Dynamics of Serum-Neutralizing Antibody Responses in Vaccinees through Multiple Doses of the BNT162b2 Vaccine

Abstract

SARS-CoV-2 mRNA vaccines are administered as effective prophylactic measures for reducing virus transmission rates and disease severity. To enhance the durability of post-vaccination immunity and combat SARS-CoV-2 variants, boosters have been administered to two-dose vaccinees. However, long-term humoral responses following booster vaccination are not well characterized. A 16-member cohort of healthy SARS-CoV-2 naïve participants were enrolled in this study during a three-dose BNT162b2 vaccine series. Serum samples were collected from vaccinees over 420 days and screened for antigen (Ag)-specific antibody titers, IgG subclass distribution, and neutralizing antibody (nAb) responses. Vaccine boosting restored peak Ag-specific titers with sustained α-RBD IgG and IgA antibody responses when measured at six months post-boost. RBD- and spike-specific IgG4 antibody levels were markedly elevated in three-dose but not two-dose immune sera. Although strong neutralization responses were detected in two- and three-dose vaccine sera, these rapidly decayed to pre-immune levels by four and six months, respectively. While boosters enhanced serum IgG Ab reactivity and nAb responses against variant strains, all variants tested showed resistance to two- and three-dose immune sera. Our data reflect the poor durability of vaccine-induced nAb responses which are a strong predictor of protection from symptomatic SARS-CoV-2 infection. The induction of IgG4-switched humoral responses may permit extended viral persistence via the downregulation of Fc-mediated effector functions.

Keywords: BNT162b2 mRNA vaccine; IgG4 subclass response; SARS-CoV-2; neutralizing antibodies; serum IgG and IgA kinetics; spike (S) and receptor-binding domain (RBD).

Figure 1

Vaccination induces durable SARS-CoV-2 spike RBD-specific serum IgG and IgA responses. The time course of BNT162b2 vaccine delivery and serum collection is shown (A). Receptor-binding domain (RBD)-reactive IgG (B) and IgA (C) levels (Wuhan-Hu-1) were measured by ELISA as described in Methods. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons of mean area under the curve (AUC) values at each sampling point. * p < 0.05, *** p < 0.001, **** p < 0.0001. ns, not significant.

Figure 2

Booster vaccinations induce IgG4-switched Ag-specific responses in serum. RBD-reactive (A) and spike-reactive (B) IgG subclass responses (Wuhan-Hu-1) were measured by ELISA as described in Methods. Statistical significance was determined as in Figure 1. For clarity, statistical comparisons only for post-vax time points 2A and later are shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001. ns, not significant.

Figure 3

Vaccination elicits potent but transient neutralizing antibody responses in serum. Neutralizing antibody responses were evaluated in SARS-CoV-2 spike pseudotyped virus inhibition assays (A) as described in Methods. Statistical significance was determined using two-way ANOVA with Tukey’s multiple comparisons of mean NT₅₀ values at each sampling point: *** p = 0.003, **** p < 0.0001. Correlations between serum NT₅₀ values and RBD-reactive IgG or IgA levels were determined at post-vax time points 2A and 2B (B,C) and post-boosting at time points 3A and 3B (D,E); Pearson coefficients of correlation (r) are listed on each graph. ns, not significant.

Figure 4

BNT162b2-induced serum IgG responses show impaired reactivity against variant SARS-CoV-2 spike RBD proteins that is improved by vaccine boosting. IgG levels against variant SARS-CoV-2 spike RBD proteins were measured by ELISA at time point 2A (A,C) and post-boosting at time point 3A (B,C) as described in Methods. Statistical significance was determined as in Figure 3. **** p < 0.0001. Reactivity for each variant at time points 2A and 3A was compared using paired t-tests. *** p = 0.0003, **** p < 0.0001.

Figure 5

SARS-CoV-2 variants are resistant to BNT162b2-induced neutralizing antibody responses but neutralization activity is improved by vaccine boosting. Neutralizing antibody responses against SARS-CoV-2 variants at time points 2A (A,C) and post-boosting at time point 3A (B,C) were evaluated in SARS-CoV-2 spike pseudotyped virus inhibition assays as described in Methods. Statistical significance was determined as in Figure 3. **** p < 0.0001. Neutralization activity for each variant at time points 2A and 3A was compared using paired t tests. * p < 0.05, ** p < 0.01. ns, not significant.