Sex-biased immunogenicity of a mucosal subunit vaccine against SARS-CoV-2 in mice

Abstract

Introduction: Current vaccines against COVID-19 administered via parenteral route have limited ability to induce mucosal immunity. There is a need for an effective mucosal vaccine to combat SARS-CoV-2 virus replication in the respiratory mucosa. Moreover, sex differences are known to affect systemic antibody responses against vaccines. However, their role in mucosal cellular responses against a vaccine remains unclear and is underappreciated.

Methods: We evaluated the mucosal immunogenicity of a booster vaccine regimen that is recombinant protein-based and administered intranasally in mice to explore sex differences in mucosal humoral and cellular responses.

Results: Our results showed that vaccinated mice elicited strong systemic antibody (Ab), nasal, and bronchiole alveolar lavage (BAL) IgA responses, and local T cell immune responses in the lung in a sex-biased manner irrespective of mouse genetic background. Monocytes, alveolar macrophages, and CD103+ resident dendritic cells (DCs) in the lungs are correlated with robust mucosal Ab and T cell responses induced by the mucosal vaccine.

Discussion: Our findings provide novel insights into optimizing next-generation booster vaccines against SARS-CoV-2 by inducing spike-specific lung T cell responses, as well as optimizing mucosal immunity for other respiratory infections, and a rationale for considering sex differences in future vaccine research and vaccination practice.

Keywords: SARS-CoV-2; and sex differences; innate immunity; lung tissue-resident T cells; mucosal vaccine.

Figure 1

Vaccines induced spike-specific humoral responses in sera and bronchoalveolar lavage. (A) experimental timeline, treatment groups, animal numbers, and vaccination protocol. (B) IgG Ab titters in sera collected at two weeks post boost vaccination (day 28). (C) BAL IgA Ab responses at day 28 day. (D) comparison of male and female IgG responses. (E) BAL IgA Ab in males and females. Empty circle dots: female; Solid circle dots: male in (B, C). Non-parametric one-way ANOVA using the Kruskal-Wallis test, followed by corrected Dunn’s multiple comparisons tests, was performed to analyze serum IgG and BAL IgA responses in group1–5 (B, C). Data are representative of two independent experiments (n=4 or 5 per sex). The nonparametric Mann-Whitney test was used to compare sex differences in grp2 Ab responses (D, E). The significance levels for p-values were denoted as follows: 0.1234 (ns), 0.0332 (*), 0.0021(**), 0.0002 (***), <0.0001 (****).

Figure 2

Spike-specific T cell responses in the lungs of C57BL/6 mice. (A) representative flow cytometry plots showing spike-specific T cell responses for each animal group. (B) spike-specific CD4+ T cell responses. (C) spike-specific lung CD8+ T_RM. (D, E) total, and spike-specific lung CD4+ T_RM, respectively. (F, G) total, and spike-specific lung CD8+ T_RM, respectively. Empty circle dots: female; Solid circle dots: male in (B–G). Data are representative of two independent experiments (n=4 or 5 per sex). Non-parametric one-way ANOVA using the Kruskal-Wallis test followed by corrected Dunn’s multiple comparisons tests was conducted in Prism to analyze cellular responses across groups 1–5. The significance levels for p-values were denoted as follows: 0.1234 (ns), 0.0332 (*), 0.0021(**), 0.0002 (***), <0.0001 (****) (B–G).

Figure 3

Sex-biased mucosal T cell responses induced by the protein mucosal vaccine in the lungs of C57BL/6 mice. (A) IFNγ+ and/or TNF-α+ secreting CD4+ T cell responses in male and female mice in i.n. boosted Grp2. (B) composition of spike-specific CD4+ T cells in i.n. boosted Grp2 of both sexes. (C) IFNγ+ and/or TNF-α+ secreting CD8+ T cell responses in i.n. boosted Grp2. (D) composition of spike-specific CD8+ T cells in i.n. boosted Grp2. (E) i.m. boosted Grp3 IFNγ+ and/or TNF-α+ secreting CD8+ T cell responses. (F) i.m. boosted Grp3 composition of spike-specific CD8+ T cells. (G, H) sex differences in spike-specific lung CD4+ and CD8+ T_RM in i.n. boosted grp2, respectively. Data are representative of two independent experiments (n=4 or 5 per sex). The nonparametric Mann-Whitney test was used to compare sex differences in T cell responses (A, C, E, G, H). The significance levels for p-values were denoted as follows: 0.1234 (ns), 0.0332 (*), and 0.0021(**).

Figure 4

Spike-specific antibody responses in BALB/c mice vaccinated with mucosal boost vaccine. ELISA plates coated with S1 proteins included Wuhan, Omicron, and BA.2 strains. (A) IgG titers. (B) nasal wash IgA responses. (C) BAL IgA responses. Non-parametric one-way ANOVA using the Kruskal-Wallis test followed by corrected Dunn’s multiple comparisons tests was conducted in Prism to analyze cellular responses across vaccine and control groups (A–C). Data are the pool of two independent experiments (n= 5 per sex). The significance levels for p-values were denoted as follows: 0.1234 (ns), 0.0332 (*), 0.0021(**), and <0.0001 (****).

Figure 5

Spike-specific T cell responses in BALB/c mice. (A) representative flow cytometry plots of CD4+ T cell responses. (B) antigen-specific CD4+ T cells responses in vaccine and naïve groups. (C) sex differences in spike-specific CD4+ T cells responses. (D) spike-specific lung CD4+ T_RM in vaccine and naïve mice. (E) sex differences in spike-specific lung CD4+ T_RM in vaccinated mice. (F) representative flow cytometry plots of CD8+ T cell responses. (G) spike-specific CD8+ T cell responses in vaccine and naïve animals. (H) sex differences in spike-specific CD8+ T cells responses. The nonparametric Mann-Whitney test was used to compare the vaccine vs naïve group as well as sex differences in T cell responses (B, C, D, E, G, H). Empty circle dots represent female mice whereas solid dots represent male mice in (B, D, G). Data are the pool of two independent experiments (n= 5 per sex). The significance levels for p-values were denoted as follows: 0.1234 (ns), 0.0332 (*), 0.0021(**), and 0.0002 (***).

Figure 6

Innate immune responses of monocyte compartment in the lungs. (A) monocytes in the vaccine and naïve group. (B) comparison of the frequencies of monocytes in vaccinated males and females. (C) Spearman correlation between neutrophil frequencies and nasal IgA antibody responses against OMICRON strain. (D–F, G–I) Spearman correlation between monocyte frequencies and mucosal BAL and nasal Ab responses, respectively. (J–L, M–O) Spearman correlation between alveolar macrophage frequencies and mucosal BAL and nasal Ab responses, respectively. The nonparametric Mann-Whitney test was used to compare the vaccine vs naïve group and sex differences in monocyte responses (A, B). Data are the pool of two independent experiments (n= 5 per sex). The significance levels for p-values were denoted as follows: 0.0021(**), and 0.0002 (***). Two-tailed Spearman was applied to analyze correlations between innate cell populations and mucosal Ab responses with a 95% confidence interval.

Figure 7

Spearman correlation between innate cell frequencies and spike-specific CD4+ T cell responses. (A–C) Dendritic cell frequencies and singular or polyfunctional S1-specific CD4+ T cells. (D) correlation between CD103+ dendritic cells and S1-specific CD4+ T cells that are double positive for IFNγ and TNF-α. (E) correlation between CD103+ dendritic cells and spike-specific lung CD4+ T_RM. Data are the pool of two independent experiments (n= 5 per sex). Two-tailed Spearman analysis was applied to perform correlations between innate cell populations and mucosal Ab responses with a 95% confidence interval.