mRNA Vaccination Induces Durable Immune Memory to SARS-CoV-2 with Continued Evolution to Variants of Concern

Abstract

SARS-CoV-2 mRNA vaccines have shown remarkable efficacy, especially in preventing severe illness and hospitalization. However, the emergence of several variants of concern and reports of declining antibody levels have raised uncertainty about the durability of immune memory following vaccination. In this study, we longitudinally profiled both antibody and cellular immune responses in SARS-CoV-2 naïve and recovered individuals from pre-vaccine baseline to 6 months post-mRNA vaccination. Antibody and neutralizing titers decayed from peak levels but remained detectable in all subjects at 6 months post-vaccination. Functional memory B cell responses, including those specific for the receptor binding domain (RBD) of the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) variants, were also efficiently generated by mRNA vaccination and continued to increase in frequency between 3 and 6 months post-vaccination. Notably, most memory B cells induced by mRNA vaccines were capable of cross-binding variants of concern, and B cell receptor sequencing revealed significantly more hypermutation in these RBD variant-binding clones compared to clones that exclusively bound wild-type RBD. Moreover, the percent of variant cross-binding memory B cells was higher in vaccinees than individuals who recovered from mild COVID-19. mRNA vaccination also generated antigen-specific CD8+ T cells and durable memory CD4+ T cells in most individuals, with early CD4+ T cell responses correlating with humoral immunity at later timepoints. These findings demonstrate robust, multi-component humoral and cellular immune memory to SARS-CoV-2 and current variants of concern for at least 6 months after mRNA vaccination. Finally, we observed that boosting of pre-existing immunity with mRNA vaccination in SARS-CoV-2 recovered individuals primarily increased antibody responses in the short-term without significantly altering antibody decay rates or long-term B and T cell memory. Together, this study provides insights into the generation and evolution of vaccine-induced immunity to SARS-CoV-2, including variants of concern, and has implications for future booster strategies.

Figure 1.. SARS-CoV-2 mRNA vaccines induce robust antibody responses.

A) University of Pennsylvania COVID-19 vaccine study design and cohort summary statistics. B) anti-Spike and anti-RBD IgG concentrations over time in plasma samples from vaccinated individuals. C) Pseudovirus neutralization titers against wild-type D614G or B.1.351 variant Spike protein over time in plasma samples from vaccinated individuals. Data are represented as focus reduction neutralization titer 50% (FRNT50) values. D) Comparison of D614G and B.1.351 FRNT50 values at 6 months post-vaccination. Statistics were calculated using paired non-parametric Wilcoxon test E) Correlation between anti-Spike or anti-RBD IgG and neutralizing titers (D614G = black, B.1.351 = green; statistics were calculated using non-parametric Spearman rank correlation). Dotted lines indicate the limit of detection for the assay. For B and C, fractions above plots indicate the number of individuals above their individual baseline at memory timepoints. Decay rates were calculated using a piecewise linear mixed effects model with censoring. Decay rates were similar in SARS-CoV-2 naïve and recovered groups unless otherwise indicated. Blue and red values indicate comparisons within naïve or recovered groups.

Figure 2.. SARS-CoV-2 mRNA vaccines generate durable and functional memory B cell responses.

A) Experimental design and B) gating strategy for quantifying the frequency and phenotype of SARS-CoV-2-specific memory B cells by flow cytometry. Antigen specificity was determined based on binding to fluorophore-labeled Spike, RBD, and influenza HA tetramers. C) Frequencies of SARS-CoV-2 Spike+, Spike+ RBD+, and influenza HA+ memory B cells over time in PBMC samples from vaccinated individuals. Data are represented as a percentage of total B cells and fractions above plots indicate the number of individuals above their individual baseline at memory timepoints. Decay rates were calculated using a piecewise linear mixed effects model with censoring. D) Frequency of isotype-specific Spike+ and E) Spike+ RBD+ memory B cells over time. F) Percent CD71+ of total Spike+ memory B cells over time. G) Experimental design for differentiation of memory B cells into antibody secreting cells. H) anti-Spike IgG levels in culture supernatants over time from PBMCs stimulated with PBS control or R848 + IL-2 (n=4). I) anti-Spike IgG levels in culture supernatants after 10 days of stimulation J) Correlation of Spike+ memory B cell frequencies by flow cytometry with anti-Spike IgG levels from in vitro stimulation. K) Correlation of anti-Spike IgG levels from in vitro stimulation with RBD-binding inhibition. For J and K, correlations were calculated using non-parametric Spearman rank correlation. Statistics were calculated using unpaired non-parametric Wilcoxon test with Holm correction for multiple comparisons. Blue and red values indicate comparisons within naïve or recovered groups.

Figure 3.. Memory B cells induced by mRNA vaccination are cross-reactive to SARS-CoV-2 variants of concern and increase in frequency over time.

A) Experimental design and B) gating strategy for quantifying the frequency and phenotype of Spike component and variant-specific memory B cells by flow cytometry. Specific mutations in B.1.1.7, B.1.351, or B.1.617.2 variant RBDs are indicated. C) Frequencies of Spike+ NTD+, Spike+ WT RBD+, Spike+ RBD++++ (all variant binding), and Spike+ S2+ memory B cells over time in PBMC samples from vaccinated or convalescent individuals. Data are represented as a percentage of total B cells. D) Percent NTD+, RBD+, or S2+ of total Spike+ memory B cells over time. E) Representative plots of variant RBD cross-binding gated on Spike+ WT RBD+ cells in vaccinated or convalescent individuals. Mean and standard error values at the 6-month timepoint are indicated. F) Percent B.1.1.7+, B.1.351+, or B.1.617.2+ of WT RBD+ memory B cells over time. G) Cross-sectional analysis of variant binding as a percentage of WT RBD+ memory B cells at 6 months post-vaccination/seropositivity. Statistics were calculated using paired non-parametric Wilcoxon test with Holm correction for multiple comparisons. Blue, red, and purple values indicate comparisons within naïve, recovered, or natural infection groups.

Figure 4.. Variant-binding memory B cell clones use distinct VH genes and evolve through somatic hypermutation.

A) Experimental design for sorting and sequencing SARS-CoV-2-specific memory B cells. B) Frequency of RBD++ (cross-binding) memory B cells as a percentage of total RBD+ cells. C) Percentage of sequence copies occupied by the top 20 ranked clones (D20) across naïve B cells and different antigen-binding memory B cell populations. D) Heatmap and hierarchical clustering of VH gene usage frequencies in memory B cell clones across different antigen-binding memory B cell populations. Data are represented as column normalized values. E) Somatic hypermutation (SHM) distributions and F) boxplots of individual clones across naïve B cells and different antigen-binding memory B cell populations. Data are represented as the percent of mutated VH nucleotides. Number of clones sampled for each population is indicated. G) Venn diagram of clonal lineages that are shared between WT RBD and RBD cross-binding (RBD++) populations. Data were filtered based on larger clones with ≥ 50% mean copy number frequency in each sequencing library. H) Example lineage trees of clones with overlapping binding to WT and B.1.351 variant RBD. VH genes and CDR3 sequences are indicated. Numbers refer to mutations compared to the preceding vertical node. Colors indicate binding specificity, black dots indicate inferred nodes, and size is proportional to sequence copy number; GL = germline sequence. I) Classification of SHM within overlapping clones. Each clone was defined as having higher (or equal) SHM in WT RBD binders or RBD++ cross-binders based on average levels of SHM for all WT RBD vs. RBD++ sequence variant copies within each lineage. J) SHM levels within overlapping clones. Data are represented as the percent of mutated VH nucleotides for WT RBD and RBD++ sequence copies. Statistics were calculated using paired non-parametric Wilcoxon test, with Holm correction for multiple comparisons in C.

Figure 5.. SARS-CoV-2 mRNA vaccines generate durable memory T cell responses.

A) Experimental design and B) gating strategy for quantifying the frequency of SARS-CoV-2-specific CD4+ and CD8+ T cells by AIM assay. For CD4+ T cells, antigen specificity was defined based on co-expression of CD40L and CD200. For CD8+ T cells, antigen specificity was defined based on expression of at least 4/5 activation markers. C) Frequencies of AIM+ CD4+ T and D) AIM+ CD8+ T cells over time in PBMC samples from vaccinated individuals. Data were background subtracted using a paired unstimulated control for each timepoint and are represented as a percentage of non-naïve CD4+ or CD8+ T cells. Fractions above plots indicate the number of individuals above their individual baseline at memory timepoints. Decay rates were calculated using a piecewise linear mixed effects model with censoring. Decay rates were similar in SARS-CoV-2 naïve and recovered groups. E) AIM+ CD4+ T cell memory subsets were identified based on surface expression of CD45RA and CD27, followed by CCR7. F) Frequencies of AIM+ CD4+ T cell memory subsets over time. G) Correlation matrix of memory subset skewing at peak (1 month) response with total AIM+ CD4+ T cell durability at 3 and 6 months. Durability was measured as the percent of peak response maintained at memory timepoints for each individual. H) Correlation between percent of EM1 cells at peak response with 6-month durability. I) AIM+ CD4+ T helper subsets were defined based on chemokine receptor expression. J) Frequencies of AIM+ CD4+ T helper subsets over time. Dotted lines indicate the limit of detection for the assay. Statistics were calculated using unpaired non-parametric Wilcoxon test with Holm correction for multiple comparisons. Correlations were calculated using non-parametric Spearman rank correlation.

Figure 6.. Immune trajectories and relationships in response to SARS-CoV-2 mRNA vaccination.

A) UMAP of 12 antigen-specific parameters of antibody, memory B, and memory T cell responses to mRNA vaccination in SARS-CoV-2 naïve subjects. Data points represent individual participants and are colored by timepoint relative to primary vaccine. Triangles indicate 6-month samples. B) Kernel density plots of anti-Spike IgG, Spike+ memory B, AIM CD4+, and AIM+ CD8+ T cells. Red contours represent areas of UMAP space that are enriched for specific immune components. C) Correlation matrix of antibody and memory B cell responses over time in SARS-CoV-2 naïve subjects. D) Correlation matrix of T cell and humoral responses over time in SARS-CoV-2 naïve subjects. E) Decay kinetics of antibody, memory B cell, and memory T cell parameters over time in SARS-CoV-2 naïve and recovered vaccinees. Data are normalized to pre-vaccine levels in SARS-CoV-2 recovered individuals to evaluate the effect of boosting pre-existing immunity. F) Correlation matrix of baseline memory components with antibody recall responses after vaccination in SARS-CoV-2 recovered individuals. Recall responses were calculated as the difference between post-vaccination levels and pre-vaccine baseline. All statistics were calculated using non-parametric Spearman rank correlation. Black boxes indicate significant values after FDR correction.