Humoral and cellular response induced by a second booster of an inactivated SARS-CoV-2 vaccine in adults

Abstract

Background: The Omicron variant has challenged the control of the COVID-19 pandemic due to its immuno-evasive properties. The administration of a booster dose of a SARS-CoV-2 vaccine showed positive effects in the immunogenicity against SARS-CoV-2, effect that is even enhanced after the administration of a second booster.

Methods: During a phase-3 clinical trial, we evaluated the effect of a second booster of CoronaVac®, an inactivated vaccine administered 6 months after the first booster, in the neutralization of SARS-CoV-2 (n = 87). In parallel, cellular immunity (n = 45) was analyzed in stimulated peripheral mononuclear cells by flow cytometry and ELISPOT.

Findings: Although a 2.5-fold increase in neutralization of the ancestral SARS-CoV-2 was observed after the second booster when compared with prior its administration (Geometric mean units p < 0.0001; Geometric mean titer p = 0.0002), a poor neutralization against the Omicron variant was detected. Additionally, the activation of specific CD4⁺ T lymphocytes remained stable after the second booster and, importantly, equivalent activation of CD4⁺ T lymphocytes against the Omicron variant and the ancestral SARS-CoV-2 were found.

Interpretation: Although the neutralizing response against the Omicron variant after the second booster of CoronaVac® was slightly increased, these levels are far from those observed against the ancestral SARS-CoV-2 and could most likely fail to neutralize the virus. In contrast, a robust CD4⁺T cell response may confer protection against the Omicron variant.

Funding: The Ministry of Health, Government of Chile, the Confederation of Production and Commerce, Chile and SINOVAC Biotech.NIHNIAID. The Millennium Institute on Immunology and Immunotherapy.

Keywords: Cellular immunity; CoronaVac®; Humoral immunity; Omicron variant; SARS-CoV-2; Second booster dose.

Fig. 1

Study profile, vaccination scheme and sampling, enrolled volunteers, and cohort included in the study. (A) Schematic representation and sample distribution of performed experiments. From a total of 138 individuals that received two booster doses of CoronaVac® followed up for 4 weeks, neutralizing antibodies were analyzed in blood samples from 63 volunteers by conventional virus neutralization test (cVNT), 87 volunteers by surrogate virus neutralization test (sVNT) and by Pseudovirus-based neutralization assay (pVNT). In addition, sVNT and pVNT were used to evaluate the neutralizing response induced by a second booster dose against the Delta (B.1.617.2) and the Omicron (B.1.1.529) variants. Cellular immunity was analyzed in blood samples from 45 volunteers at each time point. (B) Blood sampling times, before the first vaccination/pre-immune (T1), at 4 weeks (+3 weeks) after the second dose (T2), before the administration of the first booster (−9 weeks) (T3), at 4 weeks (−1/+2 weeks) after the first booster (T4), before the administration of the second booster (−9 weeks) (T5) and at 4 weeks (+5 weeks) after the second booster (T6), and vaccination schedule.

Fig. 2

Humoral response of volunteers who received a second booster dose of CoronaVac.® The neutralizing capacity of circulating antibodies in adults was evaluated in blood samples collected before the first vaccination/pre-immune (T1), at 4 weeks (+3 weeks) after the second dose (T2), before the administration of the third dose (−9 weeks) (T3), at 4 weeks (−1/+2 weeks) after the first booster (T4), before the administration of the second booster (−9 weeks) (T5) and at 4 weeks (+5 weeks) after the second booster (T6), and vaccination schedule. (A) Neutralizing capacity of circulating antibodies against SARS-CoV-2 in serum of 87 volunteers was determined by a surrogate Viral Neutralization Test (sVNT) expressed as IU/ml. Numbers on top of each data set represents the Geometric mean unit (GMU), and horizontal lines represent the 95% CI. Dashed line: limit of detection: 4.036 (B) For cVNT, reciprocal dilution of sera required to prevent in vitro infection obtained sera from 63 adults required to prevent in vitro infection of Vero E6 cells. Numbers on top of each data set represents the Geometric mean titer (GMT), and horizontal lines represent the 95% CI. Dashed line: limit of detection: 1 (C) GMT of neutralizing antibodies against the WT-spike, Delta-spike and Omicron-spike proteins detected in the serum of 87 volunteers immunized with CoronaVac® through sVNT. Dashed line: limit of detection: 1. Red values under the significance line: indicate a decrease in the means of the two compared time points; Blue values: indicate an increase in the means of the two compared time points. (A–B) Data was analyzed with ANOVA with the Geisser-Greenhouse correction test followed by a post-hoc Sidak multiple test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. (C) Two-way ANOVA test followed by a post-hoc Tukey multiple test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Each dot represents a single volunteer and for each volunteer, the experimental data was obtained in a single experiment.

Fig. 3

Cellular response of volunteers that received a second booster dose of CoronaVac.® Cellular response in adults was evaluated in PBMCs collected before the first vaccination/pre-immune (T1), at 4 weeks (+3 weeks) after the second dose (T2), before the administration of the third dose (−9 weeks) (T3), at 4 weeks (−1/+2 weeks) after the first booster (T4), before the administration of the second booster (−9 weeks) (T5) and at 4 weeks (+5 weeks) after the second booster (T6), and vaccination schedule. (A) The percentage of AIM⁺ (OX40⁺CD137⁺) CD4⁺ T cells and (B) AIM⁺ (CD69⁺CD137⁺) CD8⁺ T cells was determined in PBMCs of 45 adult volunteers by flow cytometry. PBMCs were stimulated for 24h with mega-pools of peptides derived from proteins of WT SARS-CoV-2. The number of IFN-γ producing SFCs was determined by ELISPOT upon stimulation for 48h with mega-pools of (C) S + R peptides or with (D) CD8A + B peptides. Dashed line: positivity threshold for PBMCs stimulated with MP-S + R (42.34 #SFC and MP-CD8A + B (104.92 #SFC) was calculated using the median twofold standard deviation of each sample of the pre-immune group. (E) The percentage of AIM⁺ (OX40⁺CD137⁺) CD4⁺ T cells from PBMCs of 45 adult volunteers were analyzed by flow cytometry after the stimulation for 24h with mega-pools of peptides derived from the Spike protein of the WT SARS-CoV-2, the Delta and the Omicron variants. (F) The number of IFN-γ producing SFCs was determined by ELISPOT assays PBMCs of 45 adult volunteers were analyzed by flow cytometry after the stimulation for 48h with mega-pools of peptides derived from the Spike protein of the WT SARS-CoV-2, the Delta (B.1.617.2) and the Omicron (B.1.1.529) variants. Horizontal lines represent mean and standard deviation. Flow cytometry data was normalized against the DMSO control. (A–D) Data was analyzed using a non-parametric Friedman test followed by a post-hoc Dunn's test for multiple comparisons. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001. (E–F) Two-way ANOVA test followed by a post-hoc Tukey multiple test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Each dot represents a single volunteer and for each volunteer, the experimental data was obtained in a single experiment. Red values under the significance line: indicate a decrease in the means of the two compared time points; Blue values: indicate an increase in the means of the two compared time points.