A Recombinant VSV-Based Bivalent Vaccine Effectively Protects against Both SARS-CoV-2 and Influenza A Virus Infection

Abstract

COVID-19 and influenza are both highly contagious respiratory diseases that have been serious threats to global public health. It is necessary to develop a bivalent vaccine to control these two infectious diseases simultaneously. In this study, we generated three attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates against both SARS-CoV-2 and influenza viruses. These rVSV-based vaccines coexpress SARS-CoV-2 Delta spike protein (SP) bearing the C-terminal 17 amino acid (aa) deletion (SPΔC) and I742A point mutation, or the SPΔC with a deletion of S2 domain, or the RBD domain, and a tandem repeat harboring four copies of the highly conserved influenza M2 ectodomain (M2e) that fused with the Ebola glycoprotein DC-targeting/activation domain. Animal immunization studies have shown that these rVSV bivalent vaccines induced efficient humoral and cellular immune responses against both SARS-CoV-2 SP and influenza M2 protein, including high levels of neutralizing antibodies against SARS-CoV-2 Delta and other variant SP-pseudovirus infections. Importantly, immunization of the rVSV bivalent vaccines effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads. Overall, this study provides convincing evidence for the high efficacy of this bivalent vaccine platform to be used and/or easily adapted to produce new vaccines against new or reemerging SARS-CoV-2 variants and influenza A virus infections. IMPORTANCE Given that both COVID-19 and influenza are preferably transmitted through respiratory droplets during the same seasons, it is highly advantageous to develop a bivalent vaccine that could simultaneously protect against both COVID-19 and influenza. In this study, we generated the attenuated replicating recombinant vesicular stomatitis virus (rVSV)-based vaccine candidates that target both spike protein of SARS-Cov-2 Delta variant and the conserved influenza M2 domain. Importantly, these vaccine candidates effectively protected hamsters or mice against the challenges of SARS-CoV-2 Delta variant and lethal H1N1 and H3N2 influenza viruses and significantly reduced respiratory viral loads.

Keywords: M2 protein; SARS-CoV-2 Delta variant; VSV vector; bivalent vaccine; ectodomain; influenza; neutralizing antibody; spike protein.

FIG 1

Construction and rescue of rVSV Delta SP and influenza M2e bivalent vaccines. (A) Schematic diagram of the Delta SPΔC and EboGPΔM-tM2e immunogens present in the bivalent vaccines. (a) SARS-CoV-2 Delta-SPΔC_A742 (SPΔC1), containing a C-terminal 17 aa (DEDDSEPVLKGVKLHYT) deletion and an I742A mutation as indicated. The nine mutations in Delta SP are listed in the lower part. (b) Delta SPΔC2, containing the C-terminal 17 aa deletion and another 381 aa (encompassing aa744 to aa1124) deletion in the S2 domain. The eight mutations in SPΔC2, are listed in lower part. (c) EboGPΔM-RBD, the RBD of SARS-CoV-2 was used to replace the MLD domain in EboGP. (d) EboGPΔM-tM2e, four copies of influenza virus M2 ectodomain (24 aa) polypeptide (tM2e) replaced the MLD domain in EboGP. (B) The attenuated virus entry of SPΔC1. A549_ACE2 cells were infected with equal amounts of SPΔC_Delta-PVs or SPΔC1-PVs (adjusted by P24) carrying the Gluc gene, as indicated. At 48 h postinfection, the Gluc activity in the supernatant of different infected cultures was measured. Data represent the mean ± SD of two replicates from a representative experiment out of three performed. (C and D) The attenuated cell-to-cell fusion ability of SPΔC_Delta- or SPΔC1-mediated syncytia formation was analyzed by coculturing the SPΔC_Delta- or SPΔC1-expressing 293T cells with A549_ACE2 cells. The amounts of syncytia were counted after 24 h in five different views of the microscope (C) and was also imaged under bright-field microscopy (D). (E) Schematic diagram of V-EM2e/SPΔC1, V-EM2e/SPΔC2 and V-EM2e/ERBD and the virus rescuing procedures. 293T and Vero E6 coculture cells were cotransfected with V-ΔG-EM2/SPΔC1, V-ΔG-EM2/SPΔC or V-ΔG-EM2/RBD and helping plasmids (T7, N, L, P plasmids). The supernatants containing V-EM2e/SPΔC1, V-EM2e/SPΔC2, and V-EM2e/ERBD viruses were used to infect Vero E6 cells to generate the rVSV stocks.

FIG 2

Expression of V-EM2e/SPΔC1, V-EM2e/SPΔC2, or V-EM2e/ERBD in infected Vero E6 cells. (A) The infection of V-EM2/SPΔC1, V-EM2/SPΔC2 or V-EM2/ERBD in Vero E6 cells induced the cytopathic effects after 4 days postinfection. (B) Representative immunofluorescence images of Vero E6 cells infected with V-EM2e/SPΔC1, V-EM2e/SPΔC2, V-EM2e/ERBD, or mock-infected, stained with anti-SARS-CoV-2 RBD antibody (a to d) or anti-M2e antibody (i to l), and DAPI (e to h, m to p). (C) Vero E6 cells infected with the rescued V-EM2/SPΔC1, V-EM2/SPΔC, or V-EM2/ERBD were lysed and processed with SDS-PAGE followed by WB with a rabbit anti-SARS-CoV-2 RBD antibody (top panel), a mouse antibody against influenza M2e (top second gel), anti-VSV nucleocapsid (N) (top third gel), or anti-action (a cellular protein as an internal control) (low panel).

FIG 3

Characterization of the replication kinetics and the cell tropisms of bivalent rVSV vaccine candidates. (A) Each of the bivalent VSV vaccine candidates or the rVSV expressing VSV-G (rVSV-wt) was used to infect different cell lines, including A549, MRC-5, U251MG, CD4⁺ Jurkat T cells, human monocyte-derived macrophages (MDMs), and dendritic cells (MDDCs). Supernatants were collected at different time points postinfection as indicated and were titrated on Vero E6 cells. Data represent mean ± SD and were obtained from two replicates of a representative experiment out of two performed. (B) The ability of induced cytopathic effects in A549, U251MG and CD4⁺ Jurkat T cells, by each rVSV were observed after 4 days postinfection under microscopy. (a) V-EM2e/ERBD; (b) V-EM2e/SPΔC1; (c) V-EM2e/SPΔC2; (d) rVSVwt.

FIG 4

Anti-SARS-CoV-2 RBD and anti-influenza M2e immune responses induced by immunization with different bivalent VSV vaccine candidates. (A) Schematic of the bivalent rVSV vaccine candidate immunization protocol in the mouse. BALB/c mice were immunized with V-EM2e/SPΔC1, V-EM2e/SPΔC2, or V-EM2e/ERBD via intramuscular (IM) or intranasal (IN) routes, as indicated. The mice sera were collected on days 13 and 28 and were measured for anti-SARS-CoV-2 RBD IgG and IgA antibody levels (B to D) or measured for anti-M2e IgG and IgA antibody levels (F to H). (E and I) The anti-SARS-CoV-2 RBD and anti-M2e IgA antibody levels at 28 days. Data represent mean ± SD. Statistical significance was determined using an unpaired T-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

rVSV Delta SP vaccine candidates elicited neutralization antibodies. The neutralization titers (50% inhibition) in immunized mice sera against SpΔC_WT-Luc-PVs (A), SpΔC_{Delta (B.1.617.2)}-Luc-PVs (B), SpΔC_Beta’-Luc-PVs (D), SpΔC_B.1.617-Luc-PVs (E), and SpΔC_Omic-Luc-PVs (F) infections. VSV-G-Luc-PVPs (C) were used as the negative control. The mouse serum of each immunization group collected on day 28 was pooled, 2× serially diluted, and incubated with different Luc-PVs (~10⁴ RLU). Then, the mixtures were added in A549_ACE2.cell cultures and the infection of PVs was determined by Luciferase assay at approximately 48 to 66 h postinfection. The percentage of infection was calculated compared with no serum control and neutralizing titers were calculated by using sigmoid 4PL interpolation with GraphPad Prism 9.0, as described in the Materials and Methods. Data represent mean ± SD and were obtained from over three independent experiments. Statistical significance was determined using an ordinary one-way ANOVA test and Turkey’s test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

T-cell cytokine response induced by bivalent VSV vaccine candidates. (A) Splenocytes isolated from immunized mice (as described in Fig. 4A) were cultured without peptide (no peptide control, NC) (a to c), or stimulated with SARS-CoV-2 SP subunit 1 (S1) peptide pool (d to f) or influenza M2e peptide (g to i) (1 μg/mL for each peptide). After 4 days of stimulation, supernatants were collected, and the release of IFN-γ, IL-4, and IL-5 cytokines in the supernatants was quantified with an MSD U-plex mouse cytokine immunoassay kit and counted in the MESO Quickplex SQ120 instrument. Each symbol indicated one individual mouse. (B) The ratios of Th1/Th2 cytokines from splenocytes of each mouse in the same culture condition were calculated, respectively, and the representative data (IFN-γ/IL-4) were shown. Statistical significance between the two groups was determined using an unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 7

Mice immunized with V-EM2/SPΔC1 were protected against the lethal challenge of H1N1 and H3N2 influenza viruses. (A) Schematic of the bivalent VSV vaccine candidate immunization and influenza virus challenge protocol used in the study. For the H1N1 challenge experiment, the BALB/c mice were immunized with 1 × 10⁸ TCID₅₀ (IM) or 1 × 10⁵ TCID₅₀ (IN) of V-EM2e/SPΔC1 or PBS on day 0 and day 14. On day 27, the blood samples were collected and measured for anti-influenza M2e antibody levels by ELISA (B). On day 28, all the mice were challenged with 2,100 PFU of H1N1 influenza virus. Weight loss (C) and survival rates (D) of the mice were monitored daily for 2 weeks. (E) Viral loads in the lung tissue of immunized mice and PBS group at day 5 post-H1N1 challenge were measured in MDCK cell line, as described in the Materials and Methods. For the H3N2 challenge experiment, the BALB/c mice were immunized with 1 × 10⁵ TCID₅₀ (IN) of V-EM2e/SPΔC1 or PBS on day 0 (single-dose, SD), or on day 0 and 14 (double-dose, DD). On day 28, all the mice were challenged with 1.4 × 10⁴ PFU of H3N2. (F) Weight loss, (G) survive rates, and (H) viral loads in the lung tissue of immunized mice and PBS group at day 6 after H3N2 challenge.

FIG 8

V-EM2/SPΔC1 and V-EM2/SPΔC2 provided protection against SARS-CoV-2 Delta infection in Syrian Hamsters. (A) Schematic of the bivalent VSV vaccine candidate immunization and SARS-CoV-2 Delta variant challenge protocol used in the study. (B) Total serum anti-SARS-CoV-2 spike IgG titers in hamsters following prime and boost vaccination. (C) The neutralizing antibody titers in immunized mice sera against SARS-CoV-2 Delta variant were measured and neutralizing titers were calculated by using sigmoid 4PL interpolation with GraphPad Prism 9.0, as described in the Materials and Methods. (D) Weight loss in the vaccinated or the control Syrian hamsters following infection with the SARS-CoV-2 Delta variant. (E) Viral RNA levels in oral swabs on day 3 following infection with SARS-CoV-2 Delta variant. (F) Infectious SARS-CoV-2 Delta virus titers in nasal turbinates and lung tissues on day 5 following infection with SARS-CoV-2 delta. n = 10 for B (each time point), n = 10 for C, 10 through day 28, and 10 at day 42; n = 10 for D (at day 3 postinfection) and n = 5 for E (from day 5 postinfection). Statistical significance was assessed by two-way analysis of variance with multiple comparisons (A), mixed effects analysis with multiple comparisons (B), and the Kruskal-Wallis test with multiple comparisons (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. For (B), colored asterisks indicate significant differences between the same colored group compared with the PBS group. Shown are medians for each group in A, C, and D, and mean + SEM in B.