Function Is More Reliable than Quantity to Follow Up the Humoral Response to the Receptor-Binding Domain of SARS-CoV-2-Spike Protein after Natural Infection or COVID-19 Vaccination

Abstract

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.

Keywords: COVID-19 vaccine; SARS-CoV-2; neutralization; protection; serology.

Figure 1

SARS-CoV-2 specific antibody titers decline over time, while neutralization ability is retained. Panel (A) shows the anti-Spike IgG titers, from all 59 samples, measured by enzyme-linked immunosorbent assay and expressed as titers. In panels (B,C), subset of samples with a pattern of decreased (n = 41) or increased titers (n = 18) in the second sample are presented. Panel (D) shows the blocking activity of serum antibodies expressed as percentage of neutralization by using a surrogate viral neutralization test (sVNT). In panels (E,F), subset of samples with a pattern of decreased (n = 36) or increased titers (n = 23) in the second sample are presented. The threshold for the total antibodies was 0.312. The threshold for IgG titers was 1:100 and for the blocking activity was 30%. The average time between the time of the documented infection and the first samples (n = 59) was 40.37 days, the average time between the first and the second samples (n = 59) was 67.86 days and the average time between the second and the third samples (n = 12) was 99.5 days. The single fourth sample was collected three months after the third sample. Statistical significance was determined by unpaired t-test or by one-way ANOVA multiple comparisons tests and were used to test for increase or decrease among samples. Tukey’s multiple comparisons test was performed as post-hoc test. p < 0.05 was considered significant. Samples 1 and 2 include the 59 subjects in the initial cohort before vaccination. Sample 3 encompass the 15 subjects from whom a collection of a third sample was completed.

Figure 2

Naturally acquired SARS-CoV-2 infection primes an immune response superior to a single COVID-19 vaccine dose. Panel （A） shows the mean time of sample collection following natural infection (n = 25) or after the first vaccine dose (n = 20). In panels (B,C), results from the total anti-S protein and the IgG titer measured by enzyme-linked immunosorbent assay and expressed as OD or titers, respectively, are presented. The threshold for the total antibodies was 0.312 and the threshold for IgG titers was 1:100. All participants with previous exposure to SARS-CoV-2 except one showed detectable antibodies and measurable titers at baseline. Because the threshold 1:100 of our titration assay, the IgG titers at baseline in the unexposed subjects—which had no detectable S-specific antibodies—were set arbitrarily to 50. Panel (D) shows the blocking activity of serum antibodies expressed as percentage of neutralization by using a surrogate viral neutralization test (sVNT). The cutoff for this assay was 30%. As is shown, only one sample in the pre-exposed group contained antibodies below the threshold reported as more than 30% of neutralization. Furthermore, while the distribution of antibodies and titers covers the full Y axis, values in both panel B and C, and in panel D same samples are grouped on the top values area. Statistical significance was determined by one-way ANOVA multiple comparisons test, unpaired t-test or Wilcoxon–Mann–Whitney test, to compare the means and used to test for increases or decreases among samples. p < 0.05 was considered significant. Naturally infected participants (n = 25) out of the 59 with the first sample collected between 12 and 39 days after confirmed infection with SARS-CoV-2 were selected for comparison with the 21 unexposed vaccinated subgroup (healthy vaccinated).

Figure 3

Functional neutralization assays are better predictors of the humoral immune response to COVID-19 mRNA-based vaccinations. Samples are described as the 1st or 2nd samples after 1st or 2nd vaccine dose (1 S-P1 st-vd, 1 S-P2 nd-vd or 2 S-P2 nd-vd) and the mean time of samples collection is shown. Panels (A,B) show the total antibody and IgG titers, respectively, after full vaccination with two vaccine doses. Antibody levels and titers significantly decline in both groups in a second sample collected after the second vaccine (average of 60.3 and 100.5 days after the first vaccine dose for the unexposed and pre-exposed groups, respectively). Despite the difference in sampling time between the two groups, there were no significant differences in the levels of antibodies or titers between groups in the 2 S-P2 nd-vd. Panel (C) shows antibody-blocking capabilities measured by a surrogate viral neutralization assay (sVNT). Highly relevant is the finding that the blocking baseline activity of the pre-exposed individuals is significantly higher than the basely blocking activity induced by the first vaccine dose in unexposed individuals. In addition, two vaccine doses were necessary in the unexposed cohort to induce same percentage of neutralization achieved by just the first dose in the pre-exposed group. The magnitude of neutralization remained at similar levels until the last time point evaluated in both groups, confirming that the surrogate neutralization test is more suitable to determine the efficacy of the humoral immune response to the vaccine. The threshold for the total antibodies was 0.312. The threshold for IgG titers was 1:100 and for the blocking activity was 30%. Statistical significance was determined by one-way ANOVA multiple comparisons test or unpaired t-test to test for an increase or decrease among samples. p < 0.05 was considered significant. The black arrows indicate the moment of vaccine administration related to the timing of sample collection. Healthy vaccinated (n = 21); pre-exposed vaccinated (n = 10).