Booster Gam-COVID-Vac vaccination elicited high titers of serum Ad26-neutralizing antibodies
Fifty-nine individuals were recruited to our study. All study participants were vaccinated with two injections of Gam-COVID-Vac in January-April, 2021. The first dose was rAd26, followed by the rAd5-based second dose 21 days later. After 9 months [range 249–300 days, Table 1], all participants received their booster vaccination. In the core cohort (n = 48), rAd26-based first component of the Gam-COVID-Vac served as a booster, and in the comparison cohort (n = 11), the boosters were Pfizer-BioNTech’s Comirnaty (n = 10), or Janssen’s Ad26.COV2.S (n = 1).
Table 1
Regimen of vaccination | Homologous | Heterologous |
Number of participants | 48 | 11 |
Age, years, median (range) | 27 (18–73) | 24 (23–39) |
Female Male | 23 25 | 4 7 |
First dose Gam-COVID-Vac (Ad26) | 48 | 11 |
Second dose Gam-COVID-Vac (Ad5) | 48 | 11 |
Booster vaccine (% of total) Gam-COVID-Vac (Ad26) Pfizer J&J | 48 (100) 0 0 | 0 10 (91) 1 (9) |
Time between prime vaccination and booster days, median (range) | 273 (249–300) | 284 (267–328) |
Probe sampling after booster, days, median (range) | 33 (29–41) | 43 (38–49) |
Fever Injection site reactions Weakness Headache | 14 (29) 22 (46) 21 (43) 6 (13) | 6 (55) 9 (82) 7 (64) 2 (18) |
Sera samples were collected at baseline (T0), one, three and six months later (T1, T3, and T6), and then immediately before, and one and four months after the booster immunization (T9, T10, and T14, respectively) (Fig. 1a).
The outline of Ad26-neutralization assay used in our work is presented in Fig. 1b. To model Ad26 infection and neutralization in vitro, human lung epithelial cancer cell line A549 was used as the target. These cells were infected with rAd26 vector encoding the full-length SARS-CoV-2 glycoprotein S. The percentage of Spike+ A549 cells measured 24 h later served as readout of successful rAD26-mediated infection.
First of all, we measured the titers of anti-vector antibodies before (T9) and after (T10) the booster Gam-COVID-Vac vaccination, in a core cohort with a homologous regimen of revaccination (i.e. in study participants who received Gam-COVID-Vac as both the prime and the boost). To do so, we assessed the ability of pre- and post-boost serum samples to block the infection of recombinant Ad26 virus. Sera obtained from the vaccinated individuals inhibited Ad26 vector entry in a concentration-dependent manner (Fig. 1c) and the level of Ad26 NAb was quantified as neutralization half-maximal inhibitory serum dilution (ID50).
Sera from the vaccinated individuals sampled at a pre-boost timepoint T9 were found to noticeably inhibit infection of A549 cells with Ad26 vector (median = 278, Fig. 1d), with 90% (27/30) of the samples displaying anti-Ad26 NAb levels above the baseline (ID50 for healthy non-vaccinated donors, median = 56). These numbers are consistent with the fact that all study participants had their first dose of Gam-COVID-Vac, which is rAd26-based, 9 months earlier. Following revaccination, Ad26-NAb titers increased 7.6-fold (median ID50 = 2105, P < 0.0001), compared to the pre-boost levels, and the anti-Ad26 seroprevalence reached 100%.
Individuals from the comparison arm received a heterologous boost (i.e. different platform/delivery vector/Spike). The core and comparison cohorts displayed comparable levels of anti-Ad26 NAbs at pre-boost. All participants who received an mRNA vaccine booster ((n = 10), had unaltered Ad26 NAb levels (P = 0.0645). In contrast, a 14-fold increase in Ad26 NAb activity was noted in a single recipient of the Ad26.COV2.S boost.
Thus, Ad26-based but not mRNA-based revaccination predictably led to the formation of anti-vector responses in all of the vaccinees. This is consistent with the idea that increase in Ad26-neutralizing titers is due to the administration of rAd26-based vector, rather than the vaccination per se.
Figure 1: Booster vaccination with rAd26-vectored Gam-COVID-Vac elicited high titers of serum Ad26-specific NAbs. a Timeline showing Gam-COVID-Vac vaccination and blood sampling timepoints, labeled according to the number of months elapsed after the boost. b Outline of the rAd26 virus neutralization assay. In a subpanel, representative flow plot shows Spike expression on A549 cells after rAd26-Spike infection (violet) and a negative control staining with isotype-matched irrelevant mouse monoclonal antibody (blue). c Representative rAd26 neutralization curves for serum samples before (T9) and one month after (T10) the Gam-COVID-Vac boost. Ad26 neutralizing activity was defined by the percentages of Spike+ A549 cells relative to no antibody controls. d Serum Ad26 virus-neutralizing antibody titers before and one month after booster Gam-COVID-Vac vaccination. In violet, blue, and orange are Gam-COVID-Vac vaccinated individuals that received a boost of Gam-COVID-Vac (violet, homologous revaccination, left panel), and mRNA (blue) or Ad26.COV2.S (orange) (heterologous revaccination, right panel) vaccine, correspondingly. The dotted lines indicate the pre-prime baseline. Medians are plotted and statistical significance was determined using Wilcoxon matched-pairs signed-rank test. Data are presented as median values and interquartile ranges (IQR). **** P < 0.0001.
RBD-binding and SARS-CoV-2-neutralizing antibody titers are independent on the level of pre-boost anti-Ad26 antibodies.
Of all Spike-specific antibodies, RBD-targeting antibodies are known to constitute the vast majority of SARS-CoV-2 NAbs 17, hence in order to measure recall humoral responses after booster vaccination we first analyzed the levels of anti-RBD IgGs. Nine months after the prime and immediately before the booster shot (timepoint T9), study participants displayed pronounced RBD-specific IgG titers (median = 231 ng/mL). Seroprevalence of anti-RBD IgG was 58%. As expected, Gam-COVID-Vac revaccination resulted in a 2.5-fold increase in RBD-specific IgG levels (median = 582 ng/mL, P < 0.0001) and seroprevalence 96% (46/48) (Fig. 2a).
To evaluate whether the level of pre-boost Ad26-specific immunity may affect the levels of post-boost Spike-specific antibodies, we tested for associations between the levels of RBD-specific IgGs at T10 vs Ad26-specific NAbs at T9 (Fig. 2b). The Spearman’s correlation analysis did not reveal any association, indicating that pre-boost anti-Ad26 NAbs did not affect the production of RBD-binding IgGs.
While RBD-binding activity of antibodies is known to be important for protective immunity against SARS-CoV-2, their functional activity, i.e. the ability to neutralize the virus, may serve as better predictor of protection. To quantify the SARS-CoV-2-neutralizing activity of sera from vaccinated individuals, virus neutralization assay was performed using lentiviral particles pseudotyped with Spike from SARS-CoV-2 WT (ancestral variant) or VOCs (Fig. 2c).
Before the booster at T9, serum samples demonstrated ID50 ~ 50 for WT, Alfa, and Epsilon VOCs of SARS-CoV-2. In addition, these sera also neutralized Delta and Omicron variant entry, albeit the latter was neutralized at a lower potency compared to WT (P < 0.0001) (Fig. 2d).
Much as was observed for RBD-specific IgG responses, the SARS-CoV-2 WT neutralizing capacity of the sera significantly increased after the booster shot (T10) compared with the pre-booster (T9) levels (P < 0.0001). Furthermore, this was accompanied with higher neutralizing activities against Alfa, Epsilon, Delta, and Omicron VOCs (fold increase 4, 2, 2, and 4, respectively) (Supplementary Fig. 1).
We next sought for possible associations between the immunological parameters assayed. As expected, a good correlation between RBD-specific IgGs and SARS-CoV-2 WT NAbs at T10 was observed (Spearman r = 0.8031, P = 0.0001, respectively, Fig. 2e). At the same we failed to detect any association between the levels of Ad26-specific NAbs at T9 and SARS-CoV-2-neutralizing activities against WT or other tested VOCs (Alfa, Epsilon, Delta, and Omicron) (Spearman r = 0.022, P = 0.8835; r = -0.0098, P = 0.9477; r = 0.0230, P = 0.8780; r = -0.0082, P = 0.9563; r = -0.0592, P = 0.6926, respectively, Fig. 2f). These data suggest that both RBD-binding and SARS-CoV-2 NAb recall responses are independent of the pre-boost levels of Ad26-specific NAbs.
Figure 2: Humoral anti-SARS-CoV-2 recall responses after booster Gam-COVID-Vac vaccination. a Serum anti-RBD IgG levels before and one month after the booster injection measured by ELISA. b Spearman’s correlation between pre-boost (T9) Ad26-specific nAb values and the serum levels of anti-RBD IgGs at T10. c Schematic representation of the SARS-CoV-2 virus neutralization assay. d Neutralizing antibody titer (ID50) against SARS-CoV-2 WT and Alfa, Epsilon, Delta, or Omicron VOCs. On the left and right subpanels, absolute ID50 values and fold increase after the booster vaccination are shown. Numbers indicate medians. e Spearman’s correlation between RBD-specific IgG and SARS-CoV-2 WT NAbs at T10. f Association of pre-boost (T9) Ad26-specific NAb levels with post-boost (T10) SARS-CoV-2 NAb titers against WT, Alfa, Epsilon, Delta, or Omicron VOCs, calculated using Spearman’s rank correlation. Participant IDs are shown. The dotted lines indicate the threshold for positivity.
Dynamics of Ad26 virus-neutralizing, SARS-CoV-2 RBD-binding antibodies, and SARS-CoV-2 NAbs
Our previous analysis has established the increase in anti-Ad26 antibody titers following homologous revaccination. We asked when during the course of vaccinations these antibodies appeared and how long would these antibodies persist at detectable levels.
In a subcohort of donors (n = 11), Ad26-specific NAb levels were monitored longitudinally from the first dose of the vaccine and up to 14 months at 7 timepoints. Figure 3 illustrates the dynamics of Ad26 NAbs, RBD-binding and SARS-CoV-2 WT neutralizing antibodies across six time intervals.
Pre-vaccine median level of Ad26 NAbs was on the baseline. It must be noted though that one individual in our cohort had high pre-existing Ad26 NAb titers. One month after the prime vaccination, the levels of Ad26 NAbs demonstrated a ~ 10-fold increase, and slightly declined thereafter, yet persisted for the duration of 6 months of the follow-up. During the study, Ad26 NAb seroprevalence was invariably above 64%. Booster immunization restored Ad26 NAb levels back to the maximum levels, which was again followed by a minor decline. The magnitudes of Ad26 NAb peaks following prime and booster vaccination were generally very similar.
The kinetics of RBD-specific IgG and SARS-CoV-2 WT NAb responses followed the pattern observed for Ad26 NAbs (Fig. 3b-c). Namely, after the second shot, RBD-binding IgG and SARS-CoV-2 WT NAb levels progressively declined for 6 months and reached a minimum before revaccination. Notably, RBD-binding antibody levels demonstrated the strongest decline, with nearly background levels found by the 9th month of study. Nonetheless, SARS-CoV-2 WT NAb activity remained clearly detectable. Based on these numbers, setting a booster shot at 6–9 months appears well-substantiated, as this offers the advantage of declining Ad26 NAb titers with persisting SARS-CoV-2 humoral immunity.
Figure 3: Longitudinal analysis of humoral responses to Gam-COVID-Vac vaccination. Ad26-specific NAb (a), anti-RBD IgG (b) and SARS-CoV-2 WT NAb (c) responses before the first dose of Gam-COVID-Vac and up to 14 months later. Colored lines represent the dynamics of median values. Arrows indicate immunization timepoints.