Evaluation of Long-Term Adaptive Immune Responses Specific to SARS-CoV-2: Effect of Various Vaccination and Omicron Exposure

Abstract

The immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) becomes increasingly complex as individuals receive different combinations of vaccine doses and encounter breakthrough infections. Our study focused on the immunogenicity observed over a two-year period in healthy individuals who completed a two-dose series and then experienced booster and/or Omicron infection. In June 2023, we recruited 78 healthcare workers who had previously participated in clinical research initiated in March 2021 at a single medical center in South Korea. At 1, 5, 11, and 25 months after a second dose, we assessed SARS-CoV-2-specific humoral and cellular immune responses. Longitudinal monitoring revealed a significant decline in humoral immunity levels after the second vaccine dose, followed by a substantial increase post-third vaccination or breakthrough infection. In contrast, stable cellular immune responses were consistently observed, with peak humoral and cellular immune measures reached at 25 months after the second dose. Among infection-naïve participants, three-dose vaccinated individuals had decreased neutralizing activity against wild-type (WT) and negative activities against Omicron subvariants BA.2 and BA.4/5, whereas those who received a fourth dose of bivalent BNT had significantly increased neutralizing activity (p < 0.05). All immune metrics tended to increase as the number of vaccine doses increased. Among participants with 4-exposure, homologous vaccination (mRNA × 4) led to higher humoral immunity, whereas heterologous vaccination (ChAd × 2/mRNA × 2) induced stronger cellular responses against multiple SARS-CoV-2 variants by enzyme-linked immunospot assays (p < 0.05). Immune responses from bivalent vaccines or Omicron infection did not show statistically significant differences among exposure number-matched participants (p > 0.05). Omicron exposure significantly increased cross-neutralizing activity, but magnitude of cellular immunity was not significantly altered by Omicron exposure. Our longitudinal study highlights the evolving complexity of SARS-CoV-2 immune responses, showing enhanced immunity with multiple vaccine doses and robust cellular responses from heterologous vaccination. These findings emphasize the need for ongoing surveillance to optimize vaccination strategies against emerging variants.

Keywords: Omicron; SARS-CoV-2; breakthrough infection; cellular immunity; humoral immunity; long-term; vaccine.

Figure 1

Timeline illustrating vaccination, infection and sampling timepoints of the study participants. The icons depicted in the figure represent the following: A syringe indicates one vaccine dose administration, blood droplets denote blood sampling, and a virus signifies breakthrough infection. Parentheses accompanying the virus figures indicate that some participants in the group experienced either a third or second infection.

Figure 2

Longitudinal kinetics of SARS-CoV-2–specific anti-S/RBD (A), neutralizing activity to WT (B), and the magnitude of T cell response by IFN-γ ELISpot (C). All 78 participants of this study are presented as T4 results. The available previous measurements of immunogenicity parameters are presented as T1, T2, and T3, as defined in Supplementary Table S1. The individual dots represent an assay result at each sampling point. The horizontal dotted line in (B) indicates the manufacturer’s suggested cut-off, while ns, not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Wilcoxon sign-rank test.

Figure 3

Effect of the number of vaccine doses on SARS-CoV-2–specific humoral and cellular immune response, stratified by the number of breakthrough infections. Humoral responses were measured using anti-S/RBD levels (A), neutralizing activity against WT (B), BA.2 (C), and BA.4/5 (D). The magnitudes of T-cell responses were measured using an enzyme-linked immunospot assay (E–H). The number of vaccine doses is color-coded, as indicated at the right corner. The horizontal line indicates the median and the error bars indicate interquartile ranges. The horizontal dotted line in (B–D) indicates the manufacturer’s suggested cut-off. * p < 0.05 by Mann–Whitney test, otherwise non-significant.

Figure 4

Effect of heterologous versus homologous vaccination on SARS-CoV-2–specific humoral and cellular immune response among participants with 4-exposure. Anti-S/RBD (A); neutralizing activity against WT, BA.2, and BA.4/5 (B); magnitude of T-cell responses using an enzyme-linked immunospot assay (C). The horizontal line indicates median and the error bars indicate interquartile ranges. The horizontal dotted line in B indicates the manufacturer’s suggested cut-off, while ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 by Mann–Whitney test.

Figure 5

Effect of breakthrough infection and bivalent vaccination on SARS-CoV-2–specific humoral and cellular immune response, stratified by the number of total antigenic exposure. Anti-S/RBD (A); neutralizing activity against WT (B), BA.2 (C), and BA.4/5 (D); magnitude of T-cell responses using an enzyme-linked immunospot assay (E–H). The horizontal line indicates median and the error bars indicate interquartile ranges. The horizontal dotted line in (B–D) indicates the manufacturer’s suggested cut-off. Color coded as the following: green, infection-naïve; red, infected once; blue, infected two or more times. * p < 0.05 by Mann–Whitney test, otherwise non-significant.

Figure 6

Breadth of SARS-CoV-2–specific adaptive immune response stratified by the number of total antigenic exposures. Neutralizing activity against WT and Omicron subvariants BA.2 and BA.4/5 (A); magnitude of T-cell responses against S1 peptide pools of various SARS-CoV-2 subvariants (B). The horizontal line indicates median and the error bars indicate interquartile ranges. The horizontal dotted line in A indicates the manufacturer’s suggested cut-off, while ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Kruskal–Wallis test (orange-colored) with post hoc Dunn’s multiple comparisons test (gray-colored).