Heterologous prime-boost vaccination drives stromal activation and adaptive immunity against SARS-CoV-2 variants

Abstract

Heterologous vaccination strategies have shown superior efficacy over homologous regimens in clinical studies, but the underlying immunological mechanisms remain incompletely understood. Using a mouse model, we investigated the immune responses induced by heterologous prime-boost vaccination with adenoviral and mRNA vaccines. Heterologous vaccination (adenoviral prime, mRNA boost) elicited higher neutralizing antibody titers and stronger CD8⁺ T cell responses against Delta and Omicron-BA.5 variants compared to homologous regimens. Single-cell transcriptomic analysis of injection-site tissues revealed that adenoviral priming induced minimal changes in cellular composition but established a pre-conditioned innate immune environment. This effect was further amplified upon mRNA boosting, particularly through fibroblast-driven chemokine responses that promoted immune cell recruitment. These findings suggest that adenoviral priming enhances local immune activation upon boosting, contributing to the heightened adaptive immune response observed in heterologous vaccination. This study provides mechanistic insights into the immunological effects of heterologous prime-boost strategies against SARS-CoV-2 variants.

Keywords: COVID-19; adenoviral vaccine; cross immunity; heterologous vaccination; immune response; mRNA vaccine; single cell transcriptional analysis.

Figure 1

Experimental design and sampling schedule (created with BioRender.com). BALB/c mice were intramuscularly immunized with two doses of phosphate-buffered saline (PBS), lipid nanoparticle (LNP), or 5 μg of mRNA vaccine (mRNA) and 2 x 10⁸ adenovirus-vector vaccine (Ad(S)) for the first dose, followed by 5 μg of mRNA vaccine for the second dose. Muscle tissues at the injection site used for single-cell RNA sequencing were sampled 16 h after the first vaccination and 16 h after the booster vaccination. Spleen and blood samples were collected 3 weeks after the boost vaccination and were evaluated to confirm cellular response (ELISpot, FACS) and antigen-specific humoral response (PRNT, ELISA), respectively. ELISpot, enzyme-linked immunospot; ELISA, enzyme-linked immunosorbent assay; PRNT, plaque reduction neutralization test.

Figure 2

Heterologous vaccination enhances humoral immunity in BALB/c mice. (A), Kinetics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike specific binding antibody responses against total immunoglobulin G (IgG). The blood samples of vaccinated mice were collected 3 weeks after the booster vaccination. (B), Sera of vaccinated mice were analyzed for SARS-CoV-2 specific total IgG using enzyme-linked immunosorbent assay (ELISA). (C–E), The neutralizing antibody titers in the serum of the vaccinated mice were analyzed via a plaque reduction neutralization test (PRNT) using the SARS-CoV-2 virus variants against (C) D614, (D) Delta, and (E) BA.5. P-values were determined using two-way analysis of variance (ANOVA) and Tukey’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 3

Heterologous vaccination induces robust CD8⁺ T cell responses in BALB/c mice. (A), IFN-γ ELISpot analysis was measured for T cell responses. Splenocytes from immunized mice were collected 3 weeks after the final vaccination and stimulated with D614, Delta, and BA.5 spike glycoprotein peptide pool and compared to those in unstimulated cells. Sample wells with spots were measured using a CTL Immunospot reader. (B, C), Flow cytometry analysis of intracellular cytokines (B) IFN-γ, (C) TNF-α. Antigen-specific CD8 T cells were obtained from mouse spleen samples on day 42 post-vaccination. The IFN-γ, TNF-α CD3⁺CD8⁺ cells were stimulated with D614, Delta, and BA.5 spike glycoprotein peptide pool. (D), The IgG subtypes in the sera of vaccinated mice were assessed by ELISA for SARS-CoV-2 spike-specific IgG1 and IgG2a. Endpoint titers and endpoint titer ratios of IgG2a to IgG1 were calculated. P-values were determined using two-way ANOVA and Tukey’s multiple comparison test (ns: nonsignificant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Figure 4

Single-cell transcriptomic analysis reveals distinct injection-site immune responses. (A), UMAP visualization of injection site cells across all vaccine groups. Clusters are numbered on the plot, with corresponding cell types annotated in the accompanying panel based on canonical marker expression. (B), Major cell type markers are presented in a dot plot. Colors indicate min-max normalized average expression levels in each cell type, and dot sizes represent the ratio of cells expressing marker genes in each cell type. (C), Proportion of cell types according to the injection type. Proportions of each cell type are presented in the left panel, and the right panels show differential abundances (log2 fold changes) of cell types in each injection type (compared to the PBS injection condition). (D), Target molecule detection rate across samples. The mRNA and adenoviral vaccines in the homologous vaccination group represent the combined results of prime and boost, while the heterologous vaccination group includes results of the boosting of adenovirus vaccination with the mRNA vaccine. For each group, spike-positive cell counts and total cell counts are shown on the bars. (E), Target enrichment profile across cell types. (F), Comparison of target enrichment pattern in mRNA and adenoviral injection condition.

Figure 5

Adenoviral priming conditions the injection site for enhanced immune activation upon boosting. (A), Single-cell transcriptomic landscape of injection sites according to the injection conditions. (B), Number of differentially expressed genes (DEGs) across cell types in each injection condition (right). Total number of cells analyzed per group after quality control filtering (left). Number of distinct cell types identified in each group (middle). Number of differentially expressed genes (DEGs) across cell types in each injection condition (right). (C), DEG vectors projected on principal component (PC) axes of injection site responses. Each dot represents the DEG vector of cell types in each injection condition. (D), PC projections of DEG vectors according to the injection conditions.

Figure 6

Heterologous vaccination amplifies fibroblast-driven inflammation and stromal activation. (A), Number of differentially expressed genes (prime vs boost shot) across cell types in each injection condition. (B), Gene set enrichment analysis using MSigDB Hallmark gene sets. Pathway enrichment analyses were conducted on the upregulated genes in fibroblasts: (top) adenoviral vector boost shot injection vs adenoviral vector prime shot, (bottom) cross vaccination vs boost shot injection of mRNA vaccine. (C), Cytokine gene expression changes in fibroblasts in multiple comparison conditions. Indicated values are log2-fold changes of gene expressions in each comparison.