Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo

Abstract

The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)⁺ CD8⁺ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.

Keywords: COVID-19; DNA vaccine; SARS-CoV-2; adjuvant; chemokine; mucosal.

Graphical abstract

Figure 1

pCTACK enhances IFNγ secretion in vivo (A) Mice were immunized twice, separated by 3 weeks, with 40 μg of empty plasmid (pVax) or 10 μg of SARS-CoV-2 spike DNA vaccine alone or co-immunized with pS and 30 μg cutaneous T cell-attracting chemokine DNA (+pCTACK). (B–E) Lymphocytes in the spleens (B–D) and lungs (C–E) were stimulated with overlapping peptide pools representing the full-length spike glycoprotein and subjected to an IFNγ ELISpot assay on day 11 after the second immunization. (F–I) Lung (F and G) and spleen (H and I) lymphocytes were subjected to SARS-CoV-2 peptide stimulation for 6 h, and intracellular cytokine staining was used to detect the frequencies of α4β7⁺ T cells (F–H) and α4β7⁺ IFNγ-secreting CD8⁺ T cells (G and I) in these compartments. Bars represent the mean, and error bars represent SD (B and C). Symbols represent individual animals, and bars represent SD (F–I). Data are representative of 2 (B–E) or 1 (F–I) independent experiment(s) (n = 5–20 mice/group). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA (B and C) or Kruskal-Wallis ANOVA (F–I).

Figure 2

pCTACK enhances anti-SARS-CoV-2 mucosal humoral responses (A–E) Mice were immunized as shown in Figure 1A, and SARS-CoV-2 receptor binding domain (RBD)-specific IgG1 (A), IgG2a (B), IgG2b (C), total IgG (D), and IgA (E) were quantified by ELISA. (F) SARS-CoV-2 serum pseudovirus neutralization ID50 titer. (G and H) SARS-CoV-2 Washington (G) and Delta variant (H) live virus neutralization. (I and J) SARS-CoV-2 RBD binding IgG in bronchoalveolar lavage (BAL) (I) and cecal extracts (J). (K and L) Spearman correlations between serum IgG and cecal extract IgG (K) and BAL fluid (L). (M and N) SARS-CoV-2 RBD binding IgA in BAL (M) and cecal extract (N). (O and P) Spearman correlation between serum IgA and cecal extract (O) and BAL IgA (P). (Q and R) BAL (Q) and cecal extract (R) pseudoviral neutralization ID50 values. (S and T) Quantification of small intestinal (SI) Peyer’s patches (S) and representative Peyer’s patches (blue arrows) (T). Each symbol is the average of duplicate assays for one mouse (A–R) or a single mouse (S and T). Horizontal lines represent the mean, and error bars represent SEM (A–R) or SD (S). Data are representative of 2 independent experiments (n = 5–20 mice/group). ∗p < 0.05, ∗∗p < 0.01 by Kruskal-Wallis ANOVA.

Figure 3

pCTACK protects against morbidity and mortality in heterologous SARS-CoV-2 challenge (A) Mice were immunized twice as in Figure 1 and rested for 3 months prior to challenge with 1 × 10⁵ TCID50 SARS-CoV-2 (B.1.617.2; hCoV-19/Canada/ON-NML-63169/2021) and weight loss and survival were monitored longitudinally. (B and C) SARS-CoV-2 wild-type (B) and Delta (B.1.617) (C) pseudovirus neutralization ID50 titers at day 72 post-2nd immunization. Four animals per group were euthanized at day 4 post-challenge to quantify viral loads. (D and E) Replication-competent virus (D) and viral RNA (E) in the lungs 4 days post-infection. (F–H) Weight loss as percent of initial weight for pVax (F), pS (G), and +pCTACK (H) co-immunized mice. (I and J) Summary weight loss (I) and probability of survival (J) post challenge. (K) Pathological scoring of lung sections from four mice. (L) H&E and SARS-CoV-2 nucleocapsid staining of lung sections. Each point represents the average of duplicate assays for an individual animals, horizontal lines represent the mean, and error bars represent the SEM (B–E). Lines represent individual animal (F–H) or averaged group weights (I). For (K), symbols represent individual animals, horizontal lines represent the mean, and error bars represent SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by Kruskall-Wallis ANOVA (B–E, K), Dunnett’s multiple comparison test (I), or Mantel-Cox Log rank analysis (J). Data are representative of one experiment with n = 10 per group pre challenge, n = 4 per group at day 4 post-challenge, and n = 6 per group for weight loss and survival curves. Scale bars are 500 μm in low magnification and 200 μm in high magnification.