Anti-SARS-CoV-2 Immunoglobulin Isotypes, and Neutralization Activity Against Viral Variants, According to BNT162b2-Vaccination and Infection History

Abstract

Purpose: To compare SARS-CoV-2 antigen-specific antibody production and plasma neutralizing capacity against B.1 wild-type-like strain, and Gamma/P.1 and Delta/B.1.617.2 variants-of-concern, in subjects with different Covid-19 disease and vaccination histories.

Methods: Adult subjects were: 1) Unvaccinated/hospitalized for Covid-19; 2) Covid-19-recovered followed by one BNT162b2 vaccine dose; and 3) Covid-19-naïve/2-dose BNT162b2 vaccinated. Multiplex Luminex^® immunoassays measured IgG, IgA, and IgM plasma levels against SARS-CoV-2 receptor-binding domain (RBD), spike-1 (S), and nucleocapsid proteins. Neutralizing activity was determined in Vero E6 cytopathic assays.

Results: Maximum anti-RBD IgG levels were similar in Covid^-19‑recovered individuals 8‒10 days after single-dose vaccination and in Covid-19-naïve subjects 7 days after 2^nd vaccine dosing; both groups had ≈2‑fold higher anti-RBD IgG levels than Unvaccinated/Covid-19 subjects tracked through 2 weeks post-symptom onset. Anti-S IgG expression patterns were similar to RBD within each group, but with lower signal strengths. Viral antigen-specific IgA and IgM levels were more variable than IgG patterns. Anti-nucleocapsid immunoglobulins were not detected in Covid-19-naïve subjects. Neutralizing activity against the B.1 strain, and Gamma/P.1 and Delta/B.1.617.2 variants, was highest in Covid‑19-recovered/single-dose vaccinated subjects; although neutralization against the Delta variant in this group was only 26% compared to B.1 neutralization, absolute anti-Delta titers suggested maintained protection. Neutralizing titers against the Gamma and Delta variants were 33‒77% and 26‒67%, respectively, versus neutralization against the B.1 strain (100%) in the three groups.

Conclusion: These findings support SARS-CoV-2 mRNA vaccine usefulness regardless of Covid-19 history, and confirm remarkable protection provided by a single vaccine dose in people who have recovered from Covid-19.

Keywords: COVID-19; SARS-CoV-2; bead; coronavirus; fluorescence; multiplex immunoassay; neutralization assay; serological assay.

Figure 1

Kinetic measurement of plasma anti-SARS-CoV-2 IgG, IgM, and IgA isotype levels against Receptor Binding Domain (RBD), Spike Protein-1 Subunit (S1), and the Nuclocapsid (N) proteins in different populations. Shown are values in subjects who were hospitalized for Covid-19 infection, measured at timepoints after first reported symptom onset and before vaccination (“Covid+/No Vaccine”; n=8; left column), hospital workers who had no known SARS-CoV-2 exposure and were vaccinated with the Pfizer BNT162b2 vaccine, measured at timepoints after their first vaccination dose (“No Covid/2 Vaccine Doses”; n=3; middle column), and hospital workers who had recovered from Covid-19 and then received a single dose of BNT162b2, measured at timepoints after vaccination (“Covid+/1 Vaccine Dose”; n=8; right column). All individuals in the No Covid/2 Vaccine Dose group received their second vaccination 21 ± 1 days after the first dose. In Covid+/No Vaccine individuals, IgG, IgM and IgA levels were elevated against RBD and N at 2 weeks, whereas no S1-reactivity was observed (A, D, G). The maximum anti-RBD IgG levels were seen at Day 14 after symptoms appeared (11,185 MFI). The No Covid/2 Vaccine individuals showed significantly but modestly elevated IgG (B) and transiently elevated IgM against RBD (E) at 2‒3 weeks after their first vaccination, with anti-S1 IgG upregulated by 14 days (B), but no anti-S1 IgM or IgA during this time (E, H). Starting ≈1 week after the 2nd vaccine dose, IgG against RBD rapidly increased to a maximal level at Day 28 (21,695 MFI), at which point it plateaued (B); IgG against S1 also began increasing in parallel to a maximum level at 28 days (3964 MFI) followed by a plateau through Day 42 (B). In the Covid+/1 Vaccine group, IgG against RBD and S1 began increasing at 6 days after vaccination and were maximal for both RBD (23,897 MFI) and S1 (9387 MFI) at 10 days (C). No IgM response against viral RBD, S1, or N was observed in this group through 10 days after single-dose vaccination (F). Significant IgA responses against RBD and S1 were not observed in the two groups that had been infected with SARS-CoV-2 (G, I), but were seen in Covid-naive subjects after vaccination (H). Antibody production against N was not seen in Covid-naïve groups (B, E, H). “MFI” indicates average of median fluorescence intensity units. Shown are mean values ± SEM. Asterisk *p<0.05; **p<0.01; ***p<0.001 by unpaired t-test.

Figure 2

Highest IgG levels against RBD and S1 according to infection and BNT162b2 vaccination history. Maximum average anti-RBD IgG levels were highest 14 days after symptom onset in the SARS CoV-2 infected/unvaccinated subjects, 10 days after single vaccination in the Covid recovered/1 Vaccine Dose group, and 28 days after 1st vaccination (7 days after 2nd vaccine dose injection) in the No Covid/2 Vaccine Doses group. Anti-RBD IgG levels were significantly elevated in both vaccinated groups compared to in SARS-CoV-2 infected/unvaccinated individuals, with levels in both No Covid/2 Vaccine subjects and Covid+/1 Vaccine subjects elevated approximately 2-fold. A similar relative increase in anti-S1 IgG levels after vaccination also occurred, with levels in No Covid/2 Vaccine subjects over 3 fold higher and levels in Covid+/1 Vaccine subjects more than over 8-fold higher, respectively, compared to anti-S1 IgG measured in unvaccinated Covid-infected individuals. With S1 but not RBD, a single vaccination after previous Covid-19 infection resulted in significantly higher S1 specific IgG levels than occurred in Covid-naïve individuals who received the complete 2 dose vaccination series. Shown are mean values ± SEM for 3‒8 subjects/group.

Figure 3

Neutralization of SARS-CoV-2 infectivity by plasma, according to subject infection and BNT162b2 vaccination history. Three viral variants (B.1 wild type-like early strain, Gamma, and Delta) were pre-incubated with serial dilutions of subject plasma before evaluating their infectivity of Vero E6 cells (ATCC #CRL-1586; African green monkey kidney epithelium). Y-axes denote plasma neutralization titers. All three experimental groups displayed variably increasing neutralization activity against the three SARS-CoV-2 variants, with timing of maximal observed neutralization approximating that when maximum anti-RBD and anti-S1 IgG levels were measured (detailed in Figure 1 ). The highest mean neutralization titers (and anti-RBD and anti-S1 IgG levels) were observed in individuals who received a single vaccine dose after recovering from Covid-19.

Figure 4

Neutralization of SARS-CoV-2 infectivity according to viral variant and subject infection and BNT162b2 vaccination history. (A) In unvaccinated individuals hospitalized with SARS-CoV-2 (n=8), mean neutralization effectiveness against the B.1 strain (set as 100%; titer=225 ± 135 fold dilution) was reduced by 33% against the Gamma variant (titer=150 ± 90) and by 67% against the Delta variant (titer=75 ± 45). In SARS-CoV-2-naïve subjects (n=8) who received the 2-dose BNT162b2 vaccination series, neutralizing effectiveness was similar against the three SARS-CoV-2 variants with inhibitory activity against the B.1 strain (set at 100%; titer=115 ± 39) maintained at 100% against the Gamma variant (titer=115 ± 39), and modestly 23% lower against the Delta variant (titer=90 ± 30). By far the strongest neutralizing ability was elicited in volunteers (n=3) who had previously been diagnosed with and recovered from Covid-19 and then received a single BNT162b2 vaccine dose. In this Covid+/1 Vaccine Dose group, the average maximal neutralizing titer against the B.1 strain (set at 100%; titer=1360 ± 349) were reduced by 29% against the Gamma variant (titer=960 ± 240; p>0.05) and by 74% against the Delta variant (titer=360 ± 69; p=0.048). (B) Individuals who had previously recovered from Covid-19 and then received a single vaccine dose developed significantly higher neutralization titers against B.1 and Gamma strains when compared to Covid-naïve individuals who received 2 vaccine doses. Neutralization of the Delta variant was also numerically 4-fold higher in Covid+/1 Vaccine Dose subjects versus Covid-naïve/2 Vaccine Dose subjects, and 5-fold higher than in unvaccinated individuals who were hospitalized for Covid-19, but these differences did not attain statistical significance. Neutralization capacity of the plasma of subjects hospitalized with Covid-19 did not remarkably differ from neutralization by Covid-naïve individuals who received the both vaccine doses, for all SARS-CoV-2 strains studied. Shown are mean values ± SEM.