Cellular and humoral immune responses and breakthrough infections after three SARS-CoV-2 mRNA vaccine doses

Abstract

Background: SARS-CoV-2 vaccination has proven the most effective measure to control the COVID-19 pandemic. Booster doses are being administered with limited knowledge on their need and effect on immunity.

Objective: To determine the duration of specific T cells, antibodies and neutralization after 2-dose vaccination, to assess the effect of a third dose on adaptive immunity and to explore correlates of protection against breakthrough infection.

Methods: 12-month longitudinal assessment of SARS-CoV-2-specific T cells, IgG and neutralizing antibodies triggered by 2 BNT162b2 doses followed by a third mRNA-1273 dose in a cohort of 77 healthcare workers: 17 with SARS-CoV-2 infection prior to vaccination (recovered) and 60 naïve.

Results: Peak levels of cellular and humoral response were achieved 2 weeks after the second dose. Antibodies declined thereafter while T cells reached a plateau 3 months after vaccination. The decline in neutralization was specially marked in naïve individuals and it was this group who benefited most from the third dose, which resulted in a 20.9-fold increase in neutralization. Overall, recovered individuals maintained higher levels of T cells, antibodies and neutralization 1 to 6 months post-vaccination than naïve. Seventeen asymptomatic or mild SARS-CoV-2 breakthrough infections were reported during follow-up, only in naïve individuals. This viral exposure boosted adaptive immunity. High peak levels of T cells and neutralizing antibodies 15 days post-vaccination associated with protection from breakthrough infections.

Conclusion: Booster vaccination in naïve individuals and the inclusion of viral antigens other than spike in future vaccine formulations could be useful strategies to prevent SARS-CoV-2 breakthrough infections.

Keywords: COVID-19; SARS-CoV-2; breakthrough infection; hybrid immunity; immune response; third dose; vaccination.

Figure 1

Development and maintenance of SARS-CoV-2-specific cellular and humoral immunity after 3 mRNA vaccine doses. (A) Study design and cohorts, samples were collected pre-vaccine, 21 days after the first dose, 15, 30, 90 and 180 days after the second dose and 30 days after third boost dose. (B) Dynamics of vaccine-triggered S1-IFN-γ-producing T cell response in SARS-CoV-2 naïve individuals (in blue). Data is represented as spot forming unit (sfu) per million PBMC. Dashed lines represent the positivity cut-off established by using a non-infected control group: >25 sfu/10⁶ PBMC. (C) anti-S1 IgG levels elicited by mRNA vaccination in SARS-CoV-2 naïve individuals. Dashed line represents the established cut-off of positivity: OD ratio ≥1.1. (D) Vaccine-triggered neutralizing activity in SARS-CoV-2-naïve serum samples, represented as International Units per ml (IU/ml). (E–G) Dynamics of S1-IFN-γ-producing T cell (E), anti-S1 IgG (F) and neutralizing antibody (G) responses elicited by mRNA vaccination in SARS-CoV-2 recovered individuals (in yellow). (H–J) Comparison of S1 T cell (H), anti-S1 IgG (I) and neutralizing (J) responses between SARS-CoV-2 naïve and recovered individuals. Green arrows represent the time of SARS-CoV-2 infection. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann Whitney, Wilcoxon signed rank or Kruskal-Wallis tests, ns: not statistically significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2

Factors associated with protection against breakthrough infection after SARS-CoV-2 vaccination. (A, B) Flowchart of SARS-CoV-2 naïve (A) and recovered (B) individuals included in the study and the frequency of SARS-CoV-2 infection reported after the second or third vaccine dose. (C, D) SARS-CoV-2-specific IFN-γ-producing T cell responses reactive to the S1, M and N proteins in subjects who remained SARS-CoV-2 naïve during follow-up (C) and in recovered (D) individuals. (E) Clustering based on S1-IFN-γ-producing T cell average and neutralizing titer average 15 days after the second BNT162b2 dose, when the vaccine-elicited immune response peaked, in naïve individuals. Green arrows represent the time of SARS-CoV-2 infection. Black crosses represent SARS-CoV-2-infected subjects after mRNA vaccination. Horizontal bars and whiskers represent median values and interquartile ranges, respectively. The significance between groups was determined using Mann-Whitney or Kruskal-Wallis tests, ns: not statistically significant, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 3

Effect of breakthrough infections on existing SARS-CoV-2 immunity. (A) Comparison of S1-IFN-γ-producing T cells among naïve individuals who remain naïve (in blue) and those with breakthrough infections after 2^nd (in grey) and 3^rd dose (in purple). Out of the 5 subjects who had SARS-CoV-2 infection after the 2^nd dose only 3 subjects received the third vaccine dose. (B, C) Longitudinal data on the dynamics of specific T cells in the 5 subjects infected by SARS-CoV-2 after the 2^nd dose (B) and in the 12 individuals infected after the 3^rd dose (C). (D) Comparison of anti-S1IgG levels among SARS-CoV-2 naïve individuals and breakthrough infections after 2^nd and 3^rd dose. (E, F) Dynamics of antibody levels in subjects infected after the 2^nd (E) and the 3^rd dose (F). (G) Comparison of neutralizing activity among SARS-CoV-2 naïve individuals and breakthrough infections after 2^nd and 3^rd dose. (H, I) Neutralizing activity in SARS-CoV-2 infected subjects after the 2^nd (H) and the 3^rd dose (I). Green arrows represent the time of SARS-CoV-2 infection. Dashed lines represent the positivity cut-off. The significance between groups was determined using Mann Whitney test, *p<0.05, **p<0.01, ***p<0.001. (See Figure 1 footnote for more detailed information).