Single-Dose Intranasal Administration of AdCOVID Elicits Systemic and Mucosal Immunity against SARS-CoV-2 and Fully Protects Mice from Lethal Challenge

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the urgent need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive strategy to prevent COVID-19 as the nasal mucosa represents the first-line barrier to SARS-CoV-2 entry. The current intramuscular vaccines elicit systemic immunity but not necessarily high-level mucosal immunity. Here, we tested a single intranasal dose of our candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (AdCOVID) in inbred, outbred, and transgenic mice. A single intranasal vaccination with AdCOVID elicited a strong and focused immune response against RBD through the induction of mucosal IgA in the respiratory tract, serum neutralizing antibodies, and CD4⁺ and CD8⁺ T cells with a T_h1-like cytokine expression profile. A single AdCOVID dose resulted in immunity that was sustained for over six months. Moreover, a single intranasal dose completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge, preventing weight loss and mortality. These data show that AdCOVID promotes concomitant systemic and mucosal immunity and represents a promising vaccine candidate.

Keywords: COVID-19; IgA; SARS-CoV-2; adenovirus vector; intranasal; mucosal immunity; receptor binding domain; vaccine; viral vector.

Figure 1

Spike-specific IgG responses in sera following a single intranasal administration of AdCOVID. (A) CD-1 and (B) C57BL/6J mice received a single intranasal administration of vehicle (Ctrl) or AdCOVID at a low, mid, or high dose as described. Sera were collected from euthanized animals between days (A) 7–21 or (B) 7–28 post-vaccination (n = 10 animals/group/timepoint) and analyzed individually for quantification of spike-specific IgG. (C) A single cohort of C57BL/6J mice (n = 20) received a single intranasal administration of AdCOVID at a dose of 3.78 × 10⁸ ifu. Sera were collected longitudinally between days 0–180 post-vaccination and analyzed individually for quantification of spike-specific IgG. All results are expressed in µg/mL. Data are the geometric mean response ± 95% confidence interval. Statistical analysis was performed with a Mann–Whitney test: * p < 0.05; ** p <0.01; **** p < 0.0001.

Figure 2

A single intranasal administration of AdCOVID elicits anti-SARS-CoV-2 neutralizing antibodies. (A) Neutralizing antibody response in C57BL/6J or CD-1 mice vaccinated 28 days earlier with the mid or high dose of AdCOVID as indicated. Results are expressed as the reciprocal of the dilution of serum samples required to achieve 50% neutralization (FRNT₅₀) of wild-type SARS-CoV-2 infection in permissive Vero E6 cells. Data are the group median value. (B,C) Correlation between neutralizing antibody response and spike-specific IgG response in serum of vaccinated animals. Correlation analysis was performed with a two-tailed Spearman test.

Figure 3

Spike-specific IgA responses in BAL following single intranasal administration of AdCOVID. (A) CD-1 mice (n = 10 animals/timepoint) received a single intranasal administration of 6.25 × 10⁸ ifu AdCOVID. BAL samples were collected at the indicated timepoints and analyzed individually for the quantification of spike-specific IgA. (B) C57BL/6J mice received a single intranasal administration of 6.25 × 10⁸ ifu AdCOVID. BAL samples were collected on days 0, 14, 21 and 28 (n = 10 animals/timepoint). In a separate study, C57BL/6J mice (n = 5) received a single intranasal administration of 3.78 × 10⁸ ifu AdCOVID on day 0 and were euthanized on day 180 for BAL collection. BAL samples were analyzed individually for the quantification of spike-specific IgA. All results are expressed in ng/mL. Data are the geometric mean response ± 95% confidence interval. Statistical analysis was performed with a Mann–Whitney test: * p <0.05; ** p <0.01.

Figure 4

Flow cytometry analysis of immune cells in lungs from C57BL/6J mice following a single intranasal dose of AdCOVID. C57BL/6J mice were given a single intranasal administration of vehicle (Ctrl) or 3.35 × 10⁸ ifu AdCOVID. Lung cells were isolated from the vaccinated mice at the timepoints indicated (10 mice/timepoint) and analyzed individually by flow cytometry using markers of (A) innate immune cells or (B) B and Tfh-like cells as described in the Materials and Methods. Results are expressed as cell number. Different Y-axis scales are used across the graphics. Data are the mean response ± SD. Statistical analysis was performed with a Mann–Whitney test: *** p < 0.001; **** p < 0.0001.

Figure 5

Intranasal AdCOVID vaccination elicits mucosal and systemic IFN-γ⁺ T cells. CD-1 mice were given a single intranasal administration of vehicle (Ctrl) or 3.78 × 10⁸ ifu AdCOVID. Lung and spleen cells were isolated on days (A) 10 and (B) 14 following vaccination, re-stimulated with an RBD peptide pool, and analyzed by IFN-γ ELISpot. Results are expressed as Spot Forming Cells (SFC) per million input cells. Data are the mean response ± SD. Statistical analysis was performed with a Mann–Whitney test: ** p < 0.01.

Figure 6

Intracellular cytokine production by pulmonary and splenic T cells 14 days after intranasal AdCOVID vaccination. CD-1 mice were given a single intranasal administration of vehicle (Ctrl) or 3.78 × 10⁸ ifu AdCOVID. Lung cells (n = 10 mice/vaccine, 3 mice/control) were isolated on day 14, re-stimulated with the RBD peptide pool for 5 h, and analyzed by flow cytometry. Results are expressed as the percentage of IFN-γ or TNF-α expressing (A) CD11a⁺CD8⁺ or (B) CD11a⁺CD4⁺ T cells for individual mice. Different Y-axis scales are used across the graphics. Data are the mean response ± SD. Statistical analysis was performed with a Mann–Whitney test: * p < 0.05; ** p < 0.01.

Figure 7

Intranasal AdCOVID vaccination elicits polyfunctional memory T cell populations in the lung 14 days after vaccination. CD-1 mice were given a single intranasal administration of vehicle (Ctrl) or 3.78 × 10⁸ ifu AdCOVID. Lung cells (n = 10 mice/vaccine, 3 mice/control) were isolated at day 14, re-stimulated with the RBD peptide pool for 5 h, and analyzed by flow cytometry to identify CD69⁺CD103⁺ resident memory T cells (Trm). Results are expressed as the percentage of IFN-γ⁺, TNF-α⁺, or double positive IFN-γ⁺/TNF-α⁺ expressing (A) CD8⁺ or (B) CD4⁺ Trm cells for individual mice. Different Y-axis scales are used across the graphics. Data are the mean response ± SD for the groups. Statistical analysis was performed with a Mann–Whitney test: * p < 0.05; ** p < 0.01.

Figure 8

A single intranasal dose of AdCOVID completely protects K18-hACE2 mice from lethal SARS-CoV-2 challenge. K18-hACE2 mice were given a single intranasal administration of vehicle (Ctrl, n = 5) or AdCOVID (6 × 10⁸ ifu, n = 10). All mice were intranasally challenged 28 days post-vaccination with 1.4 × 10⁴ FFU of 2019-nCoV/USA-AZ1/2020 and monitored for change of body weight and mortality for 24 days. (A) Percent weight change from baseline and (B) group survival of 2019-nCoV/USA-AZ1/2020 challenged mice. Weight loss is presented as the mean ± SEM. Survival data is presented as Kaplan–Meier survival curves and analysis was performed by the Log-rank (Mantel–Cox). **** p < 0.0001.