Over 50% of the world's population has acquired immunity to SARS-CoV-2 through infection or vaccination, creating a complex immunological landscape. While both infection and vaccination induce protection, the trajectory of adaptive immune responses and long-term immunity with repeated antigen exposure remain unclear. mRNA vaccines effectively induce humoral and cellular immunity, preventing severe disease. High levels of anti-Spike antibodies are initially induced, but they wane over time, with cellular immunity playing a critical role in long-term protection. Specifically, CD4+ T follicular helper (Tfh) cells are crucial for B cell maturation, and CD4+ Th1 cells support antiviral effector memory functions. While mRNA vaccines successfully induce coordinated immune responses, the long-term impact of recurrent boosters is less understood, especially in individuals with hybrid immunity (a combination of infection and vaccination). This study aimed to characterize the quality, magnitude, and durability of immune memory after three doses of the Pfizer BNT162b2 mRNA vaccine in both SARS-CoV-2-naive and previously infected individuals, comparing their long-term immune responses.

Extensive research demonstrates that mRNA vaccines effectively induce robust humoral and cellular immunity against SARS-CoV-2, preventing severe disease. High levels of anti-Spike antibodies are initially produced, but these wane and plateau over time. Despite this decline, cellular immunity, particularly from CD4+ and CD8+ T cells, is crucial for long-term protection. Studies have shown that mRNA vaccines elicit functional T cell immunity, with CD4+ and CD8+ T cell subsets contributing to protective responses and long-term memory. Previous research has shown an enhanced response shortly after the initial mRNA vaccination in previously infected individuals, but longitudinal data examining multiple immune compartments simultaneously are limited. The existing literature highlights the need for a comprehensive understanding of long-term immune responses to repeated mRNA vaccinations, particularly in diverse immunological settings resulting from varying exposure histories to SARS-CoV-2.

This study utilized samples from the SPARTA cohort, focusing on 40 participants: 19 with prior SARS-CoV-2 infection (preimmune) and 21 SARS-CoV-2-naive individuals. Participants received three doses of the Pfizer BNT162b2 monovalent mRNA vaccine. Blood samples were collected at six time points: pre-vaccination, and at 30, 90, and 180 days after the second dose, and at 30 and 180 days after the third dose. Several assays were performed: ELISA to measure anti-RBD IgG antibody binding; a cytopathic effect-based assay to assess SARS-CoV-2 virus neutralization; a FluoroSpot assay to quantify Spike and RBD-specific antibody-secreting cells (ASCs) of different isotypes (IgG, IgM, IgA); an activation-induced marker (AIM) assay using flow cytometry to assess Spike-specific CD4+ and CD8+ T cells, analyzing memory subsets (naive, CM, EM, TEMRA); and intracellular cytokine staining to analyze cytokine production (IFN-γ, IL-2, TNF-α) by CD4+ and CD8+ T cells. Statistical analyses included Mann-Whitney U or Wilcoxon tests for comparisons between two groups, and Kruskal-Wallis and Dunn's post-test for multiple group comparisons.

The study found that vaccination induced robust antibody responses in both groups, with preimmune individuals exhibiting significantly higher antibody concentrations after the second dose (p=0.005). However, this difference diminished by six months post-second dose (p<0.0001). The third dose significantly boosted antibody levels in both groups (p<0.0001), resulting in comparable peak levels. Neutralizing antibody titers followed a similar pattern, with preimmune individuals exhibiting higher levels initially, but both groups achieving similar levels after the third dose. Regarding antibody-secreting cells (ASCs), preimmune individuals had higher IgG Spike-specific ASCs before vaccination (p=0.03), but after the third dose, both groups displayed similar frequencies. Concerning T cells, preimmune participants had higher Spike-specific CD4+ T cell populations before vaccination (p=0.01), but differences between groups diminished after the second dose. The third dose boosted T cell responses in both groups but did not significantly increase the difference between groups. Similar results were observed for CD8+ T cells, with prior infection resulting in more detectable CD8+ T cells initially. Memory T cell subset analysis revealed primarily central memory (CM) CD4+ T cells and effector memory (EM) and TEMRA CD8+ T cells in both groups. Circulating T follicular helper (cTfh) cells were significantly higher in preimmune individuals prior to and after the second vaccination, correlating with antibody levels. Preimmune individuals also had a higher frequency of Th1 cytokine-producing CD4+ T cells initially, but differences between groups diminished after the second dose. Finally, early CD4+ T cell responses (from infection or initial vaccination) positively correlated with neutralizing antibody titers at later time points.

This study demonstrates that prior SARS-CoV-2 infection enhances the initial mRNA vaccine response, particularly the humoral response, but this benefit is less pronounced in the long term. The third dose of the vaccine effectively boosted both humoral and cellular immunity in both groups, leading to comparable levels of long-term memory. The enhanced initial response in preimmune individuals is likely due to the recall of pre-existing immunity. The correlation between early CD4+ T cell responses and later neutralizing antibody titers highlights the importance of cellular immunity in shaping the humoral response. The results support the use of booster doses to maintain robust humoral immunity and suggest that future vaccine strategies should focus on enhancing cellular immunity, specifically Th1 cells, cTfh cells, and EM cell subsets, to ensure long-lasting protection.

Prior SARS-CoV-2 infection significantly enhances the initial response to mRNA vaccination, mainly improving antibody responses. However, the long-term benefit is modest. Three doses of the vaccine effectively boost both humoral and cellular immunity, resulting in comparable levels of long-term memory in both naive and preimmune individuals. Future research should focus on enhancing T cell responses, particularly Th1 cells, cTfh cells, and EM cell subsets to improve long-term protection against SARS-CoV-2 variants.

The study is limited by its focus on peripheral blood samples, which may not fully reflect the immune responses in other tissues. The use of peptide pools for T cell stimulation may underestimate the frequency of epitope-specific T cells. The study's observation period of one year may not be sufficient to assess the very long-term durability of the immune response. The study population was largely composed of white participants, potentially limiting the generalizability of the findings to other ethnicities.