INTRODUCTION
Antibodies play an important role in neutralizing virus and provide protection to the host against viral re-infection. The antibody response to SARS-CoV-2 infection has been extensively studied in the blood (serum, plasma) of COVID-19 patients in order to gain insights into the host immune response. Antibody levels to the spike protein are particularly important since this large trimeric glycoprotein harbors the receptor-binding domain (RBD). The RBD facilitates SARS-CoV-2 access to human cells by binding to its counter receptor angiotensin-converting enzyme 2 (ACE-2) (
1), and neutralizing antibodies have been shown to target the RBD (
2). Most studies agree that the IgG antibodies to SARS-CoV-2 spike and RBD antigens are detected in the blood of greater than 90% of subjects by 10–11 days post-symptom onset (PSO) (
3–
7). However, whether levels of IgG specific for SARS-CoV-2 antigen persist (
8–
13), or alternatively decay (
14), remains a debated issue. Examination of different biofluids from multiple cohorts, and attention to the antigens tested, is required to resolve this extremely important issue that has high relevance to vaccine design.
Another gap in our knowledge is that we know very little about the local antibody response at the site of infection. SARS-CoV-2 enters the naso- and oro-pharyngeal tracts where it subsequently replicates (
15). For this reason, nasopharyngeal and throat swabs are used to test for virus using reverse transcriptase quantitative PCR (RT-qPCR) to detect viral RNA. However, saliva has also been shown to be an effective biofluid for testing for the presence of SARS-CoV-2 mRNA (
16–
19). This makes sense given that pharyngeal SARS-CoV-2 shedding precedes viral replication in the lungs (
15), and, like cytomegalovirus (
20,
21), the salivary glands themselves can be a reservoir for the virus (
22). Yet in spite of the oral cavity being a site for viral replication, few studies have examined anti-SARS-CoV-2 antibodies in this compartment.
In this study, we examined the anti-SARS-CoV-2 antibody response over a 115-day period in the serum and saliva from n=439 (serum) and n=128 (saliva) patients with COVID-19, compared to controls. Antigen-specific IgG in both biofluids was maximally detected by 16–30 days PSO and did not drastically decline in relative level as late as 105-115 days PSO. In contrast, antigen-specific IgM and IgA were rapidly induced but subsequently declined in both serum and saliva. In serum, neutralizing antibodies reached their maximum by 31–45 days PSO and slowly declined up to 105 days, with a more pronounced drop in the last blood draw (105–115 days PSO) Importantly, IgG and IgM levels against both antigens were strongly correlated across paired serum and saliva samples (n=71), indicating that saliva can be used for monitoring the immune response to SARS-CoV-2 infection. Taken together, the systemic and mucosal IgG response to SARS-CoV-2 is sustained over a 3-month period, while the IgM and IgA response occurs early and is transient.
DISCUSSION
Antibodies are key components in the arsenal of protective immunity against novel viral infections such as SARS-CoV-2. Understanding their durability and their system compartmentalization across a diverse population are critical pieces of data informing our ability to monitor seroprevalence in communities, to select plasma donors for treatment, and to design vaccines against COVID-19. We examined the stability of antibody levels over the first three months after infection in both the serum and the saliva. We observed no drastic decline in levels of anti-spike, anti-RBD or anti-NP IgG levels over a 3-month period. The same was true for the antigen-specific measurements in saliva (anti-spike and anti-RBD IgG). On the other hand, similar to other findings (
28,
29), IgA and IgM responses to SARS-CoV-2 antigens were found to decline in both serum and saliva. In summary, our data show that a durable IgG response against SARS-CoV-2 antigens is generated in both the saliva and serum in most patients with COVID-19. Of the three isotypes measured, the IgA response correlates the least between serum and saliva, particularly for the RBD antigen. This may suggest some compartmentalization of the IgA response in the oral cavity versus the periphery.
Given the presence of SARS-CoV-2 RNA in saliva, it is reasonable to hypothesize that, like other viruses such as rubella (
26), 229E alpha-coronavirus (
30), and MERS beta-coronavirus (
31), the mucosae and draining lymph nodes of the oro- and nasopharyngeal tracts serve as a site for initiation of an immune response to SARS-CoV-2. If so, then plasma cells (PC) that produce antibodies to SARS-CoV-2 will migrate back to the oro- and nasopharyngeal mucosae and produce antibodies that should be detectable in the saliva, a fluid that already has high levels of IgA (
32). With time, this response will be detected in the systemic circulation, possibly due to migration of PC into new niches as we have previously described in mice (
33). Indeed, we and two other groups have observed SARS-CoV-2 specific antibodies in saliva (
34,
35). There are some variations between study protocols that are important to consider: Randad
et al. applied a brush on the gum line as a means to capture IgG from the blood, heat inactivated this material, and performed multiplex antibody immunoassays using Luminex technology to detect antigen-specific antibody levels (
35). In contrast, our strategy was to collect saliva in a manner that best approximates the immune response that takes place in the local mucosa. In this way, our study more resembles that of Faustini
et al., who used ELISA technology on whole saliva, amplifying the signal with an additional antibody step (
34). Although Faustini
et al. employed saliva dilutions in the same range as what we used (1:5 to 1:20), the degree of correlation between the serum and saliva for each antibody/antigen ELISA pair was less obvious in that study than in ours (
34). Whether these discrepancies are methodological (i.e., detection of specific versus total Igs) and/or relate to the higher number of asymptomatic subjects in the Faustini
et al. study remains to be determined.
While the specificity of the saliva assays was very good for anti-spike and anti-RBD IgG responses based on ROC curves, this was less true for IgA, particularly the anti-RBD IgA response. This is because some of our negative controls, irrespective of whether they were collected during the pandemic (unexposed negatives) or prior to the pandemic, exhibit levels of anti-RBD IgA that approach 50% of the pooled control saliva (see
Fig. 4H). It is unclear why this would occur for only the IgA/RBD combination. Presumably these are cross-reactive IgA that bind to SARS-CoV-2 RBD. Of interest, thus far SARS-CoV-2 neutralizing antibodies appear to have limited somatic hypermutation (
36,
37), suggesting that they may originate from a naïve repertoire or from B cells that have been activated in extrafollicular responses where somatic hypermutation is limited. It is tempting to speculate that these pre-existing IgA antibodies may provide some stop-gap protection against SARS-CoV-2 in the oral cavity, and if so, it is essential to ascertain their original antigenic specificity. Future work is required to confirm these results in a greater array of subjects and using different sources of RBD antigen.
Our findings that the IgG response to SARS-CoV-2 antigens is stable over a 3-month period are consistent with other studies who likewise noted durability in the IgG response to the spike trimer (
8–
13). These data and ours contrast with those of Long
et al., who showed rapid decay of antibody levels when profiling the response to a linear peptide motif of the C-terminal part of the spike protein (
14) instead of the spike trimer used here, and it is possible that the antigen selection accounts for some of the differences. However, this does not explain discrepant results with respect to the anti-NP response in the serum, which we find also largely persisted over the 3-month period. One potential difference that could explain these divergent results is that we employed a sensitive and robust chemiluminescence plate-based ELISA whereas Long
et al. employed magnetic chemiluminescence enzyme immunoassay kits with immobilized recombinant or peptide antigens.
A limitation of our study is that we have not looked beyond the day 115 PSO – our collections began in mid-March 2020 – and it is entirely plausible that antigen-specific IgG levels will eventually wane with time. Although IgG antibodies to spike remained fairly stable, even at day 115 PSO, our surrogate neutralization assay revealed a dip in activity in the last time interval bin (days 116 - 115 PSO), consistent with some previous studies (
9,
13,
14). This final collection interval is not as well powered as the other bins, thus this requires further investigation. Nevertheless, a dip in neutralization activity using the surrogate assay does mirror the significant reduction in antigen-specific IgA (and IgM). The contributions of these isotypes to the overall neutralization activity at different time points after infection remains to be assessed. Indeed, IgA is an important mediator of protection against gastrointestinal viruses (
38), is essential in achieving immunity against avian viruses (
39), has been shown to contribute to the neutralizing antibody (nAb) response to SARS-CoV-2 (
28), and may even be a more potent nAb isotype than IgG (
40). In addition, a monoclonal antibody cloned from B cells derived from SARS-CoV-infected humanized mice was found to provide cross-reactive neutralizing activity to SARS-CoV-2 when engineered on the IgA backbone, and this neutralizing activity was further enhanced if the IgA was co-expressed with J chain to produce dimeric IgA and secretory component to produce secretory IgA – the form of IgA that is secreted at mucosal surfaces (
41). Although Sterlin
et al. show that the initial IgA plasmablast response quickly declines, IgA-producing plasma cells have been shown to persist for decades in the gut mucosae of humans (
42), and these will not be readily measurable in the blood. Indeed, we found that of all 3 isotypes measured, antigen-specific IgA levels in the saliva exhibited the poorest correlation with antigen-specific IgA levels in the serum. When combined with the parallel formation of re-activatable memory B cells (
43), many of which will be tissue-resident (
25), the host has excellent mechanisms for mounting swift and robust humoral immunity upon pathogen re-exposure that may be missed using blood-based measurements. An epidemiological study that prospectively follows confirmed COVID-19 cases for several months will determine if these immunological principles hold true in the context of SARS-CoV-2 infection.
In conclusion, our study provides evidence that the IgG response to SARS-CoV-2 spike persists in the saliva and the serum, and that this response can be correlated between the two biofluids, particularly for IgG. Given that the virus can also be measured in saliva by PCR (
16–
19), using saliva as a biofluid for both virus and antibody measurements may have some diagnostic value. Since SARS-CoV-2 initially replicates in the oro- and nasopharyngeal tracts, in the future it will be critical to characterize the nature and kinetics of salivary antibodies at the earliest time points post-infection in contact-traced individuals in order to determine if there are correlates of protection that impact viral setpoint and COVID-19 disease progression.
MATERIALS AND METHODS
Study Design
This observational study focused on monitoring the levels of antibodies to SARS-CoV-2 antigens in serum and saliva of patients with confirmed SARS-CoV-2 infection. At the onset of the study, we set to determine: 1) what are the kinetics of antibody production and decline in saliva and serum specimens from patients with COVID-19 during the first 3+ months of infection; 2) whether these levels are affected by disease severity, sex, or age; 3) whether saliva can be used as an alternative biofluid for monitoring the immune response in patients with COVID-19. Assay development was performed for each individual ELISA by assessing the classification of positives and negative samples (see definition below for serum and saliva assays) at each of the observed colorimetric or chemiluminescent values, and setting a threshold (1% for serum and 2% for saliva, respectively) for definition of positives. Irrespective of this positive/negative definition, all values are reported. The protein-based surrogate neutralization ELISA (snELISA) development and benchmarking against viral neutralization assay was described previously (
24). Samples for profiling were recruited through the Toronto Invasive Bacterial Diseases Network in metropolitan Toronto; all samples for which a PCR positive result and for which the biofluid (serum or saliva) was available were included. Data was analyzed without exclusion of outliers to avoid biasing the study. For the saliva and snELISA assays, each sample was analyzed once, through a multipoint dilution curve; for the serum-based ELISA, a single-point ELISA was performed in duplicates, and the results averaged. No randomization was performed, since this is an observational study.
Recruitment and participants – COVID19 patients
Acute and convalescent serum and saliva samples were obtained from patients identified by surveillance of COVID-19 (confirmed by PCR; in- and out-patients) by the Toronto Invasive Bacterial Diseases Network in metropolitan Toronto and the regional municipality of Peel in south-central Ontario, Canada (REB studies #20-044 Unity Health Network, #02-0118-U/05-0016-C, Mount Sinai Hospital). Consecutive consenting patients admitted to four TIBDN hospitals were enrolled: these patients had serum and saliva collected at hospital admission, and survivors were asked to submit repeat samples at 4-12 weeks PSO. Consecutive out-patients diagnosed at the same 4 hospitals prior to March 15th and on a convenience sample of later days were approached for consent to collect serum and saliva at 4-12 weeks PSO. Patients were interviewed and patient charts reviewed to determine age, sex, symptom onset date, and disease severity (mild, moderate, and severe). For this study, disease was considered mild if it did not require hospitalization, moderate if it required hospitalization but not intensive care unit (ICU) admission, and severe if it required ICU care. Specimens were considered acute if they were collected less than 21 days PSO, and convalescent if they were collected 21 or more days PSO. From March 10-April 14, patients were asked to provide a 5 ml sample of saliva in a sterile specimen container, and 2.5 mls of phosphate buffered saline was added to reduce viscosity for PCR testing. From April 16th on, saliva specimens were collected in Salivette® tubes (Sarstedt, Numbrecht, Germany). All specimens were aliquoted and stored frozen at -80°C prior to analysis.
Additional positive samples for test development were obtained through the Canadian Blood Services. Specimen-only serum donations were collected from individuals with a self-declared SARS-CoV-2-positive nucleic acid test. Collections occurred two weeks or more after cessation of clinical symptoms.
Recruitment and participants – control saliva and serum
Control saliva samples were collected from unexposed, asymptomatic individuals residing in an area of very low COVID-19 case numbers (Grey County, Ontario) and throughout the Greater Toronto Area (GTA) (REB study# 23901 University of Toronto).
Control serum samples were from patients enrolled in cancer or birth cohort studies prior to COVID-19 (prior to November 2019; REB studies #01-0138-U and #01-0347-U, Mount Sinai Hospital) and archived frozen in the LTRI Biobank, or from previous studies of the immune system or systemic lupus acquired prior to November 2019 (REB studies #31593 University of Toronto, #05-0869, University Health Network).
Study Approval
All samples were collected after Research Ethics Board (REB) review (see Sample section above for the individual REB approval numbers). The serum ELISA assays were performed at the Lunenfeld-Tanenbaum Research Institute with Mount Sinai Hospital (MSH; Toronto, ON) Research Ethics Board (REB) approval (study number: 20-0078-E). External samples were transferred through Material Transfer Agreements as appropriate. All research has been performed in accordance with relevant guidelines and regulations. All participants have provided informed consent. The samples were de-identified prior to transfer to the assay laboratory.
Sample collection, handling and viral inactivation – serum
Serum (and in some cases plasma) was collected using standard procedures at the collection sites and transferred to the testing lab on dry ice. Inactivation of potential infectious viruses in plasma or serum was performed by incubation with Triton X-100 to a final concentration of 1% for 1 hour prior to use (
44).
Antigen production – serum assays
Spike trimer was expressed as follows: the SARS-CoV-2 spike sequence (aa 1-1208 from GenBank accession number MN908947 with the S1/S2 furin site (residues 682–685) mutated [RRAR->GGAS] and K986P / V987P stabilizing mutations was codon-optimized (
Cricetulus griseus codon bias) and synthesized by GenScript. To stabilize the spike protein in a trimeric form, the cDNA was cloned in-frame with the human resistin cDNA (aa 23-108) containing a C-terminal FLAG-(His)
6 tag (
Cricetulus griseus codon bias, GenScript) into a modified cumate-inducible pTT241 expression plasmid and transfected in CHO
2353 cells followed by methionine sulfoximine selection for 14 days to generate a stable CHO pool. This CHO pool allows for cumate-inducible trimeric spike expression from the CR5 promoter as described in Poulain
et al. (
45–
47). Cell culture was harvested 8-10 days post-cumate induction and secreted spike trimer present in the clarified medium purified by immobilized metal-affinity chromatography (Ni-Excel resin; Cytiva). Purified trimeric spike was buffer exchanged in PBS and store as aliquots at -80°C. The purified spike protein integrity and purity was analyzed by SDS-PAGE and analytical size-exclusion ultra-high performance liquid chromatography (SEC-UPLC). The SEC-UPLC was run in PBS + 0.02% Tween-20 on an 4.6 × 300 mm Acquity BEH450 column (2.5 μm beads size; Waters Limited, Mississauga, ON) coupled to a MALS detector (miniDAWN
TM) and an Optilab® T-rEX
TM refractometer (both from Wyatt Technology) and the spike trimer eluted as a major (>95% integrated area) symmetrical peak of 490 kDa with less than 3% aggregates (not shown). RBD was expressed as for the saliva assay, but left non-biotinylated, as in (
24).
Nucleocapsid (aa 1-419 from the pEntry-N (closed) Open Reading Frame (a kind gift from Dr. Frederick P. Roth (
48)) was cloned into pDEST585 gift of Jim Hartley, internal ID V2097) as a HIS-GST-TEV fusion using LR-clonase. The resulting expression vector was confirmed by restriction digest, expressed in
E. coli BL21(DE3) Codon+ cells (Agilent Technologies) and induced with 0.25 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) for 16 hours at 18°C. Harvested cells were resuspended in 20 mM HEPES pH 7.5, 400 mM NaCl, 5 mM imidazole and lysed by passage through a cell homogenizer (Avestin Inc.). Following centrifugation at 30,000 g, supernatant was passed through a 0.45 μM PVDF filter and applied to a HiTrap nickel chelating HP column (GE Healthcare). Protein eluted with buffer containing 300 mM imidazole was incubated overnight with Tobacco Etch Virus (TEV) protease. Following cleavage of the His-Tag, protein was dialyzed in 20 mM HEPES pH 7.5, 50 mM NaCl and flowed over a 5 ml HiTrap nickel chelating column to remove His-GST. Nucleocapsid protein was further purified by ion exchange on a mono-S column (GE Healthcare) equilibrated in 20 mM HEPES pH 7.5, 50 mM NaCl, 1 mM DTT and eluted with a gradient to 500 mM NaCl. Purified Nucleocapsid protein was concentrated to 6 mg/mL and stored at -80°C.
Enzyme-linked immunosorbent assays for detecting antigen-specific IgG and IgA in serum or plasma
A manual colorimetric ELISA assay (similar to (
3)) was first implemented in 96-well plates using the RBD and spike non-biotinylated antigens described here for the detection of IgG (also see (
24)). Briefly, concentrations and incubation times were optimized to maximize the separation between anti-RBD or anti-spike trimer levels in convalescent plasma or serum from that of pre-COVID era banked serum while maintaining the required levels of antigens as low as possible. 75 ng and 200 ng of RBD and spike, respectively, were first adsorbed onto 96-well clear Immulon 4 HBX (Thermo Scientific, #3855) plates in PBS overnight at 4°C, then washed three times with 200 μl PBS+ 0.1% Tween-20 (PBS-T; Sigma). Plates were blocked with 3% w/v milk powder (BioShop Canada Inc., #ALB005.250, lot #9H61718) in PBS for 1–2 hours and washed three times with 200 μl PBS-T. Patient samples (pre-treated with 1% final Triton X-100 for viral inactivation) diluted 1:50 in PBS-T containing 1% w/v milk powder were then added to the plates and incubated for 2 hours at room temperature (50 μl total volume): technical duplicates were performed unless otherwise indicated. Positive and negative control recombinant antibodies and serum samples were added to each plate to enable cross-plate comparisons. Wells were washed three times with 200 μl PBS-T. Goat anti-human anti-IgG (Goat anti-human IgG Fcγ -HRP, Jackson ImmunoResearch, #109-035-098) at a 1:60,000 dilution (0.67 ng/well) in 1% w/v milk powder in PBS-T was added and incubated for 1 hour. Wells were washed three times with 200 μl PBS-T, and 50 μl of 1-StepUltra TMB-ELISA Substrate Solution (ThermoFisher, #34029) was added for 15 min at room temperature and the reaction was quenched with 50 μL stop solution containing 0.16N sulfuric acid (ThermoFisher, #N600). The plates were read in a spectrophotometer (BioTek Instruments Inc., Cytation 3) at 450 nm. For all ELISA-based assays, raw OD or luminescence values had blank values subtracted prior to analysis. All data were normalized to the positive serum control pool (single point) on each plate and expressed as a ratio to this control (ratio-converted ELISA reads). The assay performance was assessed by precision-recall analysis of ratio-expressed values (Figure S1, S3).
The assay was then re-designed to be conducted in a customized robotic platform using a 384-well plate format, first by simply scaling down the volume/amounts used, and then switching to a chemiluminescent substrate for detection, and re-optimizing the amounts per well of antigens and secondary antibodies’ dilutions to use. A chemiluminescent substrate is ideally-suited for automated ELISAs, because it offers a higher sensitivity and a better dynamic range than standard colorimetric assays. Furthermore, the reaction does not need to be stopped (e.g., with robotics-incompatible acids) and the luminescence signal is stable for at least 60 min. For all steps, liquid dispensers (Beckman Biomek NXp or ThermoFisher Multidrop Combi) and washer (Biotek 405 TS/LS LHC2) were used on a F7 robotic platform available at the Network Biology Collaborative Centre (
nbcc.lunenfeld.ca). Each step of the methods to evaluate the different antigen and antibody class combinations was optimized and routine quality control tests were performed for all dispensing steps.
For automated ELISAs, LUMITRAC 600 high-binding white polystyrene 384-well microplates (Greiner Bio-One, through VWR #82051-268) were pre-coated overnight with 10 μl/well of RBD (25 ng) or spike (50 ng). The next day, the wells were washed 4 times (a BioTek washer is used for all washing steps, and all washes are performed with 100 μl PBST). Wells were blocked for 1 hour at room temperature in 80 μl 5% BlockerTM BLOTTO (Thermo Scientific, #37530), then washed 4 times. 10 μl Triton X-100 inactivated serum (or plasma) samples diluted 1:40 in 1% BLOTTO in PBS-T were added to each well from 96-well sample source plates and incubated for 2 hours at room temperature. Positive and negative controls used on each plate are described below. After washing 4 times, 10 μl of one of the following secondary antibodies (all from Jackson ImmunoResearch) diluted in 1% BLOTTO in PBS-T were added at the indicated concentrations followed by incubation for 2 hours at room temperature: Goat anti-human IgG Fcγ – HRP (#109-035-098; 1:40,000 or 0.2 ng per well), Goat anti-human IgM Fcμ – HRP (#109-035-129; 1:12,000 or 0.66 ng per well) or Goat anti-human IgA α chain - HRP (#109-035-127; 1:10,000 or 0.8 ng per well). After 4 washes, 10 μl of SuperSignal ELISA pico Chemiluminescent substrate (diluted 1:4 in water) was added, followed by a short mix for 10s at 900 rpm, and incubation at room temperature for 5 min. Luminescence was read on an EnVision (Perkin Elmer) plate reader at 100 ms/well using an ultra-sensitive luminescence detector. All automated assays were performed in technical duplicates, processed on different days. Blank values were subtracted for all raw reads prior to data analysis, and the values were expressed as a ratio of the positive reference serum pool on the same plate (see below).
Quality controls and normalization of the samples in the automated assays were as follows: a standard curve with recombinant antibodies reacting to spike RBD or spike S1 was included on each plate. Antibodies used for the standard curves were: Human anti-spike S1 IgG (A02038, GenScript), anti-spike S1 IgM (A02046, GenScript) and Ab01680 anti-spike IgA (Ab01680-16, Absolute Antibody), all used at 0.5 to 10ng per well. Negative antibody controls were immunoglobulins from human serum (I4506 human IgG, I8260 human IgM, and I4036 human IgA, from Millipore-Sigma). A positive and negative control pool of 4 patient samples each was created and added in each plate at a single point concentration for normalization, or as a standard curve, starting with a 1:40 dilution. For all assays, a standard curve is generated by first plotting the mean of the blank-subtracted recombinant antibodies, plotted against antibody amounts (in ng) (or the positive pooled sera), and the linearity of the curve and comparison to previous runs is assessed, alongside the confirmation that the positive and negative pool sample fall within the expected range of the standard curve [%CV should be 10-15% or less]. Figure S8 displays the dilution curves for the assays shown here and Figure S9 represents the ratio distributions across the samples used here. See Table S4 for all data.
Surrogate neutralization enzyme-linked immunosorbent assays (snELISA)
To provide a measurement of the potential of the serum antibody to neutralize SARS-CoV-2, we employed a surrogate neutralization ELISA (snELISA) we described recently (
24). Briefly, 20 samples per PSO time bin were randomly selected and subjected to a four-point snELISA assay (starting with 4 μl serum) evaluating the capacity of serum antibodies to prevent the association of biotinylated ACE2 to immobilized RBD. Areas under the curves of the last two points were tabulated as an “integrated score” value: lower values correspond to a stronger displacement.
Sample collection and handling - saliva study
With the exception of some samples that were acquired early on in the pandemic (cohort 1), Salivette® tubes were used to collect samples according to manufacturer instructions (Sarstedt, Montreal, Quebec). These tubes include a cotton swab that participants are instructed to chew for set amount of time. The swab is then transferred into an inner tube which is then inserted into an outer tube that catches liquid saliva upon centrifugation at 1000 × g for 3 min (Centrifuge 5910 R, Eppendorf). Salivary flow was controlled by establishing a fixed amount of collection time (2 min) for each subject as previously recommended (
24,
49). For the early pandemic subjects that were not given Salivettes® and used in our pilot study (cohort 1), these subjects expectorated directly into a 15 mL conical tube containing 2.5 mL of phosphate-buffered saline (PBS). Prior to saliva collection, healthy subjects confirmed they had fasted, refrained from taking oral medication, and had not brushed their teeth for a minimum of 30 min.
Viral inactivation in saliva samples
Following centrifugation, all saliva samples, regardless of their SARS-CoV-2 PCR status, underwent viral inactivation by treating with Triton
® X-100 (BioShop, CAT# TRX506.100). 10% Triton X-100 (diluted 1:10 from stock) was added to all samples to a final dilution of 1% Triton X-100 and incubated for 1 hour at room temperature. Inactivated samples were immediately frozen and stored at -80°C. Heat inactivation for 30 min at 65°C was found to destroy the IgG and IgA signal against RBD and was therefore not used (Figure S5). The efficiency of virus inactivation in a saliva medium is shown in Table S3. Specifically, we assessed the treatment of saliva collected from healthy individuals using two different methods (Salivette® vs. direct saliva collection into a tube). These samples were spiked with known amounts of SARS-CoV-2 viral stock and then treated with 1% Triton X-100 for 30 min, 1 hour or 2 hours. Vero-E6 cells (ATCC
® CRL-1586
TM) were used to determine outgrowth of virus. Cells were maintained in Dulbecco’s Modified Eagle’s Media (DMEM) supplemented with L-glutamine, penicillin/streptomycin and 10% fetal bovine serum (FBS). SARS-CoV-2 virus (isolate SB3) was isolated in-house (
50). Briefly, viral stocks were created after isolation of virus from a clinical sample in Toronto, Ontario, Canada. Viral stock was expanded using Vero E6 as previously described such that stored aliquots of stock contain 2% FBS. Initial experiments were done with Triton X-100 (Sigma-Aldrich) serially diluted and applied to Vero-E6 cells in 96-well flat bottom plates to determine the minimum concentration required to prevent toxicity to cells. Furthermore, we have also determined if neat saliva itself could be cytotoxic to Vero-E6 cells by providing healthy donor saliva alone or treated with Triton X-100 ranging from final Triton-X100 concentration of 0.03%, 0.01%, 0.001% and 0.0001% (v/v). Since initial Triton X-100 experiments showed that toxicity is averted at 0.03% (v/v), we proceeded to use this concentration as the point of dilution to prevent any Triton X-100 mediated toxicity.
Antigen production – saliva assay
The expression, purification and biotinylation of the SARS-CoV-2 RBD and spike ectodomain were performed as recently described (
24,
49). The human codon optimized cDNA of the SARS-CoV-2 spike protein was purchased from Genscript (MC_0101081). The soluble RBD (residues 328-528, RFPN...CGPK) was expressed as a fusion protein containing a C-terminal 6xHis tag followed by an AviTag. The soluble trimeric spike protein ectodomain (residues 1-1211, MFVF...QYIK) was expressed with a C-terminal phage foldon trimerization motif followed by a 6xHis tag and an AviTag. To help stabilize the spike trimer in its prefusion conformation, residues 682–685 (RRAR) were mutated to SSAS to remove the furin cleavage site and residues 986 and 987 (KV) were each mutated to a proline residue (
51). Stably transfected FreeStyle 293-F cells secreting the RBD and soluble spike trimer were generated using a previously reported piggyBac transposon-based mammalian cell expression system (
52). Protein production was scaled up in 1L shake flasks containing 300 mL FreeStyle 293 medium. At a cell density of 10
6 cells/mL, 1 μg/mL doxycycline and 1 μg/mL Aprotinin were added. Every other day 150 mL of medium was removed and replaced by fresh medium. The collected medium was centrifuged at 10000 × g to remove the cells and debris and the His-tagged proteins were purified by Ni-NTA chromatography. The eluted protein was stored in PBS containing 300 mM imidazole, 0.1% (v/v) protease inhibitor cocktail (Sigma, P-8849) and 40% glycerol at -12°C. Shortly before use, the RBD and spike proteins were further purified by size-exclusion chromatography on a Superdex 200 Increase (GE healthcare) or Superose 6 Increase (GE healthcare) column, respectively. Purity was confirmed by SDS-PAGE. For the spike protein, negative stain electron microscopy was used show evidence of high-quality trimers. The Avi-tagged proteins, at a concentration of 100 μM or less, were biotinylated in reaction mixtures containing 200 μM biotin, 500 μM ATP, 500 μM MgCl
2, 30 μg/mL BirA, 0.1% (v/v) protease inhibitor cocktail. The mixture was incubated at 30°C for 2 hours followed by size-exclusion chromatography to remove unreacted biotin.
Enzyme-linked immunosorbent assays for detecting total IgA, IgG and IgM in saliva
Quantitative total IgA, IgG, and IgM analyses were performed on the same samples used for detection of anti-RBD and anti-spike Ig described below. Anti-human Ig antibody (Southern Biotech, 2010-01) diluted 1:1000 in PBS was added to 96-well Nunc MaxiSorp plates (ThermoFisher, 44-2404-21). PBS alone was added to control wells. Plates coated overnight at 4°C. Following coating, plates were blocked using 200 μl/well of 5% BLOTTO for 2 hours at 37°C. Samples were diluted in 2.5% BLOTTO, the volume of which was variable depending on the dilutions being tested per sample, per antibody isotype. Standards (purified IgA, IgG and IgM purchased from Sigma-Millipore: IgA, I4036, IgG, I2511, and IgM, I8260) were prepared in 2.5% BLOTTO ranging from 100 ng/mL to as low as 0.78 ng/mL depending on the antibody isotype being run. Upon discarding the blocking solution from the plate, 50 μl of diluted samples and 50 μl of each standard concentration solution were immediately transferred to wells and incubated for 2 hours at 37°C. Following incubation, wells were washed with 200 μL of PBS-T. HRP-conjugated secondary antibodies against IgA, IgG, and IgM (goat anti-human IgA- and IgG-HRP, Southern Biotech, IgA: 2053-05, IgG: 2044-05, IgM: 2023-05) were added to the appropriate wells at 1:1000 in 2.5% BLOTTO and incubated for 1 hour at 37°C. Development of the plate was done by adding 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) Substrate Solution (ThermoFisher, 00-4021-56) onto plates. Reaction vas then stopped by adding 50ul/well of 1N H2SO4 Optical density (OD) was read at a wavelength of 450 nm on a spectrophotometer (OD450). A four-parameter logistic curve was used to determine the line of best fit for the standard curve, and sample Ig quantities were interpolated accordingly to determine final concentrations in μg/ml. The few samples from patient or control groups that exhibited quality control issues (extremely low to negative IgA levels) were excluded from further analysis.
Enzyme-linked immunosorbent assay for detecting albumin in saliva
Salivary albumin was measured for Cohort 1 using a purchased Human Albumin ELISA Kit (Abcam, ab108788). Assay was performed according to manufacturer instructions included with the kit.
Enzyme-linked immunosorbent assays for detecting antigen-specific IgG, IgA and IgM in saliva
96-well plates pre-coated with streptavidin (ThermoFisher, 436014) were used for all assays. Without the biotin-streptavidin system, the anti-S/RBD IgG, IgA, and IgM signals obtained from COVID-19 patient saliva were undetectable. Based on titrations of antigens using saliva from convalescent COVID-19 patients, 2 μg/ml biotinylated-RBD and 20 μg/ml biotinylated-spike protein solutions were prepared in sterile PBS one day prior to starting the assay. From these dilutions, 50 μl of each were added to appropriate wells in our plates, resulting in 100 ng of biotinylated-RBD and 1 μg of biotinylated S proteins applied to the appropriate wells (see Figure S5 for RBD titration, spike titration not shown). Control wells of sterile PBS rather than biotinylated antigen were reserved for each patient and control sample. A few wells with the biotinylated antigen but with no sample added were reserved as negative internal controls for the reagents on the assay. Plates were incubated overnight at 4°C to allow sufficient coating of the antigen. 200 μL of 5% BLOTTO (5% w/vol skim milk powder (BioShop, CAT# SKI400.500) in sterile PBS) was subsequently added to each well to prevent nonspecific interactions, followed by a 2-hour incubation at 37°C. Blocking solution was discarded immediately from plates prior to addition of samples to wells. Newly thawed saliva samples were centrifuged at 8000 rpm for 4 min (Microcentrifuge 5418, Eppendorf), and appropriately diluted using 2.5% BLOTTO at dilutions ranging from 1:2 to 1:20 depending on the cohort being tested and the way in which saliva was collected. To reduce anti-streptavidin reactivity in the saliva, diluted samples were applied to streptavidin-coated plates with no antigen and allowed to incubate for 30 min at 37°C. Subsequently 50 μL of samples were transferred from the pre-adsorption plate into antigen-coated plates and incubated for 2 hours at 37°C. PBS+0.05% Tween 20 (BioShop, CAT# TWN510) (PBS-T) was used for washing plates between steps. Horseradish peroxidase (HRP)-conjugated goat anti human-IgG, IgA, and anti-IgM secondary antibodies (Southern Biotech, IgG: 2044-05, IgA: 2053-05, IgM: 2023-05) were added to wells at dilutions of 1:1000, 1:2000 and 1:1000 in 2.5% BLOTTO, respectively, and incubated for 1 hour at 37°C. Development of the plates was performed as described in the section above.
For cohort 1, because some samples had been collected in cups and were therefore diluted, normalization to a separate variable was performed. The resulting OD from antigen-specific IgA and IgG was subtracted from the OD for the PBS control wells for each sample and subsequently normalized to albumin levels or total IgA and IgG levels, respectively (see below). IgM was not calculated for cohort 1 due to lack of remaining sample from the COVID-19 patients. For cohort 1, raw OD450 measurements obtained from PBS-coated wells corresponding to each sample diluted at 1:5 (“background signal”) was subtracted from readings obtained from antigen-coated wells at each of two dilutions (1:5, 1:10). The OD from the highest concentrated saliva dilution (1:5 for samples collected by Salivette®, or 1:2 for saliva samples collected in cups and prediluted) was normalized to the total IgG, total IgA, or albumin content in each saliva sample. A small number of samples (n=9 from negative controls and n=4 from patients) exhibited high OD values that did not titrate and coincided with high OD levels when plated without antigen (PBS control). These were excluded from the analysis.
For cohort 2, raw OD450 measurements obtained from PBS-coated wells corresponding to each sample diluted at 1/5 (“background signal”) was subtracted from readings obtained from antigen-coated wells at each of three dilutions (1:5, 1:10, 1:20). For each plate, a sample of pooled saliva from COVID-19 acute and convalescent patients was likewise plated at 1/5 with no antigen (PBS control), as well as with antigens at 1:5, 1:10 and 1:20. The area under the curve was calculated based on the background subtracted values from all three dilutions for each sample. A pooled sample of positive control saliva was run on each plate and analyzed in the same manner. Each sample within a given plate was then normalized to the pooled positive control saliva for that particular plate and expressed as a percentage. For simplicity, we denoted this percentage as an “integrated score” (Figure S6A). By using the same positive control that we ran in every single plate, we determined that intra-assay precision was always greater than 90% between plates. Reproducibility between plates was determined by a coefficient of variation of less than 10% through all the plates. A small number of samples (n=6 from negative controls and n=2 from patients) exhibited high OD values that did not titrate and coincided with high OD levels when plated without antigen (PBS control) (Figure S6B). These were excluded from the final analysis.
Receiver-Operating Characteristic (ROC) curves
For serum and plasma sample analysis, samples acquired prior to November 2019 (pre-COVID) were labeled true negatives while convalescent samples from patients with PCR-confirmed COVID-19 were labeled true positives. For saliva samples, all samples from patients with PCR-confirmed COVID-19 collected more than 10 days PSO were considered true positives, and saliva collected before 2020 and from unexposed, asymptomatic individuals in March of 2020 were labeled true negative for ROC analysis. Ratio-converted ELISA reads (colorimetric or chemiluminescent) were used for ROC analysis in the easyROC webtool (v 1.3.1) with default parameters (
https://journal.r-project.org/archive/2016/RJ-2016-042/index.html). Non-parametric curve fitting was applied alongside DeLong’s method for standard error estimation and confidence interval generation.
Statistical analysis
For total IgA, IgG and IgM readouts in saliva, raw OD450 measurements obtained from PBS-coated wells (“background signal”) was subtracted from readings obtained from anti-human Ig-coated wells with saliva samples added (OD450 of sample – OD450 of PBS-coated well). Total IgA, IgG and IgM quantifications were determined relative to standard wells present on each plate. A four-parameter logistic curve was used to determine the line of best fit for the total IgA/M/G standard curves, and sample Ig quantities were interpolated accordingly, using Prism (GraphPad), Version 8.3.
For the analysis in the antigen ELISA of cohort 1, the raw OD450 measurements from the PBS-coated wells with sample added at 1/5 dilution (“background signal”) was subtracted from each of the saliva sample dilutions (1:5, 1:10) added to wells coated with protein (OD450 of sample from coated well – OD450 of PBS coated well with sample). The blank-corrected OD from the 1:5 sample dilution in the antigen-specific IgA and IgG were subsequently normalized to the concentration of total IgA and IgG, respectively, for cohort 1. An additional normalization strategy consisted of normalizing the blank corrected OD from antigen-specific IgA and IgG to the concentration of albumin for cohort 1.
For the analysis in the antigen ELISA of cohort 2, the raw OD450 measurements from the PBS-coated wells with sample added at 1:5 dilution (“background signal”) was subtracted from each of the saliva sample dilutions (1:5, 1:10, 1:20) added to wells coated with protein (OD450 of sample from coated well – OD450 of PBS coated well with sample). For cohort 2, the blank corrected OD antigen-specific IgA, IgG, and IgM OD values across three dilutions were used to calculate the integrated score for each sample. The sample area under the curve was normalized to the score of a positive pool of saliva samples used as an internal standard across all plates. The values were expressed as a percentage and denoted as an integrated score.
For serum, raw OD450 measurements for IgG, IgA and IgM on spike, RBD and NP from either the manual or automated platforms were subtracted from wells coated with PBS. A pool of serum samples that previously exhibited high levels of IgGs to all antigens was used as an internal standard across all plates, and a relative ratio between blank-adjusted measurements (OD450 or chemiluminescent reads) of patient samples and measurements of this positive pooled standard are reported as “ratio-converted ELISA reads.” Serum data were analyzed in R using version 4.0.1. Median antibody levels between negative and positive subject groups (saliva) or negative, acute and convalescent subject groups (blood) were compared using Mann Whitney U tests. These analyses were performed in Prism (GraphPad), Version 8.3.
The relationship between time PSO and antibody levels in the convalescent period was examined in multivariable linear regression models that adjusted for age, sex, and disease severity. For serum samples, seven multivariable linear regression models were constructed (one for each of anti-RBD IgA, anti-S IgA, anti-RBD IgG, anti-S IgG, anti-RBD IgM, anti-spike IgM, neutralizing antibody). Generalized estimating equations were used (proc genmod in SAS with exchangeable correlation matrices) to account for patient-level clustering. Antibody levels were transformed as appropriate to achieve heteroscedasticity, and the variance inflation factors for all covariates confirmed to be <5 to verify absence of multicollinearity. For saliva samples, six multivariable linear regression models were similarly constructed; however, only the first convalescent sample for each patient was included in the analysis (proc glm in SAS).
Acknowledgments
We thank Janet McManus at Canadian Blood Services for her technical and logistical expertise, Dzana Dervovic, Cassandra Wong, Mark Jen and Elizabeth Rubie for help with serum-based ELISAs, Joan Wither for the lupus patient samples, Frederick P. Roth for the
SARS-CoV-2 Nucleocapsid entry vector and Jim Hartley for bacteria expression vectors. We thank research associates of the Toronto Invasive Bacterial Diseases Network and Antonio Estacio, Megan Buchholz, Patti Cheatley, Rebecca Zavalunov and Katerina Pavenski for specimen collection. We thank Jeff Browning (Boston University) for critical input into the saliva experiments.
Funding: This study was supported by an
Ontario Together grant and funding from the Canadian Institutes of Health Research (CIHR; #VR1-172711, VR4–172732 and #439999). Funding for the development of the assays in the Gingras lab was provided through generous donations from the Royal Bank of Canada (RBC), Questcap and the Krembil Foundation to the Sinai Health System Foundation. The robotics equipment used is housed in the Network Biology Collaborative Centre at the Lunenfeld-Tanenbaum Research Institute, a facility supported by Canada Foundation for Innovation funding, by the Ontarian Government and by Genome Canada and Ontario Genomics (OGI-139). Indirect support for SARS-CoV-2 work in the Toronto Combined Containment Level 3 laboratory was provided by strategic research funds from the University of Toronto and the Temerity Foundation. MO is funded by: OHTN (Ontario HIV Treatment Network), CIHR and the Juan and Stefania Speck Fund. JG is a Canada Research Chair, Tier 1, in Tissue Specific Immunity and is supported by CIHR FDN15992. ACG is the Canada Research Chair, Tier 1, in Functional Proteomics and is supported by CIHR FDN143301. KTA is a recipient of an Ontario Graduate Scholarship and AJJ is supported by a Vanier Canada graduate studentship.
Competing interests: SJD has acted as a content expert for respiratory viruses for Johnson & Johnson (Janssen). Work in the Gommerman lab unrelated to this project has been funded by EMD-Serono, Roche and Novartis. The other authors declare that they have no competing interests.
Data and materials availability: Transfer of clinical samples and virus isolates required for this research to and from the hospital and university laboratories involved was covered by material transfer agreements between Sunnybrook Health Sciences Centre and the University of Toronto. All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials. 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: Dimeric IgA in nasal secretion vs monomeric IgA in serum
Thanks to Isho et al for your sincere affirmation, that we know very little about the local antibody response at the site of infection. SARS-CoV-2 enters the nasal and oropharyngeal avenue where it subsequently replicates (1) before spread inwards and outwards.
Antibodies either produced following SARS-CoV-2 infection or vaccination, play an important role in neutralizing and to protect the host against infection and severity. You have also mentioned that antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in mucosal surface antibodies. SARS-CoV-2 starts replication in nasal mucosa. However, few studies have examined anti-SARS-CoV-2 antibodies in nasal or pharyngeal mucosa.
In general, immunization or infection at mucous membranes resulted in high titer of protective antibodies at the mucosal site, however, absent or low titers in serum. Similarly, following parenteral immunization, reverse occurred (2). Necessity of isotypes of immunoglobulins differ depending on the type of viral infection and its tropism. Dimeric or polymeric IgA is produced in nasopharyngeal associated lymphoid tissue (NALT), bronchoalveolar associated lymphoid tissue (BALT), gut associated lymphoid tissue (GALT) but not in serum which can produce 15 times less effective monomeric IgA (3).
SARS-CoV-2 replicates in surface epithelial cells in nasal mucosa and shed in mucus gel to contaminate it. Mucus contaminated with SARS-CoV-2 (MCS) migrates to lung alveoli as microaspiration (3, 4). Microaspiration of MCS detonates compartmental immune response in BALT and that response might boom as pneumonia with or without cytokine storm. From SARS-CoV-2 infection at nasal mucosa to cytokine storm at alveolus; SARS-CoV-2 is on the respiratory epithelial cells. It is also found that SARS-CoV-2 do not invade lung alveoli through haematogenous spread (3). One prospective study (4) have shown that normal saline nasal spray and gargle (NSNSG) can significantly (p=0.01) wash off SARS-CoV-2, even with high virion load, from nasal and pharyngeal mucosal surface. In view of that, it is tempting to express SARS-CoV-2 as "surface virus" like other flu viruses and unlike Ebola, HIV, Dengue etc (4). So it seems that to mitigate this surface virus, SARS-CoV-2, we have to look for dimeric form with adequate titer of secretory IgA and other immunoglobulin secreted or diffused from serum in respiratory epithelial surface following vaccination.
References:
1. B. Isho et al., Sci. Immunol. 10.1126/sciimmunol.abe5511 (2020). http://immunology.sciencemag.org/content/5/52/eabe5511
2. A.J. Macpherson, F-E Johansen, P. Brandtzaeg. The immune geography of IgA induction and function. Mucosal Immunology ; 1: 2008. doi:10.1038/mi.2007.6
3. Z. Wang et al. Enhanced SARS-CoV-2 neutralization by dimeric IgA. Sci. Transl. Med. 13, eabf1555 (2021). http://stm.sciencemag.org/content/13/577/eabf1555
3. Y.J. Hou, K. Okuda, C.E. Edwards, et al. SARS-CoV-2 Reverse genetics reveals a variable infection gradient in the respiratory tract. Cell 182, 429–46 (2020). https://doi.org/10.1016/j.cell.2020.05.042
4. U. Chatterjee, A. Chakraborty, S. Naskar, B. Saha, B. Bandyopadhyay, S. Shee. Efficacy of normal saline nasal spray and gargle on SARS-CoV-2 for prevention of COVID-19 pneumonia. Research Square (2021) http://www.researchsquare.com/article/rs-153598/v1