. Peter, A. D. Marco, J. C. Nix, F. Mele, S. Jovic, B. F. Rodriguez, S. V. Gupta, F. Jin,
G. Piumatti, G. L. Presti, A. F. Pellanda, M. Biggiogero, M. Tarkowski, M. S. Pizzuto, E. Cameroni, C.
Havenar-Daughton, M. Smithey, D. Hong, V. Lepori, E. Albanese, A. Ceschi, E. Bernasconi, L. Elzi, P.
Ferrari, C. Garzoni, A. Riva, G. Snell, F. Sallusto, K. Fink, H. W. Virgin, A. Lanzavecchia, D. Corti, D.
Veesler, Mapping neutralizing and immunodominant sites on the SARS-CoV-2 spike receptor-binding
domain by structure-guided high-resolution serology. Cell (2020), doi:10.1016/j.cell.2020.09.037.
48. D. F. Robbiani, C. Gaebler, F. Muecksch, J. C. C. Lorenzi, Z. Wang, A. Cho, M. Agudelo, C. O.
Barnes, A. Gazumyan, S. Finkin, T. Hägglöf, T. Y. Oliveira, C. Viant, A. Hurley, H.-H. Hoffmann, K. G.
Millard, R. G. Kost, M. Cipolla, K. Gordon, F. Bianchini, S. T. Chen, V. Ramos, R. Patel, J. Dizon, I.
Shimeliovich, P. Mendoza, H. Hartweger, L. Nogueira, M. Pack, J. Horowitz, F. Schmidt, Y. Weisblum, E.
Michailidis, A. W. Ashbrook, E. Waltari, J. E. Pak, K. E. Huey-Tubman, N. Koranda, P. R. Hoffman, A. P.
West, C. M. Rice, T. Hatziioannou, P. J. Bjorkman, P. D. Bieniasz, M. Caskey, M. C. Nussenzweig,
Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature, 1–8 (2020).
49. J. Yu, L. H. Tostanoski, L. Peter, N. B. Mercado, K. McMahan, S. H. Mahrokhian, J. P. Nkolola, J. Liu,
Z. Li, A. Chandrashekar, D. R. Martinez, C. Loos, C. Atyeo, S. Fischinger, J. S. Burke, M. D. Slein, Y.
Chen, A. Zuiani, F. J. N. Lelis, M. Travers, S. Habibi, L. Pessaint, A. V. Ry, K. Blade, R. Brown, A. Cook, B.
Finneyfrock, A. Dodson, E. Teow, J. Velasco, R. Zahn, F. Wegmann, E. A. Bondzie, G. Dagotto, M. S.
Gebre, X. He, C. Jacob-Dolan, M. Kirilova, N. Kordana, Z. Lin, L. F. Maxfield, F. Nampanya, R.
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 128forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Nityanandam, J. D. Ventura, H. Wan, Y. Cai, B. Chen, A. G. Schmidt, D. R. Wesemann, R. S. Baric, G.

Alter, H. Andersen, M. G. Lewis, D. H. Barouch, DNA vaccine protection against SARS-CoV-2 in rhesus
macaques. Science. 369, 806–811 (2020).
50. B. Isho, K. T. Abe, M. Zuo, A. J. Jamal, B. Rathod, J. H. Wang, Z. Li, G. Chao, O. L. Rojas, Y. M. Bang,
A. Pu, N. Christie-Holmes, C. Gervais, D. Ceccarelli, P. Samavarchi-Tehrani, F. Guvenc, P. Budylowski,
A. Li, A. Paterson, F. Y. Yue, L. M. Marin, L. Caldwell, J. L. Wrana, K. Colwill, F. Sicheri, S. Mubareka, S.
D. Gray-Owen, S. J. Drews, W. L. Siqueira, M. Barrios-Rodiles, M. Ostrowski, J. M. Rini, Y. Durocher, A.
J. McGeer, J. L. Gommerman, A.-C. Gingras, Persistence of serum and saliva antibody responses to
SARS-CoV-2 spike antigens in COVID-19 patients. Sci Immunol. 5 (2020),
doi:10.1126/sciimmunol.abe5511.
51. C. W. Davis, K. J. L. Jackson, A. K. McElroy, P. Halfmann, J. Huang, C. Chennareddy, A. E. Piper, Y.
Leung, C. G. Albariño, I. Crozier, A. H. Ellebedy, J. Sidney, A. Sette, T. Yu, S. C. A. Nielsen, A. J. Goff, C.
F. Spiropoulou, E. O. Saphire, G. Cavet, Y. Kawaoka, A. K. Mehta, P. J. Glass, S. D. Boyd, R. Ahmed,
Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection. Cell. 177, 1566-1582.e17
(2019).
52. A. Z. Wec, D. Haslwanter, Y. N. Abdiche, L. Shehata, N. Pedreño-Lopez, C. L. Moyer, Z. A.
Bornholdt, A. Lilov, J. H. Nett, R. K. Jangra, M. Brown, D. I. Watkins, C. Ahlm, M. N. Forsell, F. A. Rey, G.
Barba-Spaeth, K. Chandran, L. M. Walker, Longitudinal dynamics of the human B cell response to the
yellow fever 17D vaccine. Proc National Acad Sci. 117, 6675–6685 (2020).
53. J. Neidleman, X. Luo, J. Frouard, G. Xie, G. Gill, E. S. Stein, M. McGregor, T. Ma, A. F. George, A.
Kosters, W. C. Greene, J. Vasquez, E. Ghosn, S. Lee, N. R. Roan, SARS-CoV-2-specific T cells exhibit
phenotypic features of robust helper function, lack of terminal differentiation, and high proliferative
potential. Cell Reports Medicine, 100081 (2020).
54. S. Sridhar, S. Begom, A. Bermingham, K. Hoschler, W. Adamson, W. Carman, T. Bean, W. Barclay, J.
J. Deeks, A. Lalvani, Cellular immune correlates of protection against symptomatic pandemic influenza.
Nat Med. 19, 1305–1312 (2013).
55. R. S. Akondy, M. Fitch, S. Edupuganti, S. Yang, H. T. Kissick, K. W. Li, B. A. Youngblood, H. A.
Abdelsamed, D. J. McGuire, K. W. Cohen, G. Alexe, S. Nagar, M. M. McCausland, S. Gupta, P. Tata, W.
N. Haining, M. J. McElrath, D. Zhang, B. Hu, W. J. Greenleaf, J. J. Goronzy, M. J. Mulligan, M.
Hellerstein, R. Ahmed, Origin and differentiation of human memory CD8 T cells after vaccination.
Nature. 552, 362–367 (2017).
56. S. Crotty, T Follicular Helper Cell Biology: A Decade of Discovery and Diseases. Immunity. 50,
1132–1148 (2019).
57. J. A. Juno, H.-X. Tan, W. S. Lee, A. Reynaldi, H. G. Kelly, K. Wragg, R. Esterbauer, H. E. Kent, C. J.
Batten, F. L. Mordant, N. A. Gherardin, P. Pymm, M. H. Dietrich, N. E. Scott, W.-H. Tham, D. I. Godfrey,
K. Subbarao, M. P. Davenport, S. J. Kent, A. K. Wheatley, Humoral and circulating follicular helper T cell
responses in recovered patients with COVID-19. Nat Med, 1–7 (2020).
58. S. Crotty, P. Felgner, H. Davies, J. Glidewell, L. Villarreal, R. Ahmed, Cutting Edge: Long-Term B
Cell Memory in Humans after Smallpox Vaccination. J Immunol. 171, 4969–4973 (2003).
59. X. Yu, T. Tsibane, P. A. McGraw, F. S. House, C. J. Keefer, M. D. Hicar, T. M. Tumpey, C. Pappas, L.
A. Perrone, O. Martinez, J. Stevens, I. A. Wilson, P. V. Aguilar, E. L. Altschuler, C. F. Basler, J. E. C. Jr,
Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors. Nature. 455,
532–536 (2008).
60. E. Hammarlund, M. W. Lewis, S. G. Hansen, L. I. Strelow, J. A. Nelson, G. J. Sexton, J. M. Hanifin, M.
K. Slifka, Duration of antiviral immunity after smallpox vaccination. Nature Medicine. 9, 1131–1137
(2003).
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 129forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

61. N. L. Bert, A. T. Tan, K. Kunasegaran, C. Y. L. Tham, M. Hafezi, A. Chia, M. H. Y. Chng, M. Lin, N. Tan,
M. Linster, W. N. Chia, M. I.-C. Chen, L.-F. Wang, E. E. Ooi, S. Kalimuddin, P. A. Tambyah, J. G.-H. Low,
Y.-J. Tan, A. Bertoletti, SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and
uninfected controls. Nature. 584, 457–462 (2020).
62. N. B. Mercado, R. Zahn, F. Wegmann, C. Loos, A. Chandrashekar, J. Yu, J. Liu, L. Peter, K.
McMahan, L. H. Tostanoski, X. He, D. R. Martinez, L. Rutten, R. Bos, D. van Manen, J. Vellinga, J.
Custers, J. P. Langedijk, T. Kwaks, M. J. G. Bakkers, D. Zuijdgeest, S. K. R. Huber, C. Atyeo, S.
Fischinger, J. S. Burke, J. Feldman, B. M. Hauser, T. M. Caradonna, E. A. Bondzie, G. Dagotto, M. S.
Gebre, E. Hoffman, C. Jacob-Dolan, M. Kirilova, Z. Li, Z. Lin, S. H. Mahrokhian, L. F. Maxfield, F.
Nampanya, R. Nityanandam, J. P. Nkolola, S. Patel, J. D. Ventura, K. Verrington, H. Wan, L. Pessaint, A.
V. Ry, K. Blade, A. Strasbaugh, M. Cabus, R. Brown, A. Cook, S. Zouantchangadou, E. Teow, H.
Andersen, M. G. Lewis, Y. Cai, B. Chen, A. G. Schmidt, R. K. Reeves, R. S. Baric, D. A. Lauffenburger, G.
Alter, P. Stoffels, M. Mammen, J. V. Hoof, H. Schuitemaker, D. H. Barouch, Single-shot Ad26 vaccine
protects against SARS-CoV-2 in rhesus macaques. Nature, 1–11 (2020).
63. K. S. Corbett, B. Flynn, K. E. Foulds, J. R. Francica, S. Boyoglu-Barnum, A. P. Werner, B. Flach, S.
O’Connell, K. W. Bock, M. Minai, B. M. Nagata, H. Anderson, D. R. Martinez, A. T. Noe, N. Douek, M. M.
Donaldson, N. N. Nji, G. S. Alvarado, D. K. Edwards, D. R. Flebbe, E. Lamb, N. A. Doria-Rose, B. C. Lin,
M. K. Louder, S. O’Dell, S. D. Schmidt, E. Phung, L. A. Chang, C. Yap, J.-P. M. Todd, L. Pessaint, A. V. Ry,
S. Browne, J. Greenhouse, T. Putman-Taylor, A. Strasbaugh, T.-A. Campbell, A. Cook, A. Dodson, K.
Steingrebe, W. Shi, Y. Zhang, O. M. Abiona, L. Wang, A. Pegu, E. S. Yang, K. Leung, T. Zhou, I.-T. Teng,
A. Widge, I. Gordon, L. Novik, R. A. Gillespie, R. J. Loomis, J. I. Moliva, G. Stewart-Jones, S. Himansu,
W.-P. Kong, M. C. Nason, K. M. Morabito, T. J. Ruckwardt, J. E. Ledgerwood, M. R. Gaudinski, P. D.
Kwong, J. R. Mascola, A. Carfi, M. G. Lewis, R. S. Baric, A. McDermott, I. N. Moore, N. J. Sullivan, M.
Roederer, R. A. Seder, B. S. Graham, Evaluation of the mRNA-1273 Vaccine against SARS-CoV-2 in
Nonhuman Primates. New Engl J Med (2020), doi:10.1056/nejmoa2024671.
64. L. Corey, J. R. Mascola, A. S. Fauci, F. S. Collins, A strategic approach to COVID-19 vaccine R&D.
Science. 368, 948–950 (2020).
65. J. Zhao, J. Zhao, A. K. Mangalam, R. Channappanavar, C. Fett, D. K. Meyerholz, S. Agnihothram, R.
S. Baric, C. S. David, S. Perlman, Airway Memory CD4 + T Cells Mediate Protective Immunity against
Emerging Respiratory Coronaviruses. Immunity. 44, 1379–1391 (2016).
66. W. E. Purtha, T. F. Tedder, S. Johnson, D. Bhattacharya, M. S. Diamond, Memory B cells, but not
long-lived plasma cells, possess antigen specificities for viral escape mutants. The Journal of
experimental medicine. 208, 2599–2606 (2011).
67. D. Masopust, A. G. Soerens, Tissue-Resident T Cells and Other Resident Leukocytes. Annu Rev
Immunol. 37, 521–546 (2019).
68. H. C. Whittle, P. Aaby, B. Samb, H. Jensen, J. Bennett, F. Simondon, Effect of subclinical infection
on maintaining immunity against measles in vaccinated children in West Africa. Lancet. 353, 98–102
(1999).
69. S. A. Plotkin, Vaccines: correlates of vaccine-induced immunity. Clinical infectious diseases
: an
official publication of the Infectious Diseases Society of America. 47, 401–409 (2008).
70. N. Burdin, L. K. Handy, S. A. Plotkin, What Is Wrong with Pertussis Vaccine Immunity? The Problem
of Waning Effectiveness of Pertussis Vaccines. Cold Spring Harbor perspectives in biology. 9, a029454
(2017).
71. N. van Doremalen, T. Lambe, A. Spencer, S. Belij-Rammerstorfer, J. N. Purushotham, J. R. Port, V. A.
Avanzato, T. Bushmaker, A. Flaxman, M. Ulaszewska, F. Feldmann, E. R. Allen, H. Sharpe, J. Schulz, M.
Holbrook, A. Okumura, K. Meade-White, L. Pérez-Pérez, N. J. Edwards, D. Wright, C. Bissett, C.
Gilbride, B. N. Williamson, R. Rosenke, D. Long, A. Ishwarbhai, R. Kailath, L. Rose, S. Morris, C. Powers,
Case
bioRxiv 1:21-cv-02228
preprint Document 1-1 Filed
doi: https://doi.org/10.1101/2020.11.15.383323 08/17/21
; this version USDC16,Colorado
posted November Page
2020. The copyright 130forof
holder this165
preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

J. Lovaglio, P. W. Hanley, D. Scott, G. Saturday, E. de Wit, S. C. Gilbert, V. J. Munster, ChAdOx1 nCoV-

19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature, 1–5 (2020).
72. R. L. Tillett, J. R. Sevinsky, P. D. Hartley, H. Kerwin, N. Crawford, A. Gorzalski, C. Laverdure, S. C.
Verma, C. C. Rossetto, D. Jackson, M. J. Farrell, S. V. Hooser, M. Pandori, Genomic evidence for
reinfection with SARS-CoV-2: a case study. Lancet Infect Dis (2020), doi:10.1016/s1473-
3099(20)30764-7.
73. K. K.-W. To, I. F.-N. Hung, J. D. Ip, A. W.-H. Chu, W.-M. Chan, A. R. Tam, C. H.-Y. Fong, S. Yuan, H.-
W. Tsoi, A. C.-K. Ng, L. L.-Y. Lee, P. Wan, E. Y.-K. Tso, W.-K. To, D. N.-C. Tsang, K.-H. Chan, J.-D. Huang,
K.-H. Kok, V. C.-C. Cheng, K.-Y. Yuen, COVID-19 re-infection by a phylogenetically distinct SARS-
coronavirus-2 strain confirmed by whole genome sequencing. Clin Infect Dis (2020),
doi:10.1093/cid/ciaa1275.
74. J. H. Beigel, K. M. Tomashek, L. E. Dodd, A. K. Mehta, B. S. Zingman, A. C. Kalil, E. Hohmann, H. Y.
Chu, A. Luetkemeyer, S. Kline, D. L. de Castilla, R. W. Finberg, K. Dierberg, V. Tapson, L. Hsieh, T. F.
Patterson, R. Paredes, D. A. Sweeney, W. R. Short, G. Touloumi, D. C. Lye, N. Ohmagari, M.-D. Oh, G.
M. Ruiz-Palacios, T. Benfield, G. Fätkenheuer, M. G. Kortepeter, R. L. Atmar, C. B. Creech, J. Lundgren,
A. G. Babiker, S. Pett, J. D. Neaton, T. H. Burgess, T. Bonnett, M. Green, M. Makowski, A. Osinusi, S.
Nayak, H. C. Lane, A.-1 S. G. Members, Remdesivir for the Treatment of Covid-19 — Final Report. New
Engl J Med (2020), doi:10.1056/nejmoa2007764.
75. F. Amanat, D. Stadlbauer, S. Strohmeier, T. H. O. Nguyen, V. Chromikova, M. McMahon, K. Jiang,
G. A. Arunkumar, D. Jurczyszak, J. Polanco, M. Bermudez-Gonzalez, G. Kleiner, T. Aydillo, L. Miorin, D.
S. Fierer, L. A. Lugo, E. M. Kojic, J. Stoever, S. T. H. Liu, C. Cunningham-Rundles, P. L. Felgner, T.
Moran, A. García-Sastre, D. Caplivski, A. C. Cheng, K. Kedzierska, O. Vapalahti, J. M. Hepojoki, V.
Simon, F. Krammer, A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med. 26,
1033–1036 (2020).
76. J. Mestecky, R. G. Hamilton, C. G. M. Magnusson, R. Jefferis, J. P. Vaerman, M. Goodall, G. G. de
Lange, I. Moro, P. Aucouturier, J. Radl, C. Cambiaso, C. Silvain, J. L. Preud’homme, K. Kusama, G. M.
Carlone, J. Biewenga, K. Kobayashi, F. Skvaril, C. B. Reimer, Evaluation of monoclonal antibodies with
specificity for human IgA, IgA subclasses and allotypes and secretory component Results of an
IUIS/WHO collaborative study. J Immunol Methods. 193, 103–148 (1996).
77. GraphPad, GraphPad Prism 8 Curve Fitting Guide (2020), (available at
https://www.graphpad.com/guides/prism/8/curve-fitting/index.htm).
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 131 of 165



105 105

104 104
Spike IgG (ET)

Spike IgG
103 103

102 102
LOS
101 101
LOD
100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 105  105

104 104
RBD IgG (ET)

RBD IgG
103 103

102 102

101 101

100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 104  104
PSV Neutralizing Titer
PSV Neutralizing Titer

103 103

102 102

101 101
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 
106 106
Nucleocapsid IgG (ET)

Nucleocapsid IgG

105 105

104 104

103 103

102 102

101 101

100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 106  106

105 105
Spike IgA (ET)

Spike IgA

104 104

103 103

102 102

101 101

100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 105  105

104 104
RBD IgA (ET)

RBD IgA

103 103

102 102

101 101

100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 132 of 165

Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 133 of 165


A DMSO S M N ORF3a Nsp3

p
CD69

0.029 0.30 0.63 0 28

0. 0.072 0.053
CD137
B 20 C 20
10 10

SARS-CoV-2-specific
SARS-CoV-2-specific

CD8+ T cells (%)

CD8+ T cells (%)

1 1

0.1 0.1
0.05 0.05
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
D 20 E 20
10 10

CD8+ T cells (%)

CD8+ T cells (%)

Spike-specific
Spike-specific

1 1

0.1 0.1
0.05 0.05
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
F Ag-specific/total
c
CD8+ T cells 100 TCM

80 TEM
TCM TN
CCR7

Phenotype (%)

TEMRA
60

40

TEM TEMRA 20

CD45RA 0
0 30 60 90 120 150 180 210 240
Days post symptom onset
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 134 of 165



A DMSO S M N ORF3a Nsp3

p
OX40

0.002 0.31 0.54 0.30 0.078 0.062

CD137
B 20 C 20
10 10

SARS-CoV-2-specific
SARS-CoV-2-specific

CD4+ T cells (%)

CD4+ T cells (%)

1 1

0.1 0.1

0.02 0.02
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240

D Days PSO E Days PSO

20 20
10 10
CD4+ T cells (%)
CD4+ T cells (%)

Spike-specific
Spike-specific

1 1

0.1 0.1

0.02 0.02
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Ag-specific/total
c
F CD4+ T cells
100 TCM
80 TEM
TCM TN
Phenotype (%)
CCR7

TEMRA
60

40

TEM TEMRA 20

CD45RA 0
0 30 60 90 120 150 180 210 240
Days PSO

 
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 135 of 165




106 Male ANCOVA p = 0.00019 80
Female 1-2 Mo PSO
105
Spike IgG (ET)

3-4 Mo PSO

% of subjects
60
104 5+ Months PSO

103 40

102 20
LOS
101
LOD 0
100 0 1 2 3 4 5
0 30 60 90 120 150 180 210 240 Immune memory components
Days PSO

 
B : IgA
100
B : IgG
B : CD4

Ratio (AU)
CD4 : CD8
10 CD4 : IgG

0 30 60 90 120 150 180 210 240

Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 136 of 165
 
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 137 of 165
 

% Spike-specific memory B
104
Spike-specific memory B,
# per 106 PBMC 50

103 40

binding RBD
30
102
20
101
10

<100 0
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 

Nucleocapsid-specific memory B,
104 104
RBD-specific memory B,

# per 106 PBMC

# per 106 PBMC

103 103

102 102

101 101

<100 <100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 
RBD-specific memory B, %

RBD-specific memory B, %
1
IgA+ 1
IgM+

0.1 0.1

0.01 0.01

0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 
Nucleocapsid-specific memory B, %

Nucleocapsid-specific memory B, %

1
IgA+ 1
IgM+

0.1 0.1

0.01 0.01

0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
 
1.2
Spike
105 1.0
SpikeAF647 RBD
Geometric MFI

Geometric MFI
(Normalized)

SpikeBV421 0.8 Nucleocapsid

RBDPECy7 0.6
104 NucleocapsidBV711 0.4
NucleocapsidPE
0.2

103 0.0
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 138 of 165
 

A Lymphocytes
y p y CD14-CD19- T cells CD8+ T cells

LIVE/DEAD
SSC-B-H
46 98.6 96.6 80.4 82.3 27
SSC-A

FSC-H

CD14

CD8
FSC-A FSC-A SSC-B-A CD19 CD3 CD4

B DMSO S M N ORF3a Nsp3

Unexposed

0.017 0.005 0.0 0.016 0.006 0.020

0.050 0.31 0.31 0.18 0.083 0.061

COVID-19

0.092 0.35 0.50 0.27 0.054 0.12

CD69

0.005 0.20 0.044 0.060 0.031 0.032

CD137

C 200
SARS-CoV-2-specific

100
CD8+ T cells (SI)

10

3
0 30 60 90 120 150 180 210 240
Days post symptom onset
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 139 of 165
 

A Lymphocytes
y p y CD14-CD19- T cells CD4+ T cells

LIVE/DEAD
SSC-B-H
39
9.6
6 98.4 98.1 85.1 62.6 71.6
SSC-A

FSC-H

CD14

CD8
FSC-A FSC-A SSC-B-A CD19 CD3 CD4

B DMSO S M N ORF3a Nsp3

Unexposed

0.004 0.013 0.009 0.009 0.008 0.005

0.001 0.13 0.11 0.079 0.029 0.015

COVID-19

0.004 0.17 0.21 0.14 0.013 0.017

OX40

0.004 0.21 0.097 0.037 0.009 0.014

CD137

C 200
SARS-CoV-2-specific

100
CD4+ T cells (SI)

10

2
0 30 60 90 120 150 180 210 240
Days PSO
D E
20 20
10 10
Membrane-specific

Membrane-specific
CD4+ T cells (%)

CD4+ T cells (%)

1 1

0.1 0.1

0.02 0.02
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 140 of 165
 
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 141 of 165
 
 

106
Mild
105
Spike IgG (ET)

Moderate
104 Severe
103 Asymptomatic
102
101
100
0 30 60 90 120 150 180 210 240
Days PSO

Spike-specific memory B, %

RBD-specific memory B, %
1 1

0.1 0.1

0.01 0.01

0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO


Nucleocapsid-specific memory B, %

0.1

0.01

0 30 60 90 120 150 180 210 240

Days PSO

 
20 20
10 10
SARS-CoV-2-specific
SARS-CoV-2-specific

CD4+ T cells (%)

CD8+ T cells (%)

1
1

0.1
0.1
0.05 0.02
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 142 of 165
 

107 Male ANCOVA p = 0.020 105 ANCOVA p = 0.00087
Nucleocapsid IgG (ET)
Female
106
104

RBD IgG (ET)

105
104 103

103 102
102
101
101
100 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
 
105 ANCOVA p = 0.49 104 ANCOVA p= 0.22

PSV Neutralizing Titer

RBD IgA (ET)

104 103

103 102

102 101

101 100
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 
106
ANCOVA p = 0.19 Spike-specific memory B, % ANCOVA p = 0.50
Male
Spike IgA (ET)

1 Female
104

0.1
102

0.01
100
0 30 60 90 120 150 180 210 240
Days PSO 0 30 60 90 120 150 180 210 240
Days PSO


Nucleocapsid-specific memory B, %


RBD-specific memory B, %

Male ANCOVA p = 0.92 Male ANCOVA p = 0.66

1 Female 1 Female

0.1 0.1

0.01 0.01

0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 20  20
   Female
  
10 10 Female
SARS-CoV-2-specific

SARS-CoV-2-specific

Male Male
CD8+ T cells (%)

CD4+ T cells (%)

1
1

0.1
0.1
0.05 0.02
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 143 of 165
 
 



102 10-3

RBD-specific memory B
/ Ag-specific CD8
Ag-specific CD4

/ RBD IgG
101 10-4

100 10-5

10-1 10-6
0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240
Days PSO Days PSO

 
10-2



RBD-specific memory B

10-1
10-3

Ag-specific CD4
/ RBD IgA

10-2

/ RBD IgG
10-4
10-3
10-5
10-4
10-6
0 30 60 90 120 150 180 210 240 10-5
0 30 60 90 120 150 180 210 240
Days PSO
Days PSO




101
RBD-specific memory B

100
/ Total CD4

10-1

10-2

10-3
0 30 60 90 120 150 180 210 240
Days PSO
Case 1:21-cv-02228 Document 1-1 Filed 08/17/21 USDC Colorado Page 144 of 165

EXHIBIT 8
Casepreprint
medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
doi: https://doi.org/10.1101/2021.07.25.21261093 USDC
this version posted July 26,Colorado Page
2021. The copyright 145
holder for of
this 165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.

Protection afforded by the BNT162b2 and mRNA-1273 COVID-19

vaccines in fully vaccinated cohorts with and without prior infection

Laith J. Abu-Raddad, PhD1,2,3*, Hiam Chemaitelly, MSc1,2, Houssein H. Ayoub, PhD4, Hadi M.
Yassine, PhD5,6, Fatiha M. Benslimane, PhD5,6, Hebah A. Al Khatib, PhD5,6, Patrick Tang, MD
PhD7, Mohammad R. Hasan7, Peter Coyle, MD5,8,9, Zaina Al Kanaani, PhD8, Einas Al Kuwari,
MD8, Andrew Jeremijenko, MD8, Anvar Hassan Kaleeckal, MSc8, Ali Nizar Latif, MD8,
Riyazuddin Mohammad Shaik, MSc8, Hanan F. Abdul Rahim, PhD10, Gheyath K. Nasrallah,
PhD5,6, Mohamed Ghaith Al Kuwari, MD11, Adeel A. Butt, MBBS MS3,8, Hamad Eid Al
Romaihi, MD12, Mohamed H. Al-Thani, MD12, Abdullatif Al Khal, MD8, and Roberto Bertollini,
MD MPH12
1
Infectious Disease Epidemiology Group, Weill Cornell Medicine-Qatar, Cornell University,
Doha, Qatar
2
World Health Organization Collaborating Centre for Disease Epidemiology Analytics on
HIV/AIDS, Sexually Transmitted Infections, and Viral Hepatitis, Weill Cornell Medicine–Qatar,
Cornell University, Qatar Foundation – Education City, Doha, Qatar
3
Department of Population Health Sciences, Weill Cornell Medicine, Cornell University, New
York, New York, USA
4
Department of Mathematics, Statistics, and Physics, Qatar University, Doha, Qatar
5
Biomedical Research Center, Member of QU Health, Qatar University, Doha, Qatar
6
Department of Biomedical Science, College of Health Sciences, Member of QU Health, Qatar
University, Doha, Qatar
7
Department of Pathology, Sidra Medicine, Doha, Qatar
8
Hamad Medical Corporation, Doha, Qatar
9
Wellcome-Wolfson Institute for Experimental Medicine, Queens University, Belfast, United
Kingdom
10
College of Health Sciences, QU Health, Qatar University, Doha, Qatar
11
Primary Health Care Corporation, Doha, Qatar
12
Ministry of Public Health, Doha, Qatar

Word count: Abstract: 165 words, Main Text: 1,007 words.

Number of tables: 1.
Number of figures: 3.
Running head: Effect of prior SARS-CoV-2 infection on vaccine protection.
Keywords: SARS-CoV-2; COVID-19; prior infection; vaccine; cohort study; immunity;
epidemiology.

*
Correspondence to Professor Laith J. Abu-Raddad, E-mail: lja2002@qatar-med.cornell.edu.

1
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Abstract

Effect of prior SARS-CoV-2 infection on vaccine protection remains poorly understood. Here,

we investigated whether persons vaccinated after a prior infection have better protection against

future infection than those vaccinated without prior infection

infection. Effect of prior infection was

assessed in Qatar’s population, where the Alpha (B.1.1.7) and Beta (B.1.351) variants dominate

incidence, using two national retrospective, matched-cohort studies, one for the BNT162b2

(Pfizer-BioNTech) vaccine, and one for the mRNA-1273 (Moderna) vaccine. Incidence rates of

infection among BNT162b2-vaccinated persons, with and without prior infection, were

estimated, respectively, at 1.66 (95% CI: 1.26-2.18) and 11.02 (95% CI: 9.90-12.26) per 10,000

person-weeks. The incidence rate ratio was 0.15 (95% CI: 0.11-0.20). Analogous incidence rates

among mRNA-1273-vaccinated persons were estimated at 1.55 (95% CI: 0.86-2.80) and 1.83

(95% CI: 1.07-3.16) per 10,000 person-weeks. The incidence rate ratio was 0.85 (95% CI: 0.34-

2.05). Prior infection enhanced protection of those BNT162b2-vaccinated, but not those mRNA-

1273-vaccinated. These findings may have implications for dosing, interval between doses, and

potential need for booster vaccination.

2
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Main text

Effect of prior acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection on vaccine

protection against acquisition of infection remains poorly understood1-3. Qatar launched

Coronavirus Disease 2019 (COVID-19) immunization in December 21, 2020, first using the

BNT162b24 (Pfizer-BioNTech) vaccine and subsequently adding the mRNA-12735 (Moderna)

vaccine6,7. As vaccination was scaled up following the FDA-approved protocol, the country

experienced two back-to-back SARS-CoV-2 waves from January-June, 2021, which were

dominated by the Alpha8 (B.1.1.7) and Beta8 (B.1.351) variants6,7,9-11 (Methods). This provided

an opportunity to assess whether persons vaccinated after a prior SARS-CoV-2 infection have

better protection against future infection than those vaccinated without prior infection.

Leveraging the national, federated databases that have captured all SARS-CoV-2 vaccinations

and PCR testing since the epidemic onset (Methods), we investigated this question using two

retrospective, matched-cohort studies. We compared incidence of documented SARS-CoV-2

infection in the national cohort of individuals who completed ≥14 days after the second

BNT162b2 vaccine dose, but who had experienced a prior PCR-confirmed infection, with

incidence among individuals who completed ≥14 days after the second BNT162b2 dose, but who

had not experienced a prior infection, between December 21, 2020-June 6, 2021 (Figure 1). The

same comparison was made for the mRNA-1273 vaccine (Figure 2). Cohorts were matched in a

1:1 ratio by sex, 5-year age group, nationality, and calendar week of the first vaccine dose, to

control for differences in exposure risk12,13 and variant exposure6,7,9-11. Reporting of the study

followed the STROBE guidelines (Supplementary Table 1).

Figures 1-2 show the process for identifying infections in these cohorts, and Table 1 presents

their demographic characteristics. Using the Kaplan–Meier estimator14, cumulative infection

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incidence among BNT162b2-vaccinated persons, with and without prior infection, was estimated

at 0.14% (95% CI: 0.11-0.19%) and 0.93% (95% CI: 0.83-1.04%), respectively, after 63 days of

follow-up (Figure 1). Incidence rates of infection were estimated, respectively, at 1.66 (95% CI:

1.26-2.18) and 11.02 (95% CI: 9.90-12.26) per 10,000 person-weeks. The incidence rate ratio

was estimated at 0.15 (95% CI: 0.11-0.20).

Cumulative infection incidence among mRNA-1273-vaccinated persons, with and without prior

infection, was estimated at 0.06% (95% CI: 0.03-0.12%) and 0.08% (95% CI: 0.04-0.15%),

respectively, after 63 days of follow-up (Figure 1). Incidence rates were estimated, respectively,

at 1.55 (95% CI: 0.86-2.80) and 1.83 (95% CI: 1.07-3.16) per 10,000 person-weeks. The

incidence rate ratio was estimated at 0.85 (95% CI: 0.34-2.05).

Infection incidence was low in these cohorts during a time of intense incidence in Qatar6,7,15,

indicating that both vaccines were highly effective against the Alpha and Beta variants6,7, which

dominated incidence9 (Methods). Still, prior infection of those BNT162b2-vaccinated further

enhanced protection and reduced the incidence rate by 85% (6.6-fold) compared to those without

prior infection. No evidence for such an effect was found for those mRNA-1273-vaccinated.

These findings are perhaps explained by the observed differences in effectiveness of these two

vaccines against the Alpha and Beta variants, estimated in Qatar at 89.5% (95% CI: 85.9-92.3%)

and 75.0% (95% CI: 70.5-78.9%) for BNT162b2, respectively6, and at 100% (95% CI: 91.8-

100.0%) and 96.4% (95% CI: 91.9-98.7%) for mRNA-1273, respectively7.

The differences in effectiveness could have risen for a variety of reasons, such as differences in

dosing, interval between doses, or the biology of both vaccines and their mechanisms of action.

The dose of each of these two vaccines differed—it was 30-μg per dose for BNT162b24 and 100

μg per dose for mRNA-12735. This may have resulted in a more activated immune response for

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the mRNA-1273 vaccine than the BNT162b2 vaccine, and made the existence of prior immunity

due to natural infection of no additional benefit for the mRNA-1273 vaccine. The interval

between doses also differed and was one week longer for mRNA-12735. Evidence suggests that a

longer dose interval could be associated with improved protection after receiving the second

dose16.

Limitations include identifying prior infection based on a record of a PCR-positive result,

thereby missing those who may have been infected, but were unaware of their infection, or who

did not seek testing by PCR to document the infection. Misclassification of prior infection status

could lead to underestimation of the effect size of prior infection on vaccine protection.

Depletion of the cohorts with prior infection due to COVID-19 mortality at time of the prior

infection may have biased these cohorts toward healthier individuals with stronger immune

responses. However, COVID-19 mortality has been low in Qatar’s predominantly young and

working-age population12,17, and no evidence for such bias was found in the mRNA-1273

vaccine results, where the incidence rate was similar for those with and without prior infection.

We assessed risk of only documented infections, but other infections may have occurred and

gone undocumented, perhaps because of minimal/mild or no symptoms. Our cohorts

predominantly included working-age adults; therefore, results may not necessarily be

generalizable to other population groups, such as children or the elderly. Matching was done for

age, sex, nationality, and calendar week of the first vaccine dose, and could not be done for other

factors, such as comorbidities or additional socio-demographic factors, as these were not

available to study investigators. However, matching by age and sex may have served as a proxy

given that co-morbidities are associated with older age and may be different between women and

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men. Matching by nationality may have also captured some of the occupational risk given the

distribution of the labor force in Qatar18-20.

Imperfect assay sensitivity and specificity of PCR or antibody testing could have affected current

or prior infection ascertainment. However, all PCR and serological testing was performed with

extensively used, investigated, and validated commercial platforms with essentially 100%

sensitivity and specificity (Methods). Unlike blinded, randomized clinical trials, the investigated

observational cohorts were neither blinded nor randomized.

Our results demonstrate low infection incidence among those vaccinated with BNT162b2 or

mRNA-1273, but among those vaccinated with BNT162b2, protection against infection was

further enhanced and infection incidence was further reduced by prior infection. In contrast,

those vaccinated with mRNA-1273 were as well protected as those who received the vaccine

after a prior infection. These findings may have implications for the potential need of a booster

vaccination.

6
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Acknowledgements

We acknowledge the many dedicated individuals at Hamad Medical Corporation, the Ministry of

Public Health, the Primary Health Care Corporation, and the Qatar Biobank for their diligent

efforts and contributions to make this study possible. The authors are grateful for support from

the Biomedical Research Program, the Biostatistics, Epidemiology, and Biomathematics

Research Core, and the Genomics Core, all at Weill Cornell Medicine-Qatar, as well as for

support provided by the Ministry of Public Health and Hamad Medical Corporation. The authors

are also grateful for the Qatar Genome Programme for supporting the viral genome sequencing.

The funders of the study had no role in study design, data collection, data analysis, data

interpretation, or writing of the article. Statements made herein are solely the responsibility of

the authors.

Author contributions

LJA conceived and co-designed the study, led the statistical analyses, and co-wrote the first draft

of the article. HC co-designed the study, performed the statistical analyses, and co-wrote the first

draft of the article. All authors contributed to data collection and acquisition, database

development, discussion and interpretation of the results, and to the writing of the manuscript.

All authors have read and approved the final manuscript.

Competing interests

Dr. Butt has received institutional grant funding from Gilead Sciences unrelated to the work

presented in this paper. Otherwise, we declare no competing interests.

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References

1. Reynolds, C.J., et al. Prior SARS-CoV-2 infection rescues B and T cell responses to
variants after first vaccine dose. Science (2021).
2. Muena, N.A., et al. Long-lasting neutralizing antibody responses in SARS-CoV-2
seropositive individuals are robustly boosted by immunization with the CoronaVac and
BNT162b2 vaccines. medRxiv (2021).
3. Letizia, A.G., et al. SARS-CoV-2 seropositivity and subsequent infection risk in healthy
young adults: a prospective cohort study. Lancet Respir Med 9, 712-720 (2021).
4. Polack, F.P., et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. N
Engl J Med (2020).
5. Baden, L.R., et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. N
Engl J Med 384, 403-416 (2021).
6. Abu-Raddad, L.J., Chemaitelly, H., Butt, A.A. & National Study Group for Covid-19
Vaccination. Effectiveness of the BNT162b2 Covid-19 Vaccine against the B.1.1.7 and
B.1.351 Variants. N Engl J Med (2021).
7. Chemaitelly, H., et al. mRNA-1273 COVID-19 vaccine effectiveness against the B.1.1.7
and B.1.351 variants and severe COVID-19 disease in Qatar. Nat Med (2021).
8. World Health Organization. Tracking SARS-CoV-2 variants. Available from:
https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/. Accessed on: June 5,
2021. (2021).
9. National Project of Surveillance for Variants of Concern and Viral Genome Sequencing.
Qatar viral genome sequencing data. Data on randomly collected samples.
https://www.gisaid.org/phylodynamics/global/nextstrain/. (2021).
10. Benslimane, F.M., et al. One year of SARS-CoV-2: Genomic characterization of
COVID-19 outbreak in Qatar. medRxiv, 2021.2005.2019.21257433 (2021).
11. Hasan, M.R., et al. Real-Time SARS-CoV-2 Genotyping by High-Throughput Multiplex
PCR Reveals the Epidemiology of the Variants of Concern in Qatar. medRxiv,
2021.2007.2018.21260718 (2021).
12. Abu-Raddad, L.J., et al. Characterizing the Qatar advanced-phase SARS-CoV-2
epidemic. Scientific Reports 11, 6233 (2021).
13. Ayoub, H.H., et al. Mathematical modeling of the SARS-CoV-2 epidemic in Qatar and
its impact on the national response to COVID-19. J Glob Health 11, 05005 (2021).
14. Kaplan, E.L. & Meier, P. Nonparametric estimation from incomplete observations. The
Journal of the American Statistical Association 53, 457-481 (1958).
15. Chemaitelly, H., et al. Reinfections with the SARS-CoV-2 B.1.351 variant and efficacy
of natural immunity against reinfection under review at New England Journal of
Medicine (2021).
16. Voysey, M., et al. Single-dose administration and the influence of the timing of the
booster dose on immunogenicity and efficacy of ChAdOx1 nCoV-19 (AZD1222)
vaccine: a pooled analysis of four randomised trials. Lancet 397, 881-891 (2021).
17. Seedat, S., et al. SARS-CoV-2 infection hospitalization, severity, criticality, and fatality
rates. medRxiv 2020.11.29.20240416 (non-peer-reviewed preprint) (2020).
18. Jeremijenko, A., et al. Herd Immunity against Severe Acute Respiratory Syndrome
Coronavirus 2 Infection in 10 Communities, Qatar. Emerg Infect Dis 27, 1343-1352
(2021).

8
Casepreprint
medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
doi: https://doi.org/10.1101/2021.07.25.21261093 USDC
this version posted July 26,Colorado Page
2021. The copyright 153
holder for of
this 165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.

19. Al-Thani, M.H., et al. SARS-CoV-2 infection is at herd immunity in the majority
segment of the population of Qatar. Open Forum Infectious Diseases (2021).
20. Coyle, P.V., et al. SARS-CoV-2 seroprevalence in the urban population of Qatar: An
analysis of antibody testing on a sample of 112,941 individuals. iScience, 102646 (2021).
21. World Health Organization. COVID-19 clinical management: living guidance. Available
from: https://www.who.int/publications/i/item/WHO-2019-nCoV-clinical-2021-1.
Accessed on: May 15, 2021. (2021).
22. World Health Organization. International guidelines for certification and classification
(coding) of COVID-19 as cause of death. Available from:
https://www.who.int/classifications/icd/Guidelines_Cause_of_Death_COVID-19-
20200420-EN.pdf?ua=1. Document Number: WHO/HQ/DDI/DNA/CAT. Accessed on
May 15, 2021. (2020).
23. Planning and Statistics Authority-State of Qatar. Qatar Monthly Statistics. Available
from: https://www.psa.gov.qa/en/pages/default.aspx. Accessed on: May 26, 2020. (2020).
24. Vogels, C., Fauver, J. & Grubaugh, N. Multiplexed RT-qPCR to screen for SARS-COV-
2 B.1.1.7, B.1.351, and P.1 variants of concern V.3.
dx.doi.org/10.17504/protocols.io.br9vm966. (2021).
25. Abu-Raddad, L.J., et al. Assessment of the risk of SARS-CoV-2 reinfection in an intense
re-exposure setting. Clin Infect Dis (2020).
26. Abu-Raddad, L.J., et al. SARS-CoV-2 antibody-positivity protects against reinfection for
at least seven months with 95% efficacy. EClinicalMedicine 35, 100861 (2021).
27. Al Kuwari, H.M., et al. Epidemiological investigation of the first 5685 cases of SARS-
CoV-2 infection in Qatar, 28 February-18 April 2020. BMJ Open 10, e040428 (2020).
28. Ayoub, H.H., et al. Epidemiological impact of prioritising SARS-CoV-2 vaccination by
antibody status: mathematical modelling analyses. BMJ Innovations, bmjinnov-2021-
000677 (2021).
29. Butt, A.A., et al. Hospital admission rates, length of stay, and in-hospital mortality for
common acute care conditions in COVID-19 vs. pre-COVID-19 era. Public Health 189,
6-11 (2020).
30. Saththasivam, J., et al. COVID-19 (SARS-CoV-2) outbreak monitoring using
wastewater-based epidemiology in Qatar. Sci Total Environ 774, 145608 (2021).
31. Seedat, S., et al. SARS-CoV-2 infection hospitalization, severity, criticality, and fatality
rates. medRxiv 2020.11.29.20240416 (2020).
32. Abu-Raddad, L.J., et al. Two prolonged viremic SARS-CoV-2 infections with conserved
viral genome for two months. Infect Genet Evol 88, 104684 (2020).
33. Abu-Raddad, L.J., et al. Pfizer-BioNTech mRNA BNT162b2 Covid-19 vaccine
protection against variants of concern after one versus two doses. J Travel Med (2021).
34. Nasrallah, G.K., et al. Analytic comparison between three high-throughput commercial
SARS-CoV-2 antibody assays reveals minor discrepancies in a high-incidence
population. Sci Rep 11, 11837 (2021).
35. Thermo Fisher Scientific. TaqPath™ COVID‑19 CE‑IVD RT‑PCR Kit instructions for
use. Available from: https://assets.thermofisher.com/TFS-
Assets/LSG/manuals/MAN0019215_TaqPathCOVID-19_CE-IVD_RT-
PCR%20Kit_IFU.pdf. Accessed on December 02, 2020. (2020).

9
Casepreprint
medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
doi: https://doi.org/10.1101/2021.07.25.21261093 USDC
this version posted July 26,Colorado Page
2021. The copyright 154
holder for of
this 165
preprint
(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.

36. Kalikiri, M.K.R., et al. High-throughput extraction of SARS-CoV-2 RNA from

nasopharyngeal swabs using solid-phase reverse immobilization beads. medRxiv,
2020.2004.2008.20055731 (2020).
37. Kubina, R. & Dziedzic, A. Molecular and Serological Tests for COVID-19 a
Comparative Review of SARS-CoV-2 Coronavirus Laboratory and Point-of-Care
Diagnostics. Diagnostics (Basel) 10(2020).
38. US Food and Drug Administration. Cobas® SARS-CoV-2: Qualitative assay for use on
the cobas® 6800/8800 Systems. Avilable from:
https://www.fda.gov/media/136049/download. Accessed on: December 02, 2020. (2020).
39. Muench, P., et al. Development and Validation of the Elecsys Anti-SARS-CoV-2
Immunoassay as a Highly Specific Tool for Determining Past Exposure to SARS-CoV-2.
J Clin Microbiol 58(2020).
40. The Roche Group. Roche’s COVID-19 antibody test receives FDA Emergency Use
Authorization and is available in markets accepting the CE mark. Available from:
https://www.roche.com/media/releases/med-cor-2020-05-03.htm. Accessed on: June 5,
2020. (2020).
41. Oved, K., et al. Multi-center nationwide comparison of seven serology assays reveals a
SARS-CoV-2 non-responding seronegative subpopulation. EClinicalMedicine 29,
100651 (2020).
42. StataCorp. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC.
(2021).

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Figure 1. The process for identifying SARS-CoV-2 infections in the national cohort of individuals who completed ≥14 days
after the second BNT162b2 vaccine dose and who had experienced a PCR-confirmed infection before the first dose, compared
with the process for identifying SARS-CoV-2 infections in the national cohort of individuals who completed ≥14 days after the
second BNT162b2 vaccine dose, but who had experienced no PCR-confirmed infection before the first dose. Cohorts were
matched in a 1:1 ratio by sex, 5-year age group, nationality, and calendar week of the first vaccine dose. Total follow-up time
among BNT162b2-vaccinated persons, with and without prior infection, was 308,086.0 and 305,891.9 person-weeks,
respectively.
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Figure 2. The process for identifying SARS-CoV-2 infections in the national cohort of individuals who completed ≥14 days
after the second mRNA-1273 vaccine dose and who had experienced a PCR-confirmed infection before the first dose,
compared with the process for identifying SARS-CoV-2 infections in the national cohort of individuals who completed ≥14
days after the second mRNA-1273 vaccine dose, but who had experienced no PCR-confirmed infection before the first dose.
Cohorts were matched in a 1:1 ratio by sex, 5-year age group, nationality, and calendar week of the first vaccine dose. Total
follow-up time among mRNA-1273-vaccinated persons, with and without prior infection, was 70,729.9 and 70,872 person-
weeks, respectively.
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Table 1. Demographic characteristics of matched cohorts that received the BNT162b2 and mRNA-1273 vaccines.
Characteristics Vaccination with the BNT162b2 vaccine Vaccination with the mRNA-1273 vaccine
Individuals with a Individuals with no p-value Individuals with a Individuals with no p-value
prior PCR-confirmed prior PCR-confirmed prior PCR-confirmed prior PCR-confirmed
infection infection infection infection
Median age (IQR) — years 39 (32-48) 39 (32-48) 0.972 40 (33-47) 40 (33-47) 0.869
Age group — no. (%)
<20 years 1,573 (3.1) 1,573 (3.1) 1.000 194 (0.8) 194 (0.8) 1.000
20-29 years 7,282 (14.1) 7,282 (14.1) 3,481 (14.5) 3,481 (14.5)
30-39 years 18,027 (35.0) 18,027 (35.0) 8,216 (34.2) 8,216 (34.2)
40-49 years 13,593 (26.4) 13,593 (26.4) 7,972 (33.1) 7,972 (33.1)
50-59 years 7,468 (14.5) 7,468 (14.5) 3,368 (14.0) 3,368 (14.0)
60-69 years 2,830 (5.5) 2,830 (5.5) 704 (2.9) 704 (2.9)
70+ years 713 (1.4) 713 (1.4) 117 (0.5) 117 (0.5)
Sex
Male 36,970 (71.8) 36,970 (71.8) 1.000 18,697 (77.7) 18,697 (77.7) 1.000
Female 14,516 (28.2) 14,516 (28.2) 5,355 (22.3) 5,355 (22.3)
Nationality†
Bangladeshi 3,728 (7.2) 3,728 (7.2) 1.000 2,066 (8.6) 2,066 (8.6) 1.000
Egyptian 3,470 (6.7) 3,470 (6.7) 1,748 (7.3) 1,748 (7.3)
Filipino 4,792 (9.3) 4,792 (9.3) 2,435 (10.1) 2,435 (10.1)
Indian 13,033 (25.3) 13,033 (25.3) 8,180 (34.0) 8,180 (34.0)
Nepalese 4,570 (8.9) 4,570 (8.9) 2,730 (11.4) 2,730 (11.4)
Pakistani 1,892 (3.7) 1,892 (3.7) 1,105 (4.6) 1,105 (4.6)
Qatari 9,700 (18.8) 9,700 (18.8) 1,047 (4.4) 1,047 (4.4)
Sri Lankan 1,490 (2.9) 1,490 (2.9) 1,114 (4.6) 1,114 (4.6)
Sudanese 1,259 (2.5) 1,259 (2.5) 481 (2.0) 481 (2.0)
Other nationalities 7,552 (14.7)‡ 7,552 (14.7)‡ 3,146 (13.1) 3,146 (13.1)
*Cohorts were matched in a 1:1 ratio by sex, 5-year age group, nationality, and calendar week of first vaccine dose.
†
Nationalities were chosen to represent the most numerous groups in the population of Qatar.
‡
Individuals who received the BNT162b2 vaccine in Qatar comprised 96 other nationalities, while those who received the mRNA-1273 vaccine represented 78 other nationalities.
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Figure 3. Kaplan-Meier curves showing the cumulative incidence of documented SARS-

CoV-2 infection in the national cohort of individuals who completed ≥14 days after the
second vaccine dose and who had a prior PCR-confirmed infection, compared to the
cumulative incidence of documented SARS-CoV-2 infection in the matched national cohort
of individuals who completed ≥14 days after the second vaccine dose, but without prior
PCR-confirmed infection. The curves compare vaccination with A) the BNT162b2 (Pfizer-
BioNTech) vaccine and B) the mRNA-1273 vaccine. Cohorts were matched in a 1:1 ratio by
sex, 5-year age group, nationality, and calendar week of the first vaccine dose. The curves
for a longer time of follow up for only the BNT162b2 vaccine are in Supplementary Figure
1. Vaccination with BNT162b2 started few weeks before vaccination with mRNA-1273.

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doi: https://doi.org/10.1101/2021.07.25.21261093 USDC
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Methods

Data sources and study design

Analyses were conducted using the centralized, integrated, and standardized national severe

acute respiratory syndrome coronavirus 2 (SARS-CoV-2) databases compiled at Hamad Medical

Corporation (HMC), the main public healthcare provider and the nationally designated provider

for all Coronavirus Disease 2019 (COVID-19) healthcare needs. Through a nation-wide digital

health information platform, these databases have captured all SARS-CoV-2-related data along

with related-demographic details with no missing information since the start of the epidemic,

including all records of polymerase chain reaction (PCR) testing, antibody testing, COVID-19

hospitalizations, vaccinations, infection severity classification per World Health Organization

(WHO) guidelines21 (performed by trained medical personnel through individual chart reviews),

and COVID-19 deaths, also assessed per WHO guidelines22. Every PCR test conducted in Qatar,

regardless of location (outpatient clinic, drive-thru, or hospital, etc.), is classified on the basis of

symptoms and the reason for testing (clinical symptoms, contact tracing, random testing

campaigns (surveys), individual requests, routine healthcare testing, pre-travel, and port of

entry). Qatar has unique demographics by sex and nationality, since expatriates from over 150

countries comprise 89% of the population12,23.

The nature of circulating SARS-CoV-2 virus was informed by weekly rounds of viral genome

sequencing and multiplex, quantitative, reverse-transcription PCR (RT-qPCR) variant

screening24 of randomly collected clinical samples6,7,9-11, as well as by the results of deep

sequencing of wastewater samples9. The weekly rounds of viral genome sequencing from

January 1-May 19, 2021 identified Beta (n=623; 50.9%), Alpha (n=193; 15.8%), Delta (n=43;

3.5%), and wild-type/undetermined variants (n=366; 29.9%) in 1,225 randomly collected, PCR-

15
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medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
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positive specimens9,10. Meanwhile, the weekly rounds of multiplex RT-qPCR variant screening

from March 23-May 10, 2021 identified Beta-like (n=2,605; 66.4%), Alpha-like (n=970; 24.7%),

and “other” variants (n=349; 8.9%) in 3,924 randomly collected PCR-positive specimens9,11.

Sanger sequencing of the receptor binding domain of SARS-CoV-2 spike protein on 109 “other”

specimens confirmed that 103 were Delta-like, 3 were B.1-like, and 3 were undetermined9,11.

All records of PCR testing in Qatar were examined in this study. Every individual that met the

inclusion criteria in the national database, that is being vaccinated with BNT162b2 or mRNA-

1273 and completing ≥14 days after the second vaccine dose, for each of these cohort studies,

was classified based on infection status (with or without PCR-positive swab before the start of

the study). Individuals were matched based on infection status on a 1:1 ratio by sex, 5-year age

group, nationality (>75 nationality groups), and calendar week of first vaccine dose to control for

differences in exposure risk12,13 and variant exposure6,7,9-11. Only matched samples were included

in the analysis.

Further background on Qatar’s epidemic, such as on reinfections25,26, national seroprevalence

surveys12,18-20, PCR surveys12, and other epidemiological studies can be found in previous

publications on this epidemic6,7,12,13,27-34.

Laboratory methods

Nasopharyngeal and/or oropharyngeal swabs (Huachenyang Technology, China) were collected

for PCR testing and placed in Universal Transport Medium (UTM). Aliquots of UTM were:

extracted on a QIAsymphony platform (QIAGEN, USA) and tested with real-time reverse-

transcription PCR (RT-qPCR) using TaqPath™ COVID-19 Combo Kits (100% sensitivity and

specificity35; Thermo Fisher Scientific, USA) on an ABI 7500 FAST (ThermoFisher, USA);

extracted using a custom protocol36 on a Hamilton Microlab STAR (Hamilton, USA) and tested

16
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medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
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using AccuPower SARS-CoV-2 Real-Time RT-PCR Kits (100% sensitivity and specificity37;

Bioneer, Korea) on an ABI 7500 FAST; or loaded directly into a Roche cobas® 6800 system and

assayed with a cobas® SARS-CoV-2 Test (95% sensitivity, 100% specificity38; Roche,

Switzerland). The first assay targets the viral S, N, and ORF1ab regions. The second targets the

viral RdRp and E-gene regions, and the third targets the ORF1ab and E-gene regions.

Antibodies against SARS-CoV-2 in serological samples were detected using a Roche Elecsys®

Anti-SARS-CoV-2 assay (99.5% sensitivity39, 99.8% specificity39,40; Roche, Switzerland),

an electrochemiluminescence immunoassay that uses a recombinant protein representing the

nucleocapsid (N) antigen for antibody binding. Results were interpreted according to the

manufacturer’s instructions (reactive: optical density (proxy for antibody titer41) cutoff index

≥1.0 vs. non-reactive: optical density cutoff index <1.0).

All PCR tests were conducted at the Hamad Medical Corporation Central Laboratory or Sidra

Medicine Laboratory, following standardized protocols.

Statistical analysis

Descriptive statistics (frequency distributions and measures of central tendency) were used to

characterize study samples. Significant associations were determined using two-sided p-values.

The Kaplan–Meier estimator method14 was used to estimate the cumulative risk of documented

infection. Cumulative risk was defined as the proportion of individuals identified with an

infection during the study period among all eligible individuals in each cohort.

Incidence rates of documented infection in each cohort were calculated by dividing the number

of infection cases identified during the study by the number of person-weeks contributed by all

eligible individuals in the cohort. Incidence rates and corresponding 95% CIs were estimated

17
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using a Poisson log-likelihood regression model with the STATA 17.042 stptime command.

Follow-up person-time was calculated from the day each person completed 14 days after the

second vaccine dose up to the infection swab, all-cause death, or end-of-study censoring (June 6,

2021). The incidence rate ratio and corresponding 95% CI were calculated using the exact

method.

Statistical analyses were conducted in STATA/SE version 17.042.

Ethical approvals

The study was approved by the Hamad Medical Corporation and Weill Cornell Medicine-Qatar

Institutional Review Boards with waiver of informed consent.

Data availability

The dataset of this study is a property of the Qatar Ministry of Public Health that was provided to

the researchers through a restricted-access agreement that prevents sharing the dataset with a

third party or publicly. Future access to this dataset can be considered through a direct

application for data access to Her Excellency the Minister of Public Health

(https://www.moph.gov.qa/english/Pages/default.aspx). Aggregate data are available within the

manuscript and its Supplementary information.

18
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medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
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Supplementary Material

1
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Supplementary Table 1. STROBE checklist for cohort studies.

Main Text page
Item No Recommendation
no
Title and abstract 1 (a) Indicate the study’s design with a commonly used term in the title or the abstract 1
(b) Provide in the abstract an informative and balanced summary of what was done and 2
what was found
Introduction
Background/rationale 2 Explain the scientific background and rationale for the investigation being reported 3
Objectives 3 State specific objectives, including any prespecified hypotheses 3
Methods
Study design 4 Present key elements of study design early in the paper 3
Setting 5 Describe the setting, locations, and relevant dates, including periods of recruitment, 15-18
exposure, follow-up, and data collection
Participants 6 (a) Give the eligibility criteria, and the sources and methods of selection of participants. 15-17
Describe methods of follow-up
(b) For matched studies, give matching criteria and number of exposed and unexposed 16
Variables 7 Clearly define all outcomes, exposures, predictors, potential confounders, and effect 16
modifiers. Give diagnostic criteria, if applicable
Data sources/ 8* For each variable of interest, give sources of data and details of methods of assessment 15-16
measurement (measurement). Describe comparability of assessment methods if there is more than one
group
Bias 9 Describe any efforts to address potential sources of bias 16
Study size 10 Explain how the study size was arrived at 16 & Figures 1-2
Quantitative variables 11 Explain how quantitative variables were handled in the analyses. If applicable, describe 16-17
which groupings were chosen and why
Statistical methods 12 (a) Describe all statistical methods, including those used to control for confounding 16-17
(b) Describe any methods used to examine subgroups and interactions NA
(c) Explain how missing data were addressed NA, see p.15
(d) If applicable, explain how loss to follow-up was addressed NA
(e) Describe any sensitivity analyses NA
Results
Participants 13* (a) Report numbers of individuals at each stage of study—eg numbers potentially eligible, Figures 1-2
examined for eligibility, confirmed eligible, included in the study, completing follow-up,
and analysed
(b) Give reasons for non-participation at each stage
(c) Consider use of a flow diagram
Descriptive data 14 (a) Give characteristics of study participants (eg demographic, clinical, social) and Table 1
information on exposures and potential confounders
(b) Indicate number of participants with missing data for each variable of interest NA, see p.15
(c) Summarise follow-up time (eg, average and total amount) Table 1
Outcome data 15 Report numbers of outcome events or summary measures over time 3-4, Figure 3, and
Supplementary
Figure 1
Main results 16 (a) Give unadjusted estimates and, if applicable, confounder-adjusted estimates and their 3-4, Figure 3, and
precision (eg, 95% confidence interval). Make clear which confounders were adjusted for Supplementary
and why they were included Figure 1
(b) Report category boundaries when continuous variables were categorized 16
(c) If relevant, consider translating estimates of relative risk into absolute risk for a NA
meaningful time period
Other analyses 17 Report other analyses done—eg analyses of subgroups and interactions, and sensitivity NA
analyses
Discussion
Key results 18 Summarise key results with reference to study objectives 4-5
Limitations 19 Discuss limitations of the study, taking into account sources of potential bias or 5
imprecision. Discuss both direction and magnitude of any potential bias
Interpretation 20 Give a cautious overall interpretation of results considering objectives, limitations, 5-6
multiplicity of analyses, results from similar studies, and other relevant evidence
Generalisability 21 Discuss the generalisability (external validity) of the study results 5
Other information
Funding 22 Give the source of funding and the role of the funders for the present study and, if Acknowledgements
applicable, for the original study on which the present article is based
Abbreviations: NA: not applicable;

2
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medRxiv 1:21-cv-02228 Document 1-1 Filed; 08/17/21
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Supplementary Figure 1. Kaplan-Meier curves showing the cumulative incidence of

documented SARS-CoV-2 infection in the national cohort of individuals who completed
≥14 days after the second vaccine dose and who had a prior PCR-confirmed infection,
compared to the cumulative incidence of documented SARS-CoV-2 infection in the
matched national cohort of individuals who completed ≥14 days after the second vaccine
dose, but without prior PCR-confirmed infection. The curves compare vaccination with A)
the BNT162b2 (Pfizer-BioNTech) vaccine and B) the mRNA-1273 vaccine. Cohorts were
matched in a 1:1 ratio by sex, 5-year age group, nationality, and calendar week of the first
vaccine dose. The cumulative infection incidence among the BNT162b2-vaccinated persons,
with and without prior infection, was estimated at 0.16% (95% CI: 0.11-0.23%) and 1.45%
(95% CI: 1.20-1.76%), respectively, after 132 days of follow-up.