SUMMARY
The SARS-CoV-2 virus is responsible for the current worldwide coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing hundreds of thousands of deaths. The Spike glycoprotein of SARS-CoV-2 mediates viral entry and is the main target for neutralizing antibodies. Understanding the antibody response directed against SARS-CoV-2 is crucial for the development of vaccine, therapeutic and public health interventions. Here we performed a cross-sectional study on 98 SARS-CoV-2-infected individuals to evaluate humoral responses against the SARS-CoV-2 Spike. The vast majority of infected individuals elicited anti-Spike antibodies within 2 weeks after the onset of symptoms. The levels of receptor-binding domain (RBD)-specific IgG persisted overtime, while the levels of anti-RBD IgM decreased after symptoms resolution. Some of the elicited antibodies cross-reacted with other human coronaviruses in a genus-restrictive manner. While most of individuals developed neutralizing antibodies within the first two weeks of infection, the level of neutralizing activity was significantly decreased over time. Our results highlight the importance of studying the persistence of neutralizing activity upon natural SARS-CoV-2 infection.
MAIN
The first step in the replication cycle of coronaviruses (CoV) is viral entry. This is mediated by their trimeric Spike (S) glycoproteins. Similar to SARS-CoV, the S glycoprotein of SARS-CoV-2 interacts with angiotensin-converting enzyme 2 (ACE2) as its host receptor1–3. During entry, the Spike binds the host cell through interaction between its receptor binding domain (RBD) and ACE2 and is cleaved by cell surface proteases or endosomal cathepsins1,4,5, triggering irreversible conformational changes in the S protein enabling membrane fusion and viral entry6,7. The SARS-CoV-2 Spike is very immunogenic, with RBD representing the main target for neutralizing antibodies8–11. Humoral responses are important for preventing and controlling viral infections12,13. However, little is known about the chronology and durability of the human antibody response against SARS-CoV-2.
Here we analyzed serological samples from 98 SARS-CoV-2-infected individuals at different times post-infection and 10 uninfected individuals for their reactivity to SARS-CoV-2 S glycoprotein, cross-reactivity with other human CoV (HCoV), as well as virus neutralization. Samples were collected from COVID-19 positive individuals starting on March 2020 or healthy individuals before the COVID-19 outbreak (COVID-19 negative). Cross-sectional serum samples (n= 71) were collected from individuals presenting typical clinical symptoms of acute SARS-CoV-2 infection (Extended Table 1). All patients were positive for SARS-CoV-2 by RT-PCR on nasopharyngeal specimens. The average age of the infected patients was 56 years old, including 31 males and 40 females. Samples were classified into 3 different time points after infection: 24 (11 males, 13 females) were obtained at 1-7 days (T1, median = 3 days), 20 (9 males, 11 females) between 8-14 days (T2, median = 11 days) and 27 (20 males, 7 females) between 16-31 days (T3, median = 23 days). Samples were also obtained from 27 convalescent patients (20 males, 7 females, median = 42 days), who have been diagnosed with or tested positive for COVID-19 with complete resolution of symptoms for at least 14 days.
We first evaluated the presence of RBD-specific IgG and IgM antibodies by ELISA14,15. The level of RBD-specific IgM peaked at T2 and was followed by a stepwise decrease over time (T3 and Convalescent) (Figure 1). Three quarter of the patients had detectable anti-RBD IgM two weeks after the onset of the symptoms. Similarly, 75% of patients in T2 developed anti-RBD IgG, reaching 100% in convalescent patients. In contrast to IgM, the levels of RBD-specific IgG peaked at T3 and remained relatively stable after complete resolution of symptoms (convalescent patients).
We next used flow cytometry to examine the ability of sera to recognize the full-length SARS-CoV-2 Spike expressed at the cell surface. Briefly, 293T cells expressing SARS-CoV-2 S glycoproteins were stained with samples, followed by incubation with secondary antibodies recognizing all antibody isotypes. As presented in Figure 2, 54.2% of the sera from T1 already contained SARS-CoV-2 full Spike-reactive antibodies. Interestingly, the majority of patients from T2, T3 and convalescent groups were found to be seropositive. Antibody levels targeting the SARS-CoV-2 Spike significantly increased from T1 to T2/T3 and remained relatively stable thereafter. As expected, the levels of antibodies recognizing the full Spike correlated with the presence of both RBD-specific IgG and IgM (Extended Figure 1). We also evaluated potential cross-reactivity against the closely related SARS-CoV Spike. None of the COVID-19 negative samples recognized the SARS-CoV Spike. While the reactivity of COVID-19+ samples to SARS-CoV S was lower than for SARS-CoV-2 S, it followed a similar progression and significantly correlated with their reactivity to SARS-CoV-2 full Spike or RBD protein (Figure 2 and Extended Figure 1). This indicates that SARS-CoV-2-elicited antibodies cross-react with human Sarbecoviruses. This was also observed with another Betacoronavirus (OC43) but not with Alphacoronavirus (NL63, 229E) S glycoproteins, suggesting a genus-restrictive cross-reactivity (Figure 2c and Extended Figure 1). This differential cross-reactivity could be explained by the high degree of conservation in the S protein, particularly in the S2 subunit among Betacoronaviruses16–18.
We next measured the capacity of patient samples to neutralize pseudoparticles bearing SARS-CoV-2 S, SARS-CoV S or VSV-G glycoproteins using 293T cells stably expressing ACE2 as target cells (Figure 3 and Extended Figure 2). Neutralizing activity, as measured by the neutralization half-maximum inhibitory dilution (ID50) or the neutralization 80% inhibitory dilution (ID80), was detected in most patients within 2 weeks after the onset of symptoms (T2, T3 and Convalescent patients) (Figure 3). SARS-CoV-2 neutralization was specific since no neutralization was observed against pseudoparticles expressing VSV-G. The capacity to neutralize SARS-CoV-2 S-pseudotyped particles significantly correlated with the presence of RBD-specific IgG/IgM and anti-S antibodies (Extended Figure 3). While the percentage of patients eliciting neutralizing antibodies against SARS-CoV-2 Spike remained relatively stable 2 weeks after disease symptom onset (T2, T3 and Convalescent patients), neutralizing antibody titers significantly decreased after the complete resolution of symptoms as observed in the convalescent patients (Figure 3g,h). Cross-reactive neutralizing antibodies against SARS-CoV S protein (Figure 2b) were also detected in some SARS-CoV-2-infected individuals, but with significantly lower potency and also waned over time. We note that around 40% of convalescent patients did not exhibit any neutralizing activity. This suggests that the production of neutralizing antibodies is not a prerequisite to the resolution of the infection and that other arms of the immune system could be sufficient to control the infection in an important proportion of the population.
This study helps to better understand the kinetics and persistence of humoral responses directed against SARS-CoV-2 (Figure 4). Our results reveal that the vast majority of infected individuals are able to elicit antibodies directed against SARS-CoV-2 Spike within 2 weeks after symptom onset and persist after the resolution of the infection. Accordingly, all tested convalescent patients were found to be seropositive. As expected, RBD-specific IgM levels decreased over the duration of the study while IgG remained relatively stable. Our results highlight how SARS-CoV-2 Spike, like other coronaviruses, appears to be relatively easily recognized by Abs present in sera from infected individuals. This was suggested to be linked to the higher processing of glycans compared to other type I fusion protein, such as HIV-1 Env, Influenza A HA or filoviruses GP19,20. The ease of naturally-elicited Abs to recognize the Spike might be associated with the low rate of somatic hypermutation observed in neutralizing Abs9. This low somatic hypermutation rate could in turn explain why the majority of the SARS-CoV-2 infected individuals are able to generate neutralizing antibodies within only two weeks after infection (Figure 3). In contrast, the development of potent neutralizing antibodies against HIV-1 Env usually requires 2-3 years of infection and require a high degree of somatic hypermutation21. Nevertheless, in the case of SARS-CoV-2 infection, the neutralization capacity decreases significantly 6 weeks after the onset of symptoms, following a similar trend as anti-RBD IgM (Figure 4). Interestingly, anti-RBD IgM presented a stronger correlation with neutralization than IgG (Extended Figure 3a), suggesting that at least part of the neutralizing activity is mediated by IgM. However, it remains unclear whether this reduced level of neutralizing activity would remain sufficient to protect from re-infection.
AUTHOR CONTRIBUTIONS
J.Prévost, J.R., B.S., R.B., M.R. and A.F. conceived the study. J.Prévost, J.R., A.F. designed experimental approaches; J.Prévost, G.B.B., R.G., A.Laumaea, J.R., S.P.A., G.G., S.D., T.T., J.Perreault, A.Lewin., R.D. R.B., M.R., and A.F. performed, analyzed and interpreted the experiments; J.Prévost, G.B.B., J.R., H.M., G.G.-L., M.D., P.T., G.T.G.M., M.Côté and A.F. contributed novel reagents; N.G., M.Carrier, D.M., A.P., M.L., A.B., V.L., G.B., C.T., R.B. and M.R. collected clinical samples; J.Prévost, J.R. and A.F. wrote the paper. Every author has read edited and approved the final manuscript.
Competing interests
The authors declare no competing interests.
METHODS
Ethics statement
All work was conducted in accordance with the Declaration of Helsinki in terms of informed consent and approval by an appropriate institutional board. In addition, this study was conducted in accordance with the rules and regulations concerning ethical reviews in Quebec, particularly those specified in the Civil Code (http://legisquebec.gouv.qc.ca/fr/ShowDoc/cs/CCQ-1991) and in subsequent IRB practice. Informed Consent was obtained for all participating subjects and the study was approved by Quebec Public health authorities. Convalescent plasmas were obtained from donors who consented to participate in this research project (REB # 2020-004). The donors were recruited by Héma-Québec and met all donor eligibility criteria for routine apheresis plasma donation, plus two additional criteria: previous confirmed COVID-19 infection and complete resolution of symptoms for at least 14 days.
Plasmids
The plasmids expressing the human coronavirus Spikes of SARS-CoV-2, SARS-CoV, NL63 and 229E were previously reported1,22. The OC43 Spike with an N-terminal 3xFlag tag and C-terminal 17 residue deletion was cloned into pCAGGS following amplification of the spike gene from pB-Cyst-3FlagOC43SC17 (kind gift of James M. Rini, University of Toronto, ON, Canada). The plasmid encoding for SARS-CoV-2 S RBD (residues 319-541) fused with a hexahistidine tag was reported elsewhere15. The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described23. The lentiviral packaging plasmids pLP1 and pLP2, coding for HIV-1 gag/pol and rev respectively, were purchased from Invitrogen. The transfer plasmid encoding for human angiotensin converting enzyme 2 (ACE2) fused with a mGFP C-terminal tag and a puromycin selection marker was purchased from OriGene. The lentiviral vector to produce pseudoparticles was pNL4.3 R-E- Luc.
Cell lines
293T human embryonic kidney cells (obtained from ATCC) were maintained at 37°C under 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent) containing 5% fetal bovine serum (VWR) and 100 μg/ml of penicillin-streptomycin (Wisent). For the generation of 293T cells stably expressing human ACE2, transgenic lentivirus were produced in 293T using a third-generation lentiviral vector system. Briefly, 293T cells were co-transfected with two packaging plasmids (pLP1 and pLP2), an envelope plasmid (pSVCMV-IN-VSV-G) and a lentiviral transfer plasmid coding for human ACE2 (OriGene). Supernatant containing lentiviral particles was harvested and purified on a 20% sucrose cushion gradient. Purified lentiviral particles were used to infect 293T cells and stably transduced cells were enriched upon puromycin selection. 293T-ACE2 cells were cultured in a medium supplemented with 2 μg/ml of puromycin (Sigma)
Sera and antibodies
Sera from SARS-CoV-2-infected and uninfected donors were collected, heat-inactivated for 1 hour at 56 °C and stored at −80°C until ready to use in subsequent experiments. The monoclonal antibody CR3022 was used to as a positive control in ELISA assays and was previously described 8,24,25. Horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Invitrogen) or for the Fc region of human IgM (Invitrogen) were used as secondary antibodies to detect sera binding in ELISA experiments. Alexa Fluor-647-conjugated goat anti-human IgG (H+L) Abs (Invitrogen) were used as secondary antibodies to detect sera binding in flow cytometry experiment. Polyclonal goat anti-ACE2 (RND systems) and Alexa-Fluor-conjugated donkey anti-goat IgG Abs (Invitrogen) were used to detect cell-surface expression of human ACE2.
Protein expression and purification
FreeStyle 293F cells (Invitrogen) were grown in FreeStyle 293F medium (Invitrogen) to a density of 1 × 106 cells/mL at 37°C with 8 % CO2 with regular agitation (150 rpm). Cells were transfected with a plasmid coding for SARS-CoV-2 S RBD using ExpiFectamine 293 transfection reagent, as directed by the manufacturer (Invitrogen). One week later, cells were pelleted and discarded. Supernatants were filtered using a 0.22 μm filter (Thermo Fisher Scientific). The recombinant RBD proteins were purified by nickel affinity columns, as directed by the manufacturer (Invitrogen). The RBD preparations were dialyzed against phosphate-buffered saline (PBS) and stored in aliquots at −80°C until further use. To assess purity, recombinant proteins were loaded on SDS-PAGE gels and stained with Coomassie Blue. For cell-surface staining, RBD proteins were fluorescently labelled with Alexa Fluor 594 (Invitrogen) according to the manufacturer’s protocol.
ELISA assay
Recombinant SARS-CoV-2 S RBD proteins (2.5 μg/ml), or bovine serum albumin (BSA) (2.5 μg/ml) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4°C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween 20 and 2% [wt/vol] BSA) for 1 h at room temperature. Wells were then washed four times with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween 20). Anti-SARS-CoV-2 RBD CR3022 mAb (50 ng/ml) or sera from SARS-CoV-2-infected or uninfected donors (1:100, 1:250, 1:500, 1:1000, 1:2000, 1:4000 dilution) were diluted in blocking buffer and incubated with the RBD-coated wells for 1 h at room temperature. Plates were washed four times with washing buffer followed by incubation with secondary Abs (diluted in blocking buffer) for 1 h at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB 941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each serum and were then normalized to the signal obtained with CR3022 mAb present in each plate. The seropositivity threshold was established using the following formula: mean RLU of all COVID-19 negative sera normalized to CR3022 + (3 standard deviations of the mean of all COVID-19 negative sera).
Flow cytometry analysis of cell-surface staining
Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES-GFP) was transfected into 2 × 106 293T cells. At 48h post transfection, 293T cells were stained with sera from SARS-CoV-2-infected or uninfected individuals (1:250 dilution). The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on the basis of viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA). The seropositivity threshold was established using the following formula: (mean of all COVID-19 negative sera + (3 standard deviation of the mean of all COVID-19 negative sera) + inter-assay coefficient of variability).
Virus neutralization assay
Target cells were infected with single-round luciferase-expressing lentiviral particles. Briefly, 293T cells were transfected by the calcium phosphate method with the lentiviral vector pNL4.3 R-E- Luc (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike, SARS-CoV Spike or VSV-G at a ratio of 5:4. Two days after transfection, cell supernatants were harvested and stored in aliquots at –80°C until use. 293T-ACE2 target cells were seeded at a density of 1 × 104 cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 h before infection. Luciferase-expressing recombinant viruses in a final volume of 100 μl were incubated with the indicated sera dilutions (1/50; 1/250; 1/1250; 1/6250; 1/31250) for 1h at 37°C and were then added to the target cells followed by incubation for 48 h at 37°C; the medium was then removed from each well, and the cells were lysed by the addition of 30 μl of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB 941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 μl of luciferin buffer (15 mM MgSO4, 15 mM KPO4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 μl of 1 mM d-luciferin potassium salt (Prolume). The neutralization half-maximal inhibitory dilution (ID50) or the neutralization 80% inhibitory dilution (ID80) represents the sera dilution to inhibit 50% or 80% of the infection of 293T-ACE2 cells by recombinant lentiviral viruses bearing the indicated surface glycoproteins.
Time series visualization
Area graphs were generated using RawGraphs with DensityDesign interpolation and the implemented normalization using vertically un-centered values26.
Statistical analyses
Statistics were analyzed using GraphPad Prism version 8.0.2 (GraphPad, San Diego, CA, (USA). Every data set was tested for statistical normality and this information was used to apply the appropriate (parametric or nonparametric) statistical test. P values <0.05 were considered significant; significance values are indicated as * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.
Supplemental Information
Supplemental information includes 2 tables and 3 figures, and can be found online.
Extended Table 1. Cross-sectional SARS-CoV-2 cohort clinical characteristics
Extended Table 2. Serological analysis of samples from SARS-CoV-2 infected individuals
ACKNOWLEDGMENTS
The authors thank the CRCHUM BSL3 and Flow Cytometry Platforms for technical assistance. We thank Dr Florian Krammer (Icahn School of Medicine at Mount Sinai, NY) for the plasmid expressing the SARS-CoV-2 RBD domain, Dr Stefan Pöhlmann (Georg-August University, Germany) for the plasmids coding for SARS-CoV S, SARS-CoV-2 S and hCoV 229E and NL63 S glycoproteins and Dr M. Gordon Joyce (U.S. MHRP) for the monoclonal antibody CR3022. We also thank Danka K Shank and Melina Bélanger Collard from the Laboratoire de Santé Publique du Québec for their help in preparing the specimens. This work was supported by le Ministère de l’Économie et de l’Innovation du Québec, Programme de soutien aux organismes de recherche et d’innovation to A.F and by the Fondation du CHUM. This work was also supported by a CIHR foundation grant #352417 to A.F. Development of SARS-CoV-2 reagents was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS) contract HHSN272201400008C. A.F. is the recipient of a Canada Research Chair on Retroviral Entry # RCHS0235 950-232424. R.D. was supported by NIH grant R01 AI122953-05. M.C. is the recipient of a Tier II Canada Research Chair in Molecular Virology and Antiviral Therapeutics and an Ontario’s Early Researcher Award. J.P., G.B.B. and S.P.A are supported by CIHR fellowships. R.G. is supported by a MITACS Accélération postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.