Antibodies are being developed as therapeutics to combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (
1). Antibodies against some other viruses can be rendered ineffective by viral mutations that are selected during treatment of infected patients (
2,
3) or that spread globally to confer resistance on entire viral clades (
4). Therefore, determining which SARS-CoV-2 mutations escape key antibodies is essential for assessing how mutations observed during viral surveillance may affect the efficacy of antibody treatments.
Most leading anti–SARS-CoV-2 antibodies target the viral receptor binding domain (RBD), which mediates binding to the angiotensin-converting enzyme 2 (ACE2) receptor (
5,
6). We recently developed a deep mutational scanning method to map how all mutations to the RBD affect its function and recognition by antiviral antibodies (
7,
8). This method involves creating libraries of RBD mutants, expressing them on the surface of yeast, and using fluorescence-activated cell sorting and deep sequencing to quantify how each mutation affects RBD folding, ACE2 affinity (measured across a titration series), and antibody binding (fig. S1A). In this study, we used the duplicate mutant libraries described in (
7), which consist of barcoded RBD variants that cover 3804 of the 3819 possible amino acid mutations. Our libraries were made in the genetic background of the RBD from the early isolate Wuhan-Hu-1, which still represents the most common RBD sequence, although several mutants are currently increasing in frequency (
9,
10). We mapped how the 2034 mutations that do not strongly disrupt RBD folding and ACE binding (
7) affected binding by recombinant forms of the two antibodies in Regeneron’s REGN-COV2 cocktail (REGN10933 and REGN10987) (
11,
12) and Eli Lilly’s LY-CoV016 antibody (also known as CB6 or JS016) (
13) (fig. S1B). REGN-COV2 was recently granted emergency use authorization for treatment of COVID-19 (
14), whereas LY-CoV016 is currently in phase 3 clinical trials (
15).
We completely mapped RBD mutations that escape binding by the three individual antibodies as well as the REGN-COV2 cocktail (
Fig. 1, A and B, and zoomable maps at
https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/). REGN10933 and REGN10987 are escaped by largely nonoverlapping sets of mutations in the RBD’s receptor binding motif (
Fig. 1A), consistent with structural work showing that these antibodies target distinct epitopes in this motif (
11). But unexpectedly, one mutation [Glu
406→Trp (E406W)] strongly escapes the cocktail of both antibodies (
Fig. 1A). The escape map for LY-CoV016 also reveals escape mutations at a number of different sites in the RBD (
Fig. 1B). Although some escape mutations impair the RBD’s ability to bind ACE2 or be expressed in properly folded form, many come at little or no cost to these functional properties, according to prior deep mutational scanning measurements using yeast-displayed RBD (
7) (color gradient in
Fig. 1, A and B, indicates loss of ACE2 affinity and in fig. S2 indicates decrease in RBD expression).
To validate the antigenic effects of key mutations, we performed neutralization assays using spike-pseudotyped lentiviral particles and found concordance between the antibody binding escape maps and neutralization assays (
Fig. 1C and fig. S3). As expected from the maps for the REGN-COV2 antibodies, a mutation at site 486 escaped neutralization only by REGN10933, whereas mutations at sites 439 and 444 escaped neutralization only by REGN10987—and so none of these mutations escaped the cocktail. But E406W escaped both individual REGN-COV2 antibodies and thus also strongly escaped the cocktail. Structural analyses and viral-escape selections led Regeneron to posit that no single amino acid mutation could escape both antibodies in the cocktail (
11,
12), but our complete maps identify E406W as a cocktail escape mutation. E406W affects the REGN-COV2 antibodies in a relatively specific way and does not grossly perturb the function of the RBD, given that it only mildly reduces neutralization by LY-CoV016 (
Fig. 1C) and the titers of spike-pseudotyped lentiviral particles (fig. S3F).
To explore whether our escape maps are consistent with how the virus evolves under antibody selection, we first examined data from Regeneron’s viral escape–selection experiments in which spike-expressing vesicular stomatitis virus (VSV) was grown in cell culture in the presence of either REGN10933, REGN10987, or the REGN-COV2 cocktail (
12). That work identified five escape mutations from REGN10933, two from REGN10987, and none from the cocktail (
Fig. 2A). All seven cell culture–selected mutations were prominent in our escape maps while also being accessible by just a single-nucleotide change to the wild-type codon in the Wuhan-Hu-1 RBD sequence (
Fig. 2B), demonstrating concordance between the escape maps and viral evolution under antibody pressure in cell culture. Notably, E406W is not accessible by a single-nucleotide change, which may explain why it was not identified by the Regeneron cocktail selections despite being relatively well tolerated for RBD folding and ACE2 affinity.
To determine whether the escape maps could inform analysis of viral evolution in infected humans, we examined deep sequencing data from a persistently infected immunocompromised patient who was treated with REGN-COV2 at day 145 after diagnosis with COVID-19 (
16). The late administration of the treatment allowed ample time for the patient’s viral population to accumulate genetic diversity, some of which could have been driven by immune pressure, because the patient mounted a weak autologous neutralizing antibody response before treatment (
16). Administration of REGN-COV2 was followed by rapid changes in the frequencies of five amino acid mutations in the RBD (
Fig. 2C and fig. S4). Our escape maps showed that three of these mutations escaped REGN10933 and that one escaped REGN10987 (
Fig. 2B). Notably, the mutations did not all sweep to fixation after antibody treatment; instead, there were competing rises and falls (
Fig. 2C). This pattern has been observed in the adaptive within-host evolution of other viruses (
17,
18) and can arise from genetic hitchhiking and competition among viral lineages. Both these forces appear to be at play in the persistently infected patient (
Fig. 2C and fig. S4C): E484A (not an escape mutation in our maps) hitchhikes with F486I (which escapes REGN10933) after treatment, and the viral lineage carrying N440D and Q493K (which escape REGN10987 and REGN10933, respectively) competes first with the REGN10933 escape-mutant Y489H and then with the lineage carrying E484A and F486I and the Q493K lineage.
Three of the four escape mutations in the REGN-COV2–treated patient were not identified in Regeneron’s viral cell culture selections (
Fig. 2B), illustrating an advantage of complete maps. Viral selections are incomplete in the sense that they only identify whatever mutations are stochastically selected in that particular cell culture experiment. In contrast, complete maps annotate all mutations, which could include mutations that arise for reasons unrelated to treatment but incidentally affect antibody binding.
Of course, viral evolution is shaped by functional constraints as well as pressure to evade antibodies. The mutations selected in cell culture and the patient consistently met the following criteria: They escaped antibody binding, were accessible via a single-nucleotide change, and imposed little or no cost on ACE2 affinity [as measured by prior deep mutational scanning using yeast-displayed RBD (
7)] (
Fig. 2D and fig. S5). Therefore, complete maps of how mutations affect key biochemical phenotypes of the RBD (e.g., ACE and antibody binding) can be used to assess likely paths of viral evolution. One caveat is that over longer evolutionary time frames, the space of tolerated mutations could shift as a result of epistatic interactions, as has been observed in viral immune and drug escape (
19–
21).
The complete maps enable us to assess what escape mutations are already present among circulating SARS-CoV-2. We examined all human-derived SARS-CoV-2 sequences available as of 11 January 2021 and found a substantial number of RBD mutations that escaped one or more of the antibodies (
Fig. 3). However, the only escape mutations present in >0.1% of sequences were the REGN10933 escape-mutant Y453F [0.3% of sequences; see also (
12)], the REGN10987 escape-mutant N439K [1.7% of sequences; see also
Fig. 1C and (
22)], and the LY-CoV016 escape-mutant K417N (0.1% of sequences; see also
Fig. 1C). Y453F is associated with independent outbreaks linked to mink farms in the Netherlands and Denmark (
23,
24); notably, the mink sequences themselves sometimes also contain other escape mutations, such as F486L (
24). N439K is prevalent in Europe, where it has constituted a large percentage of sequences from regions including Scotland and Ireland (
22,
25). K417N is present in the B.1.351 lineage first identified in South Africa (
10). Another mutation of current interest is N501Y, which is present in B.1.351 and also the B.1.1.7 lineage originally identified in the United Kingdom (
9). Our maps indicate that N501Y has no effect on either of the REGN-COV2 antibodies and has only a modest effect on LY-CoV016 (
Fig. 3).
To determine whether the escape maps could be rationalized from the structural interfaces of the antibodies and RBD, we projected the maps onto crystal or cryo–electron microscopy structures (
Fig. 4A; interactive versions at
https://jbloomlab.github.io/SARS-CoV-2-RBD_MAP_clinical_Abs/). As might be expected, escape mutations generally occur in the antibody-RBD interface. However, structures alone are insufficient to predict which mutations mediate escape. For example, LY-CoV016 uses both its heavy and light chains to bind a wide epitope overlapping the ACE2-binding surface, but escape is dominated by mutations at RBD residues that contact the heavy-chain complementarity-determining regions (
Fig. 4A and fig. S6, E to G). In contrast, escape from REGN10933 and REGN10987 mostly occurs at RBD residues that pack at the antibody heavy- and light-chain interface (
Fig. 4A and fig. S6, A to D). The E406W mutation that escapes the REGN-COV2 cocktail occurs at a residue not in contact with either antibody (
Fig. 4, A and B). Although E406 is in closer structural proximity to LY-CoV016 (
Fig. 4B and fig. S6H), the E406W mutation has a much smaller impact on this antibody (
Fig. 1, B and C), suggesting a long-range structural mechanism specific to the REGN-COV2 antibodies (fig. S6I). Taken together, mutations at RBD residues that contact antibody do not always mediate escape, and several prominent escape mutations occur at residues not in contact with antibody (
Fig. 4B and fig. S6, D and G).
In this study, we have completely mapped mutations that escape three leading anti–SARS-CoV-2 antibodies. These maps demonstrate that prior characterization of escape mutations was incomplete, having identified neither a single amino acid mutation that escapes both antibodies in the REGN-COV2 cocktail nor most mutations that arose in a persistently infected patient treated with the cocktail. Of course, our maps still do not answer the most pressing question: Will SARS-CoV-2 evolve widespread resistance to these antibodies? But certainly, it is concerning that so many escape mutations impose little cost on RBD folding or receptor affinity and that some are already present at low levels among circulating viruses. Ultimately, it will be necessary to wait and see what mutations spread as SARS-CoV-2 circulates in the human population. Our work will help with the “seeing,” by enabling immediate interpretation of the effects of the mutations cataloged by viral genomic surveillance.
Acknowledgments
We thank K. Crawford for help with neutralization assays; A. Feder, D. Veesler, N. King, and D. Ellis for helpful comments; and the Fred Hutch Flow Cytometry and Genomics facilities for assistance.
Funding: This work was supported by the NIAID (R01AI127893 and R01AI141707 to J.D.B. and T32AI083203 to A.J.G.), the Gates Foundation (INV-004949 to J.D.B.), and the Massachusetts Consortium for Pathogen Readiness through grants from the Evergrande Fund (to J.Z.L.). Scientific computing at Fred Hutch is supported by ORIP grant S10OD028685. T.N.S. is a Washington Research Foundation Innovation Fellow at the University of Washington Institute for Protein Design and an HHMI Fellow of the Damon Runyon Cancer Research Foundation (DRG-2381-19). J.D.B. is an investigator of the Howard Hughes Medical Institute.
Author contributions: T.N.S., A.J.G., A.S.D., and J.D.B. designed the study. T.N.S., A.J.G., A.A., and A.S.D. performed the experiments. T.N.S., A.J.G., and J.D.B. analyzed the experimental data. J.Z.L. and M.C.C. sequenced the persistent infection, and W.W.H. analyzed that data. T.N.S., A.A., W.W.H., and J.D.B. wrote the initial draft, and all authors edited the final version.
Competing interests: J.Z.L. has consulted for Abbvie and Jan Biotech. The other authors declare no competing interests.
Data and materials availability: Raw sequencing data are on the NCBI SRA under BioProject PRJNA639956/BioSample SAMN16850904 (escape mapping) and BioProject PRJNA681234 (patient sequencing). Computer code and processed data for the escape mapping are at (
27) and
https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_clinical_Abs. Code and data for the patient sequencing are at (
28) and
https://github.com/jbloomlab/SARS-CoV-2_chronic-infection-seq. The sequences of the antibodies are provided via citations in the materials and methods, the cells and plasmids for the neutralization assays are available in BEI Resources and AddGene (see materials and methods for details), and the yeast mutant libraries are available to academic researchers upon request with a completed material transfer agreement. We gratefully acknowledge the authors from the originating laboratories and the submitting laboratories, who generated and shared via GISAID genetic sequence data on which this research is based (all contributors listed at
https://github.com/jbloomlab/SARS-CoV-2-RBD_MAP_clinical_Abs/blob/main/data/gisaid_hcov-19_acknowledgement_table_2021_01_11.pdf). This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit
https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.
RE: Viral Mutants Escaping Antibodies for Treating COVID-19
An impressive team of medical experts report on the prospective mapping of viral mutations that escape antibodies for treating COVID-19.
Antibodies can be therapeutic for the SARS-CoV-2 virus that causes COVID-19, but the virus can escape and evolve into mutated strains and variants.
Mapping the movements and trajectories of the original wild-type SARS-CoV-2 and the mutated strains can be used to identify the escaped mutants, which may be present in the mutated strains that are presently in circulation, such as those from the UK, South Africa, Brazil, and the USA, predominantly in California.
In addition to aiding interpretation and analysis, complete escape maps might also be able to predict how the mutated strains evolve, whether the range of approved vaccines that are presently being distributed globally will be safe, effective, and durable against the mutated strains, and whether the virus and its mutated variants evolve to develop widespread resistance to the antibodies.
Examining antibodies to map the original virus and its mutated variants may provide a range of options for medical researchers and healthcare professionals in anticipating the range of PPE that might be necessary to treat patients infected with possibly unknown mutations.
Such informative mapping would also be useful for vaccine producers to incorporate the knowledge regarding mutant strains in developing safes, and more effective and durable vaccines.
Effect of RBD (Y453F) in spike glycoprotein of SARS-CoV-2 variant on COVID-19 vaccine
Mustelidae animals such as mink and ferrets can be infected with SARS-CoV-2 relatively easily compared to other mammals. It is not clear why SARS-CoV-2 is so contagious to these animals (1). It is clear that when SARS-CoV-2 is infected with farmed minks that is kept in large quantities in a high density environment, SARS-CoV-2 proliferates in large quantities in the mink body. As a result, humans may pose a high risk of infection with SARS-CoV-2 as well as farmed minks.
Natural selection "adaptation" in the coronavirus can occur during coronavirus amplification in vivo in farmed mink (2). Natural selection in such viruses is observed by the appearance of mutations in SARS-CoV-2 that are not observed during the growth process in humans (2,3). Infection with a RBD mutant (Y453F) of SARS-CoV-2 from farmed mink is widespread among people (4).
Recent studies have revealed the following matters (5).
The Y453F mutation does not affect the three-dimensional structure of conventional SARS-CoV-2 spike glycoproteins. It was clarified that the binding property of RBD Y453F mutant in the spike glycoprotein to human ACE2 was slightly weaker than that of the conventional SARS-CoV-2 spike glycoprotein. It was revealed that the affinity between RBD Y453F mutant in the spike glycoprotein and 4 of the 6 neutralizing antibodies examined was clearly weak compared to the conventional SARS-CoV-2 spike glycoprotein.
Therefore, we investigated the affinity of IgG in the serum of COVID-19-positive patients and IgG in the serum of healthy subjects for RBD or RBD Y453F mutant (5). The affinity of IgG in the serum of 21 COVID-19-positive patients and IgG in the serum of 10 COVID-19 negative subjects with RBD or RBD Y453F mutant was examined by Enzyme-Linked Immuno Sorbent Assay (5). As a result, a strong affinity for RBD was shown in IgG in the serum of 14 of the 21 COVID-19-positive patients (5). However, no affinity for RBD Y453F mutant was shown in the serum IgG of 19 of the 21 COVID-19-positive patients (5). Affinity for both RBD and RBD Y453F mutant was shown in IgG in the serum of 2 of the 21 COVID-19-positive patients. No affinity for RBD or RBD Y453F mutant was shown in IgG in the sera of all 10 COVID-19 negative subjects (5). From these results, the mutation of tyrosine at amino acid residue 453 of RBD to phenylalanine eliminated the inhibitory effect of the neutralizing antibody on binding between ACE2 and RBD.
It is considered that the affinity between the appropriate amino acid residues in the variable region of the antibody and the spike glycoprotein RBD F453 has diminished as cause of weak recognition of monoclonal antibodies for spike glycoproteins. Subspecies of SARS-CoV-2 derived from farmed mink has been observed example of infected peoples, the virus mutants are inherited by infected individuals.
Since the mutations have occurred in the SARS-CoV-2, subspecies of SARS-CoV-2, which are susceptible to human and animal infection, easily propagate in the host. The spread of SARS-CoV-2 variants mediated by millions of farmed minks is uncontrolled. As a result, there is concern that SARS-CoV-2 mutants, which cause serious symptoms in humans, may be spread all over the world.
Disclosure
The authors declare no potential conflicts of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments
We thank all the medical staffs and co-medical staffs for providing and helping medical research at National Hospital Organization Kyoto Medical Center.
Reference
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