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. 2024 Aug 30:15:1430928.
doi: 10.3389/fimmu.2024.1430928. eCollection 2024.

Intranasal HD-Ad-FS vaccine induces systemic and airway mucosal immunities against SARS-CoV-2 and systemic immunity against SARS-CoV-2 variants in mice and hamsters

Peter Zhou #  1 , Jacqueline Watt #  1 , Juntao Mai  1 , Huibi Cao  2 , Zhijie Li  1 , Ziyan Chen  2   3 , Rongqi Duan  2 , Ying Quan  1 , Anne-Claude Gingras  1   4 , James M Rini  1   5 , Jim Hu  2   3 , Jun Liu  1
Affiliations

Intranasal HD-Ad-FS vaccine induces systemic and airway mucosal immunities against SARS-CoV-2 and systemic immunity against SARS-CoV-2 variants in mice and hamsters

Peter Zhou et al. Front Immunol. .

Abstract

The outbreak of coronavirus disease 19 (COVID-19) has highlighted the demand for vaccines that are safe and effective in inducing systemic and airway mucosal immunity against the aerosol transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, we developed a novel helper-dependent adenoviral vector-based COVID-19 mucosal vaccine encoding a full-length SARS-CoV-2 spike protein (HD-Ad-FS). Through intranasal immunization (single-dose and prime-boost regimens), we demonstrated that the HD-Ad-FS was immunogenic and elicited potent systemic and airway mucosal protection in BALB/c mice, transgenic ACE2 (hACE2) mice, and hamsters. We detected high titers of neutralizing antibodies (NAbs) in sera and bronchoalveolar lavages (BALs) in the vaccinated animals. High levels of spike-specific secretory IgA (sIgA) and IgG were induced in the airway of the vaccinated animals. The single-dose HD-Ad-FS elicited a strong immune response and protected animals from SARS-CoV-2 infection. In addition, the prime-boost vaccination induced cross-reactive serum NAbs against variants of concern (VOCs; Beta, Delta, and Omicron). After challenge, VOC infectious viral particles were at undetectable or minimal levels in the lower airway. Our findings highlight the potential of airway delivery of HD-Ad-FS as a safe and effective vaccine platform for generating mucosal protection against SARS-CoV-2 and its VOCs.

Keywords: COVID-19; HD-Ad; SARS-CoV-2; adenoviral vector; intranasal delivery; vaccine.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
HD-Ad-FS induced efficient systemic and mucosal immunities in BALB/c mice. (A) Schematic of HD-Ad-FS vaccine. ITR, inverted terminal repeats; Ψ, adenoviral packaging signal; CBA, promoter of chicken beta-actin gene; UbC intron, first intron of ubiquitin C gene; FS, full-length spike gene of SARS-CoV-2 ancestral strain; bGH PolyA, polyadenylation tail of bovine growth hormone gene. (B) Western blot analysis of FS protein expression. IB3-1 cells were transduced with HD-Ad-FS at the indicated MOI, and proteins were harvested at day 3 post-transduction. (C) Schematic timelines of BALB/c mouse experiments. BALB/c mice were intranasally administered with HD-Ad-FS or HD-Ad-control at single-dose (5x109 vp) and prime-boost regimens (5x109 + 5x109 vp, three weeks interval). Three weeks after the last administration, animal samples were harvested. (D) Levels of FS-specific sIgA responses in BALs were determined with Enzyme-linked immunosorbent assay (ELISA). The starting dilution factor was 1:1. (E, F) Neutralization of live SARS-CoV-2 and VOCs infection by sera (E) and BALs (F). The Omicron used in this study was BA.1.18. The starting dilution factors were 1:50 and 1:5 for sera and BALs, respectively. (G) CD4+ T cell responses in the lungs of single-dose mice were measured by the production of IFN-γ (left), TNF (middle), and double-positive IFN-γ and TNF (right) at 3 weeks post-immunization, following ex vivo stimulation with spike antigen. (H) The production of IFN-γ (left), TNF (middle), and double-positive IFN-γ and TNF (right) in CD4+ T cells in the spleens of single-dose mice were measured at 3 weeks post-immunization, following ex vivo stimulation with spike antigen. (I) IFN-γ producing CD8+ T cells in the lungs of single-dose mice were measured by ex vivo stimulation with spike antigen. In all figures, dots represent individual mice (n=4 or 5). The dotted lines represent the limit of detection (LOD) of the assays. Statistical analyses were performed by one-way ANOVA. Bars and errors represent the geometric mean with geometric SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.
Figure 2
Figure 2
Vaccination of single-dose HD-Ad-FS protected hACE2 mice from SARS-Cov2 in the upper airway and lungs. (A) Schematic timeline of single-dose hACE2 experiment. The hACE2 mice were immunized with a single-dose of HD-Ad-FS (single-FS) or HD-Ad-control (single-control) at 5x109 vp via intranasal delivery. Three weeks after immunization, the hACE2 mice were intranasally challenged with SARS-CoV-2 at 1x105 TCID50. Animal samples were harvested at day 3 post-infection (dpi). (B) RNA levels of SARS-CoV-2 in oropharyngeal swabs were determined with RT-qPCR. (C) RNA levels of SARS-CoV-2 in the lungs were determined with RT-qPCR. (D) The titers of infectious SARS-CoV-2 in the lungs were measured with TCID50 assay. (E) RNA levels of SARS-CoV-2 in the spleens (left) and hearts (right) were measured with RT-qPCR. (F) FS-specific IgG response in serum were determined with ELISA. The starting dilution was 1:25. (G) Serum neutralizing activities against SARS-CoV-2 and VOCs were measured with neutralization assays. Dots represent individual mice (n=6). The dotted lines represent the LOD of the assays. For RT-qPCR, the LOD was set to 100 E copies/sample. Statistical analyses were performed by Mann-Whitney test, two-tailed. Bars and errors represent geometric mean with geometric SD. *p<0.05, **p<0.01, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.
Figure 3
Figure 3
Prime-boost vaccination of HD-Ad-FS protected hACE2 mice from SARS-CoV-2 VOCs in the lungs. (A) Schematic timeline of prime-boost hACE2 experiment. The mice were intranasally administered with a prime-boost regimen of HD-Ad-FS or HD-Ad-control (5x109 + 5x109 vp, three-week interval). Three weeks after the boost dose, the mice were intranasally challenged with a SARS-CoV-2 variant (Beta, Delta, or Omicron) at 1x105 TCID50. Animal samples were harvested at 4 dpi. (B) Body weight was recorded at 0 (pre-challenge) and 4 dpi after challenge. (C) SARS-CoV-2 variant RNA levels in the lungs were measured with RT-qPCR. (D) The titers of infectious SARS-CoV-2 variant in the lungs were determined with TCID50 assays. (E, F) The titers of FS-specific IgG in sera (E) and BALs (F) were determined with ELISA. The starting dilution factors were 1:25 and 1:5 for sera and BALs, respectively (G) The titers of FS-specific sIgA in BALs were measured with ELISA. The starting dilution factor was 1:1. (H, I) Neutralizing activities in sera (H) and BALs (I) against SARS-CoV-2 variants were determined with neutralization assays. Dots represent individual mice (n=4, 5, or 6). For (C-F, H, I), the horizontal dotted lines represent the LOD of the assays. For (B), statistical analyses were performed by two-way ANOVA. Error bars represent mean ± s.e.m. For (C-I), statistical analyses were performed by Mann-Whitney test, two-tailed. Error bars represent geometric mean with geometric SD. *p<0.05, **p<0.01, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.
Figure 4
Figure 4
Single-dose vaccination of HD-Ad-FS protected hamsters against SARS-CoV-2 in the upper airway and lungs. (A) Schematic timeline of single-dose hamster experiment. Hamsters were immunized with a single-dose of HD-Ad-FS or HD-Ad-control at 5x109 vp via intranasal route. Three weeks after immunization, hamsters were intranasally challenged with SARS-CoV-2 at 1x105 TCID50. Animal samples were harvested at 4 and 14 dpi. (B, C) Body weight was monitored at the indicated days after challenging with SARS-CoV-2. For (C), the change of weight in male and female hamsters were analyzed separately. (D, E) RNA levels of SARS-CoV-2 from oropharyngeal swabs were determined with RT-qPCR. Swabs were collected at the indicated time points. For (E), the levels of viral RNA in male and female hamsters were analyzed separately. (F) RNA levels of SARS-CoV-2 in lungs were determined with RT-qPCR at 4 dpi. (G) The titers of infectious SARS-CoV-2 in lungs were determined with TCID50 assays at 4 dpi. (H) RNA levels of SARS-CoV-2 in spleens (left) and hearts (right) were measured with RT-qPCR at 4 dpi. (I) The titers of FS-specific IgG in sera were measured with ELISA at 4 dpi. The starting dilution factor was 1:1000. (J) Serum NAb activities against SARS-CoV-2 and variants were determined with neutralization assays at 14 dpi. Dots represent individual hamsters (4 dpi, n=6; 14 dpi, n=9 or 10). For (D-J), the horizontal dotted lines represent the LOD of the assays. For (B, C), statistical analyses were performed by two-way ANOVA; error bars represent mean ± s.e.m. For (D, E), statistical analyses were performed by two-way ANOVA; error bars represent geometric mean with geometric SD. For (F-J), statistical analyses were performed by Mann-Whitney test, two-tailed. Error bars represent geometric mean with geometric SD. For (C), the asterisk signs represent statistical analyses in single-FS hamsters, and the pound signs represent statistical analyses in single-control hamsters. */# p<0.05, **/## p<0.01, ***<0.001, ****/####p<0.0001, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.
Figure 5
Figure 5
Histopathology of SARS-CoV-2 infection in the lungs of single-dose HD-Ad-FS immunized hamsters. (A, B) The presence of SARS-CoV-2 RNA in lung sections was detected with RNAscope in situ hybridization at 4 (A) and 14 dpi (B). Hematoxylin and eosin staining (H&E, 5th row) was performed with lung sections of the indicated conditions. Continuous lung sections were used for staining. Scale bars = 200 (grey bar) or 1000 (black bar) µm. Images are representatives of n=2 per group. Data represent one independent animal experiment with indicated biological replicates.
Figure 6
Figure 6
Prime-boost vaccination of HD-Ad-FS protected hamster against SARS-CoV-2 VOCs in the lungs. (A) Schematic timeline of the hamster experiment. Hamsters were intranasally immunized with a prime-boost regimen of HD-Ad-FS or HD-Ad-control (5x109 + 5x109 vp, three-week interval). Three weeks after the second dose, the hamsters were intranasally challenged with SARS-CoV-2 variants (Beta, Delta, or Omicron) at 1x105 TCID50. (B-D) Hamster body weight was monitored at the indicated days after challenging with SARS-CoV-2 variant Beta (B), Delta (C), or Omicron (D). (E) RNA levels of SARS-CoV-2 variants in the lungs were determined with RT-qPCR at 4 and 14 dpi. (F) Levels of infectious SARS-CoV-2 variants in the lungs were measured with TCID50 assay at 4 and 14 dpi. (G) The titers of FS-specific IgG in sera were measured with ELISA at 4 dpi. The starting dilution factor was 1:40. (H) The titers of FS-specific IgG in BALs were measured with ELISA at 4 dpi. The starting dilution factor was 1:10. (I) Serum NAbs against SARS-CoV-2 variants were measured with neutralization assays at 4 and 14 dpi. (J) BAL NAbs against the SARS-CoV-2 variants were determined with neutralization assay at 4 and 14 dpi. Dots represent individual hamsters (4 dpi, n=6; 14 dpi, n=9 or 10). For (E-J), the horizontal dotted lines represent the LOD of the assays. For (B-D), statistical analyses were performed by two-way ANOVA; error bars represent mean ± s.e.m. For (E, F), statistical analyses were performed by two-way ANOVA; error bars represent geometric mean with geometric SD. For (G-J), statistical analyses were performed by Mann-Whitney test, two-tailed; error bars represent geometric mean with geometric SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, and ns, not significant. Data represent one independent animal experiment with indicated biological replicates.
Figure 7
Figure 7
Histopathological analysis of SARS-CoV-2 VOC infection in the lungs of prime-boost HD-Ad-FS vaccinated hamsters. (A-C) Detection of SARS-CoV-2 variant RNA (Beta, A; Delta, B; Omicron, C) in lung sections with RNA in situ hybridization at 4 dpi. (D-F) The presence of SARS-CoV-2 variant RNA (Beta, d; Delta, e; Omicron, f) in lung sections with RNA in situ hybridization at 14 dpi. Continuous lung sections were used for H&E staining (3rd, 6th, and 9th rows). Scale bars = 200 (grey bar) or 1000 (black bar) µm. Images are representatives of n=2 per group. Data represent one independent animal experiment with indicated biological replicates.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by Canadian Institutes of Health Research (CIHR) grants, VR1-172771, VS-1-175531, GA-2-177717, and Covid-19 Immunity Task Force (CITF).

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