ChAdOx1-S adenoviral vector vaccine applied intranasally elicits superior mucosal immunity compared to the intramuscular route of vaccination

Abstract

COVID-19 vaccines prevent severe forms of the disease, but do not warrant complete protection against breakthrough infections. This could be due to suboptimal mucosal immunity at the site of virus entry, given that all currently approved vaccines are administered via the intramuscular route. In this study, we assessed humoral and cellular immune responses in BALB/c mice after intranasal and intramuscular immunization with adenoviral vector ChAdOx1-S expressing full-length Spike protein of SARS-CoV-2. We showed that both routes of vaccination induced a potent IgG antibody response, as well as robust neutralizing capacity, but intranasal vaccination elicited a superior IgA antibody titer in the sera and in the respiratory mucosa. Bronchoalveolar lavage from intranasally immunized mice efficiently neutralized SARS-CoV-2, which has not been the case in intramuscularly immunized group. Moreover, substantially higher percentages of epitope-specific CD8 T cells exhibiting a tissue resident phenotype were found in the lungs of intranasally immunized animals. Finally, both intranasal and intramuscular vaccination with ChAdOx1-S efficiently protected the mice after the challenge with recombinant herpesvirus expressing the Spike protein. Our results demonstrate that intranasal application of adenoviral vector ChAdOx1-S induces superior mucosal immunity and therefore could be a promising strategy for putting the COVID-19 pandemic under control.

Keywords: ChAdOx1-S; SARS-CoV-2; mucosal immunity; vaccination; vaccine vectors.

Figure 1

Intranasal immunization with ChAdOx1‐S adenoviral vector vaccine induces a superior IgA response and a similar IgG response to intramuscular immunization in mice. (A) BALB/c mice were vaccinated via intranasal (IN) or intramuscular (IM) route with ChAdOx1‐S and were boosted 6 weeks after the first immunization. At 3, 8, and 10 weeks after first immunization, sera were collected for analysis. (B–D) Spike‐specific IgG and IgA were assessed by ELISA (n = 5–20). Dotted lines indicate the median absorbance value of unimmunized mice sera. (E) Neutralization titers were determined by in vitro neutralization assay (all groups n = 5). Dotted lines indicate the median value of IU/mL of unimmunized mice sera. Dots represent individual data points. Data were analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; p values indicate significant differences (*p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001). All experiments (B–E) have been repeated at least two times. IU: international units.

Figure 2

Intranasal immunization with ChAdOx1‐S induces a superior mucosal immunity in the lungs compared to intramuscular immunization. (A) BALB/c mice were vaccinated via intranasal (IN) or intramuscular (IM) route with ChAdOx1‐S. (B) At 6 weeks after the first immunization, mice were boosted, and at 9 weeks mucosal, Spike‐specific immune response in BALs was assessed by ELISA (n = 3‐4). Dotted lines indicate the median absorbance value of unimmunized mice sera. Neutralization titers were determined by in vitro neutralization assay (all groups n = 5). Dotted lines indicate the median value of IU/mL of unimmunized mice sera. Dots represent individual data points. (C) Eight weeks after the first immunization, spleen and lung homogenates were restimulated with Spike‐specific peptide KNKCVNFNF (S535‐543). The responding CD8 T cells were identified by intracellular staining for accumulated IFN‐γ. Bars represent group means overlaid with individual data points (n = 10). (D) Tissue‐resident phenotypes of antigen‐experienced CD8 T cells (CD8⁺CD44⁺) were assessed by staining for CD69 and/or CD103. Data were analyzed by one‐way ANOVA followed by Tukey's multiple comparison test; p values indicate significant differences (*p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001). All experiments (B–D) have been repeated two to three times. IU: international units.

Figure 3

Intranasal immunization with ChAdOx1‐S confers excellent protection from challenge with a heterologous virus expressing SARS‐CoV‐2 S protein. (A) Schematic representation of the recombinant MCMV vector (MCMV‐S). The SARS‐CoV‐2 Spike ORF was inserted in the place of the MCMV ie2. (B) Western blots of purified virus stocks of MCMV‐S and MCMV were performed with an antibody against the SARS CoV‐2 S protein. Protein gB/M55 of MCMV was used as a loading control. (C) BALB/c mice were infected intravenously with MCMV or MCMV‐S (2 × 10⁵ PFU/mouse), and 3 weeks later, mice were boosted. (D) Four weeks after the first immunization, spleen homogenates were restimulated with Spike‐specific peptide KNKCVNFNF (S535‐543). The responding CD8 T cells were identified by intracellular staining for accumulated IFN‐γ. Bars represent group means overlaid with individual data points (n = 5). (E) Neutralization titers were determined by in vitro neutralization assay (all groups n = 5). Dotted lines indicate the median value of IU/mL of unimmunized mice sera. Dots represent individual data points. (F) BALB/c mice were vaccinated via intranasal (IN) or intramuscular (IM) route with ChAdOx1‐S. Six weeks after the first immunization, mice were boosted and 2 weeks later challenged with either intraperitoneal (IP) or intranasal (IN) inoculation of MCMV‐S (2 × 10⁵ PFU/mouse). At different time points, namely at 3 and 7 days after the intraperitoneal (G), or 5 days after the intranasal challenge (H–I), respectively, tissues were harvested, and viral titers were determined in lung, spleen, and liver homogenates (n = 4–7). (I) In some groups, CD8 T cells were depleted systemically by intraperitoneal injection of 500 µg of α‐CD8α antibody 1 day before intranasal MCMV‐S challenge. (G–I) Titers in organs of individual mice are shown (circles); horizontal bars indicate the median values. Dotted lines indicate the detection limit of the assay (DL). Data were analyzed by Mann–Whitney U test (D‐E, G‐I); p values indicate significant differences (*p < 0.05; **p < 0.01). All experiments (B, D, G‐I) have been repeated two to three times. IU: international units.