Intranasal administration of a single dose of MVA-based vaccine candidates against COVID-19 induced local and systemic immune responses and protects mice from a lethal SARS-CoV-2 infection

Abstract

Current coronavirus disease-19 (COVID-19) vaccines are administered by the intramuscular route, but this vaccine administration failed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infection in the upper respiratory tract, mainly due to the absence of virus-specific mucosal immune responses. It is hypothesized that intranasal (IN) vaccination could induce both mucosal and systemic immune responses that blocked SARS-CoV-2 transmission and COVID-19 progression. Here, we evaluated in mice IN administration of three modified vaccinia virus Ankara (MVA)-based vaccine candidates expressing the SARS-CoV-2 spike (S) protein, either the full-length native S or a prefusion-stabilized [S(3P)] protein; SARS-CoV-2-specific immune responses and efficacy were determined after a single IN vaccine application. Results showed that in C57BL/6 mice, MVA-based vaccine candidates elicited S-specific IgG and IgA antibodies in serum and bronchoalveolar lavages, respectively, and neutralizing antibodies against parental and SARS-CoV-2 variants of concern (VoC), with MVA-S(3P) being the most immunogenic vaccine candidate. IN vaccine administration also induced polyfunctional S-specific Th1-skewed CD4⁺ and cytotoxic CD8⁺ T-cell immune responses locally (in lungs and bronchoalveolar lymph nodes) or systemically (in spleen). Remarkably, a single IN vaccine dose protected susceptible K18-hACE2 transgenic mice from morbidity and mortality caused by SARS-CoV-2 infection, with MVA-S(3P) being the most effective candidate. Infectious SARS-CoV-2 viruses were undetectable in lungs and nasal washes, correlating with high titers of S-specific IgGs and neutralizing antibodies against parental SARS-CoV-2 and several VoC. Moreover, low histopathological lung lesions and low levels of pro-inflammatory cytokines in lungs and nasal washes were detected in vaccinated animals. These results demonstrated that a single IN inoculation of our MVA-based vaccine candidates induced potent immune responses, either locally or systemically, and protected animal models from COVID-19. These results also identified an effective vaccine administration route to induce mucosal immunity that should prevent SARS-CoV-2 host-to-host transmission.

Keywords: MVA; S protein; SARS-CoV-2; immunogenicity; intranasal delivery; mice; protective efficacy; vaccine candidates.

Figure 1

SARS-CoV-2-specific humoral immune responses elicited in C57BL/6 mice immunized with one IN dose of different MVA-based vaccine candidates against COVID-19. SARS-CoV-2-specific humoral immune responses were evaluated in serum and BAL samples obtained at 14 days postimmunization from C57BL/6 mice immunized with one IN dose of MVA-S, MVA-Δ-S, or MVA-S(3P). (A) Titers of binding IgG antibodies specific for the S protein (Wuhan strain), determined by ELISA in individual mouse serum samples in duplicate. Mean values and SEM are represented. The dashed line represents the limit of detection. (B) SARS-CoV-2 neutralizing antibody titers. NT₅₀ titers were determined in individual mouse serum samples by using a live virus microneutralization assay. Mean NT₅₀ values and SEM are represented. The dashed line represents the limit of detection (1:25 dilution). (C) SARS-CoV-2 neutralizing antibody titers against SARS-CoV-2 VoC. NT₅₀ titers were evaluated in pooled mouse serum samples, using VSV-based pseudoparticles expressing the SARS-CoV-2 S protein of different VoC. Mean NT₅₀ values and 95% confidence intervals are represented. The dashed line represents the limit of detection. (D) Titers of IgA antibodies specific for the S protein (Wuhan strain), determined by ELISA in pooled BAL samples in duplicate. Mean values and SEM are represented. Student’s t-test: *p < 0.05; **p < 0.005.

Figure 2

SARS-CoV-2-specific T-cellular immune responses elicited in C57BL/6 mice immunized with one IN dose of different MVA-based vaccine candidates against COVID-19. SARS-CoV-2 S-specific T-cellular immune responses were evaluated in spleens, lungs, and bronchial lymph nodes (BLNs) at 14 days postimmunization from C57BL/6 mice immunized with one IN dose of MVA-S, MVA-Δ-S, or MVA-S(3P). Cell percentages were determined by ICS. (A, B) Magnitude of S-specific CD4⁺ (A) and CD8⁺ (B) T-cell immune responses in spleens, lungs, and BLN. Percentages of CD4⁺ or CD8⁺ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against a mixture of S1 and S2 peptide pools in immunized mice. (C) Polyfunctional profiles (based on expression of selected markers CD107a, IFN-γ, TNF-α, and IL-2) of total S-specific CD8⁺ T-cell immune responses directed against a mixture of S1 and S2 peptide pools, in spleen, lungs, and BLN. (D) Magnitude of S-specific CD4⁺ Tfh cell responses in spleen. Percentages of CD4⁺ Tfh cells expressing CD40L and/or producing IFN-γ and/or IL-21 against a mixture of S protein plus S1 and S2 peptide pools in immunized mice. Polyfunctionality profile is shown on the right.

Figure 3

One IN dose of MVA-based vaccine candidates protects transgenic K18-hACE2 mice from SARS-CoV-2 infection. (A) Efficacy schedule. Female K18-hACE2 transgenic mice (n = 9 per group) were immunized by the IN route with one dose of 1 × 10⁷ PFUs of MVA-S, MVA-Δ-S, or MVA-S(3P) as indicated. At week 5 (day 35), mice were challenged intranasally with 1 × 10⁵ PFUs of SARS-CoV-2 (MAD6 isolate). MVA-WT-inoculated control mice were also challenged with SARS-CoV-2. At day 5 postchallenge, four mice per group were sacrificed and lungs, nasal washes, and serum samples were collected as indicated. Serum was also collected at 14 days after immunization in all groups and at 14 days postchallenge in groups 1, 2, and 3. (B, C) The challenged mice were monitored for change of body weight (B) and mortality (C) for 14 days. †: mice were euthanized due to loss of more than 20% of initial body weight. (D, E) Virus replication in lung samples (D) and nasal washes (E). SARS-CoV-2 subgenomic E and genomic RdRp mRNA detected by RT-qPCR at 5 days after virus infection (n = 4/group). Mean RNA levels (in arbitrary units [A.U.]) and SEM from duplicates of each lung and nasal washes samples; relative values are referred to uninfected mice. (F, G) SARS-CoV-2 infectious virus in lung samples (F) and nasal washes (G). Mean (PFUs/g of lung tissue or PFUs /ml of nasal wash) and SEM from triplicates of each sample. Student’s t-test: *p < 0.05; **p < 0.005; ***p < 0.001.

Figure 4

One IN dose of MVA-S(3P) reduced SARS-CoV-2 lung pathology in K18-hACE2 transgenic mice. (A) Lung inflammation scores (left) and percentage of lung area with lesions (right) examined in lung samples taken from mice (n = 4/group) vaccinated and infected as indicated in Figure 3A , and euthanized at 5 days postchallenge. Mean and SEM of cumulative histopathological lesion scores (left) and percentage of lung area affected by inflammatory lesions (right). Unpaired t-test: **p < 0.01. (B) Representative lung histopathological sections (H&E staining) from K18-hACE2 mice euthanized at day 5 postchallenge (magnification: 10×). Mice immunized with one dose of MVA-S (A) and MVA-Δ-S (B) displayed moderate inflammatory lung lesions that were, in general, more severe and extensive in mice immunized with MVA-WT (control infected group; d). These lesions highlighted the presence of diffuse thickening of the alveolar septae, perivascular edema (red arrowheads), mononuclear cell infiltrates within alveolar spaces (black arrowheads), large multifocal perivascular and peribronchiolar mononuclear infiltrates (black arrows), and occasional hemorrhages (white arrowheads). However, mice immunized with MVA-S(3P) (C) only displayed small lung areas with mild inflammatory lesions such as focal thickening of alveolar septae, occasional presence of mononuclear cell infiltrates within alveolar spaces (black arrowheads), and mild perivascular or peribronchiolar mononuclear infiltrates (black arrows).

Figure 5

One IN dose of MVA-S(3P) diminished levels of pro-inflammatory cytokines in K18-hACE2 transgenic mice. mRNA levels of several cytokines/chemokines were detected by RT-qPCR in lungs (A) and nasal washes (B) obtained at 5 days postchallenge (n = 4/group). Mean RNA levels (in A.U.) and SEM from duplicates of each sample; relative values are referred to uninfected mice. Student’s t-test: *p < 0.05; **p < 0.005; ***p < 0.001.

Figure 6

MVA-based vaccine candidates induced high levels of SARS-CoV-2-specific humoral immune responses in vaccinated and challenged K18-hACE2 transgenic mice. (A) Titers of IgG antibodies specific for the S protein (Wuhan strain). Determined by ELISA in individual mouse serum samples collected at day 14 postimmunization (pi) (prechallenge; n = 9/group) and at days 5 (n = 4/group) and 14 (n = 5/group) postchallenge (pc) from K18-hACE2 mice. Mean values and SEM are represented. The dashed line represents the limit of detection. (B) SARS-CoV-2 neutralizing antibody titers. NT₅₀ titers were evaluated in individual mouse serum samples collected at day 14 pi and at days 5 and 14 pc, using a live virus microneutralization assay (MAD6 strain, having D614G mutation). Mean NT₅₀ values and SEM are represented. The dotted line represents the limit of detection. (C) SARS-CoV-2 neutralizing antibody titers against SARS-CoV-2 VoC. NT₅₀ titers were evaluated in pooled mouse serum samples collected at days 14 pi (left) and 14 pc (right), using VSV-based pseudoparticles expressing the SARS-CoV-2 S protein of different VoC. Mean NT₅₀ values and 95% confidence intervals are represented. The dashed line represents the limit of detection. Student’s t-test: *p < 0.05; ***p < 0.001.