Staggered immunization with mRNA vaccines encoding SARS-CoV-2 polymerase or spike antigens broadens the T cell epitope repertoire

Abstract

Combining a T cell-targeting mRNA vaccine encoding the conserved SARS-CoV-2 RNA-dependent RNA polymerase, RdRp, with a Spike-encoding mRNA vaccine may offer an additional pathway toward COVID-19 protection. Here, we show that a nucleoside-modified RdRp mRNA vaccine raises robust and durable CD8+ T cell responses in mice. Immunization drives a CD8+ T cell response enriched toward a specific RdRp epitope. Unexpectedly, coadministration of mRNA vaccines encoding RdRp or the Spike Receptor Binding Domain (RBD) dampens RBD-specific immune responses. Contralateral administration reduces the suppression of RBD-specific T cell responses while type I interferon signaling blockade restores RBD-specific antibodies. A staggered immunization strategy maintains both RBD vaccine-mediated antibody and T cell responses as well as protection against lethal SARS-CoV-2 challenge in human ACE2 transgenic mice. In HLA-A2.1 transgenic mice, the RdRp vaccine elicits CD8+ T cell responses against HLA-A*02:01-restricted epitopes recognized by human donor T cells. These results highlight RdRp as a candidate antigen for COVID-19 vaccines. The findings also offer insights into crafting effective multivalent mRNA vaccines to broaden CD8+ T cell responses against SARS-CoV-2 and potentially other viruses with pandemic potential.

Keywords: SARS-CoV-2; T cell receptor specificity; anti-pathogen T cell response; interferon signaling; mRNA vaccines.

Fig. 1.

RdRp mRNA vaccine immunization elicits an antigen-specific CD8+ T cell response in mice. (A) Experimental design to test the T cell immunogenicity of a RdRp mRNA-iLNP vaccine in C57BL/6 mice (n = 4/group; i.m.). (B) Body weight change following immunization. (C) Intracellular cytokine staining (ICS) analysis of day 28 splenocytes restimulated with a RdRp peptide pool (mean ± SD; n = 4; one way ANOVA). (D) Experimental design to profile RdRp-specific and RBD-specific T cell responses in mice receiving two or three doses of the RBD mRNA-iLNP, the RdRp mRNA-iLNP or an empty iLNP control. (E) ICS analysis of CD3+/ CD8+ peripheral blood mononuclear cells (PBMC) from immunized mice restimulated with RdRp or RBD peptide pools (mean ±SD; one way ANOVA). (F) ELISpot analysis of day 30 splenocytes and lung cells pulsed with RdRp or RBD peptide pools. n.s.: not significant; **** P < 0.0001.

Fig. 2.

RdRp or RBD mRNA-iLNP immunization remodels the CD8+ T cell transcriptional landscape and TCR repertoire. (A) Single cell sequencing experiment to generate 5’GEX and V(D)J libraries of CD8+ splenocytes from mice immunized with two doses of RdRp or RBD mRNA-iLNP. Splenocytes from 5 animals/group on day 9 following boost immunization were pooled for enrichment and sequencing. (B) Uniform Manifold Approximation and Projection (UMAP) representation of CD8+ cell transcriptomes colored by experimental group or Louvain cluster assignment. (C) T cell effector or activation transcriptional signature enrichment across clusters. (D) Transcript level z-scores for representative T cell activation or effector genes. (E) UMAP representation and abundance of cells with the 10 most abundant TCR clonotypes in the RdRp mRNA-iLNP group. (F) UMAP representation and abundance of cells with the 10 most abundant TCR clonotypes in the RBD mRNA-iLNP group.

Fig. 3.

T cells expressing RdRp-specific TCRαβ recognize a conserved epitope and kill RdRp-expressing cells. (A) Strategy for the deconvolution of peptide antigens recognized by T cells undergoing clonal expansion in RdRp mRNA-iLNP immunized mice. (B) IFNy ELISA analysis of TCRαβ-transduced CD3+ splenocytes restimulated with a RdRp peptide pool (mean ± SD; n = 2 biological replicates). (C) IFNγ ELISA analysis of TCRαβ-transduced CD3+ splenocytes restimulated with a dose range of RdRp peptides (mean ± SD; n = 2). (D) Immunoblot analysis of murine KP4662 (KP) cells engineered to stably express trRdRp. (E) IFNγ ELISA analysis of RdRp TCRαβ-transduced CD3+ splenocytes co-cultured with RdRp-expressing KP cells at a 8:1 effector:target (E:T) ratio for 48 h (mean ± SEM; n = 2; unpaired t test). (F) Crystal violet cell survival analysis of adherent RdRp-expressing KP cells co-cultured with TCRαβ-transduced or control T cells at an 8:1 effector:target ratio for 72 h (n = 2). **** P < 0.0001.

Fig. 4.

Simultaneous RdRp and RBD mRNA vaccine immunization diminishes spike RBD-specific CD8+ T cell responses. (A) Experimental design to monitor immune response in C57BL/6 mice immunized with RBD and RdRp mRNA-iLNP individually or simultaneously (2.5 μg; i.m.). (B) ICS analysis of day 27 splenocytes from experiment in Fig. 4A pulsed with RdRp or RBD peptide pools (mean ± SD; n = 5; one way ANOVA). (C) ICS analysis of PBMC from mice immunized with RdRp or RBD mRNA-iLNP as in Fig. 1D restimulated with RdRp (STGYHFREL) or RBD (VVVLSFEL) peptides alone or together (0.25 μg/mL). PBMC were obtained from mice immunized with three doses of 2.5 μg RdRp or RBD mRNA-iLNP. PBMC from 5 immunized mice from each group were pooled before ex vivo peptide restimulation, ICS analysis, and flow cytometry. Replicates represent individual stimulations (wells) of pooled PBMC (mean ± SD; n = 2; one way ANOVA). **** P < 0.0001.

Fig. 5.

Type I IFN signaling restrains spike RBD-specific antibody responses elicited by immunization. (A) ELISA analysis of sera Spike S1-specific IgG titers at the endpoint from experiment in Fig. 4A (mean ± SD; n = 5; one way ANOVA). (B) Experimental design to monitor immune response in C57BL/6 mice immunized with RBD or RdRp mRNA-iLNP individually at separate Left (L) or Right (R) gastrocnemius muscle injection sites (2.5 μg; i.m.). A non-SARS-CoV-2-targeting mRNA-iLNP encoding hemagglutinin of influenza A virus (HA) was administered as a control. IFNAR-blocking or isotype control antibodies were administered by i.p. injection 1 d before mRNA vaccine immunization. (C) ELISA analysis of serum Spike S1-specific IgG titers at the experimental endpoint (mean±SD; n = 3 to 4; one way ANOVA).* P <0.05; ** P <0.01.

Fig. 6.

Staggered RdRp vaccine immunization preserves the immunogenicity and protection from SARS-CoV-2 challenge conferred by the RBD vaccine. (A) Experimental design to test the immunogenicity of RdRp and RBD mRNA-iLNP following staggered immunization in K18-hACE2 C57BL/6 mice (2.5 μg; i.m.). A non-SARS-CoV-2-targeting mRNA-iLNP encoding HA was administered as a control. (B) ICS and ELISpot analysis of day 50 splenocytes pulsed with RdRp or RBD peptide pools (mean ± SD; n = 5; one way ANOVA). (C) ELISA analysis of serum Spike S1-specific IgG titers at the experimental endpoint (mean ± SD; n = 5). (D) SARS-CoV-2 lethal challenge in RdRp, RBD or HA mRNA-iLNP immunized K18-hACE2 C57BL/6 mice (1e4 PFU SARS-CoV-2; 2.5 μg mRNA vaccine; i.m.; n = 5). (E) Body weight change following challenge. (F) Survival of mice following 1e4 PFU SARS-CoV-2 challenge. n.s.: not significant.