A multi-antigen-based SARS-CoV-2 vaccine provides higher immune responses and protection against SARS-CoV-2 variants

Abstract

The emergence of divergent SARS-CoV-2 variants has significantly compromised the effectiveness of first-generation COVID-19 vaccines. We investigated a prime-boost approach using bovine adenoviral (Ad) [BAd] and human Ad (HAd) vectors expressing the spike (S), membrane (M), or nucleocapsid (N) with the autophagy-inducing peptide C5 (AIP-C5) for enhanced antigen-specific immunity. The combinational vaccine formulation expressing three antigens demonstrated markedly elevated antigen-specific cell-mediated immune (CMI) responses compared to groups immunized with vectors expressing individual antigens. Furthermore, vaccinated animals exhibited 100% survival, significant reductions in lung viral titers, and no apparent signs of morbidity following challenges with Delta or Omicron variants in K18-hACE2 transgenic mice. Surprisingly, immunization with vectors expressing M and N resulted in immune suppression. However, including S with M and N overcomes this antagonistic interaction and significantly enhances immune responses and protection efficacy. Using the BAd vaccine platform in a multi-antigen approach complemented with AIP-C5 is a promising strategy for developing next-generation SARS-CoV-2 vaccines.

Fig. 1. Construction, Expression, and Immunogenicity of HAd-Vectored SARS-CoV-2 Antigens in Mice.

A Diagrammatic representation of human adenoviral (HAd) vectors. The gene cassettes of spike (S), membrane (M), or nucleocapsid (N) of SARS-CoV-2 Wuhan Hu 1 strain with or without autophagy-inducing peptide C5 (AIP-C5) were under the control of the cytomegalovirus (CMV) immediate early promotor and the bovine growth hormone (BGH) polyadenylation signal (PA). The diagram is not drawn to scale. ΔE1 deletion of E1 region, ΔE3 deletion of E3 region, HAd-ΔE1E3 HAd-5 having deletions of E1 and E3 regions, LITR left inverted terminal repeat, RITR right inverted terminal repeat. B Immunoblots confirming expression of S, M, or N with or without AIP-C5. 293Cre cells were mock-infected or infected with HAd-ΔE1E3 (empty vector), HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, or HAd-N/C5. The cell pellets were processed for immunoblotting using S-, M-, or N-specific antibodies. To confirm equal loading, immunoblotting for β-actin was also performed. C Outline of the immunogenicity study in BALB/c mice.

Fig. 2. Humoral immune responses in mice immunized with different HAd vector formulations.

Seven-week-old BALB/c mice were intranasally (i.n.) mock-inoculated or inoculated with 5 × 10⁷ plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, HAd-N/C5, HAd-S + HAd-M, HAd-S/C5 + HAd-M/C5, HAd-S + HAd-N, HAd-S/C5 + HAd-N/C5, HAd-M + HAd-N, HAd-M/C5 + HAd-N/C5, HAd-S + HAd-M + HAd-N, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval. Blood samples were collected 4 weeks post-booster inoculation. A S-specific IgG, IgG1, IgG2a, and IgA levels were assessed in serum (top panel) and lung washes (bottom panel). B N-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). C M-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). ELISA data were shown as the area under curve (AUC) with a cut-off value calculated by the average of blank wells. Each symbol represents an individual animal. Median and interquartile range (IQR) are presented. Statistical significance was assessed using non-parametric one-way ANOVA with Kruskal–Wallis test.

Fig. 3. Cell-mediated immune responses and virus-neutralization (VN) titers in mice immunized with different HAd vector formulations.

Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated, or inoculated with 5 × 10⁷ plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S, HAd-S/C5, HAd-M, HAd-M/C5, HAd-N, HAd-N/C5, HAd-S + HAd-M, HAd-S/C5 + HAd-M/C5, HAd-S + HAd-N, HAd-S/C5 + HAd-N/C5, HAd-M + HAd-N, HAd-M/C5 + HAd-N/C5, HAd-S + HAd-M + HAd-N, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval. Four weeks post-booster inoculation, splenocytes (A–C) and lung mononuclear cells (D–F) were collected and analyzed for S- (A, D), N- (B, E), or M-specific (C, F) T cell-mediated immune responses by ELISpot assay. The numbers of IFN-γ-secreting cells per 10⁶ splenocytes (A–C) or 10⁵ lung mononuclear cells (D–F) are presented. Statistical analysis was performed using U-test Mann-Whitney for non-parametric data. Significance levels were set at *p < 0.05; and **p < 0.01. VN titers against SARS-CoV-2 variants in sera of mice immunized with different HAd vector formulations. Four weeks post-booster inoculation, the serum samples were collected and used to study the development of VN titers against SARS-CoV-2 variants: hCoV-19/USA-WA1/2020 (Wuhan) (G), hCoV-19/USA/PHC658/2021 (B.1.617.2, Delta) (H), or hCoV-19/USA/GR484A/2021 (B.1.1.529, Omicron) (I) by VN assay.

Fig. 4. Protection of immunized K18-hACE2 transgenic mouse groups challenged with SARS-CoV-2.

A Outline of the protection study in K18-hACE2 mice. Five-week-old K18-hACE2 transgenic mice (3 males + 2 females per group) were intranasally (i.n.) mock-inoculated or inoculated with 5 × 10⁷ plaque-forming units (PFU) of HAd-ΔE1E3, HAd-S/C5, HAd-M/C5, HAd-N/C5, HAd-S/C5 + HAd-M/C5, HAd-S/C5 + HAd-N/C5, HAd-M/C5 + HAd-N/C5, or HAd-S/C5 + HAd-M/C5 + HAd-N/C5 twice at four weeks interval (Created by BioRender.com). Four weeks post-booster, animals were challenged with 1 × 10⁴ TCID₅₀ of hCoV-19/USA/PHC658/2021 (Lineage B.1.617.2, Delta) and monitored for B morbidity (% body weight change) or C mortality 14 days post-challenge. Statistical analysis was performed using U-test Mann-Whitney for non-parametric data. Significance levels were set at *p < 0.05 and **p < 0.01. D Lungs were collected 5 days post-challenge and RT-PCR was conducted to quantify RNA copies. The data are shown as log₁₀ RNA copy numbers and the detection limit was 500 copy numbers (2.5 log₁₀). Each symbol represents an individual animal, and the error is shown as SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001.

Fig. 5. Construction, Expression, and Immunogenicity of BAd- and HAd-Vectored SARS-CoV-2 Antigens in Mice.

A Diagrammatic representation of bovine adenoviral (BAd) or human adenoviral (HAd) vectors. The gene cassettes of the native spike (S) protein of the Delta variant or the stabilized form of S protein of the Omicron variant, membrane (M) of Wuhan Hu 1, or nucleocapsid (N) of Wuhan Hu 1 with autophagy-inducing peptide C5 (AIP-C5) were under the control of the cytomegalovirus (CMV) immediate early promotor and the bovine growth hormone (BGH) polyadenylation signal (PA) (Created by BioRender.com). The diagram is not drawn to scale. ΔE1 deletion of E1 region, ΔE3 deletion of E3 region, Ad-ΔE1E3 adenovirus having deletions of E1 and E3 regions, LITR left inverted terminal repeat, RITR right inverted terminal repeat. B Immunoblots confirming expression of S, M, or N with AIP-C5. 293Cre cells were mock-infected or infected with HAd-ΔE1E3, HAd-S/C5(Delta), HAd-S/C5(Omicron/St), HAd-M/C5, or HAd-N/C5. BHHF5 cells infected with BAd-S/C5(Delta), BAd-S/C5(Omicron/St), BAd-M/C5, or BAd-N/C5. At 48 h post-infection, the cell pellets were collected and processed for immunoblotting using S-, M-, or N-specific antibodies. To confirm equal loading, immunoblotting for β-actin was also performed. C An outline of the immunogenicity & protection studies in BALB/c & k18-hACE2 mice, respectively, is depicted (Created by BioRender.com).

Fig. 6. Humoral immune responses in mice immunized by a prime-boost approach using BAd and HAd vector formulations.

Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 10⁷ plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 10⁷ PFU (each vector). Blood samples were collected 4 weeks post-booster inoculation. A S-specific IgG, IgG1, IgG2a, and IgA levels were assessed in serum (top panel) and lung washes (bottom panel). B N-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). C M-specific IgG, IgG1, IgG2a, and IgA responses in serum (top panel) and lung washes (bottom panel). ELISA data were shown as the area under curve (AUC) with a cut-off value calculated by the average of blank wells. Each symbol represents an individual animal. Median and interquartile range (IQR) are presented. Statistical significance was assessed using non-parametric one-way ANOVA with Kruskal–Wallis test.

Fig. 7. Cell-mediated immune responses and virus-neutralization (VN) titers in mice immunized by a prime-boost approach using BAd and HAd vector formulations.

Seven-week-old BALB/c mice (3 males + 3 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 10⁷ plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 10⁷ PFU (each vector). Four weeks post-booster inoculation, splenocytes (A–C), mediastinal lymph nodes (MLN) (D–F), and lung mononuclear (MN) cells (G–I) were collected and analyzed for S- (A, D, G), N- (B, E, H), or M-specific (C, F, I) T-cell-mediated immune responses by ELISpot assay using antigen-specific peptides. Numbers of IFN-γ-secreting cells per 10⁶ splenocytes (A–C), 10⁶ MLN cells (D–F), or 10⁵ lung MN cells (G–I) are presented. *, significant at p < 0.05; and **, significant at p < 0.01. VN titers against SARS-CoV-2 variants in sera of mice immunized by a prime-boost approach using BAd and HAd vector formulations. Four weeks post-booster inoculation, the serum samples were collected and used to study the development of VN titers against SARS-CoV-2 variants: J hCoV-19/USA/PHC658/2021 (B.1.617.2, Delta) and K hCoV-19/USA/GR484A/2021 (B.1.1.529, Omicron) by VN assay.

Fig. 8. Protective Efficacy and Immune Profiling of BAd/HAd Prime-Boost SARS-CoV-2 Vaccination in K18-hACE2 Mice.

A, B Protection of mice immunized by a prime-boost approach using BAd and HAd vector formulations. Five-week-old K18-hACE2 transgenic mice (3 males + 2 females per group) were intranasally (i.n.) mock-inoculated or inoculated with BAd vector individually or various combinations as shown in Fig. 5C with a dose of 1 × 10⁷ plaque-forming units (PFU) (each vector). At four weeks post-prime, each group received a booster i.n. inoculation with HAd vector individually or in various combinations, as shown in Fig. 10C with a dose of 1 × 10⁷ PFU (each vector). Three weeks post-booster inoculation, animal groups were challenged with 10⁵ TCID₅₀ of A SARS-CoV-2 (B.1.617.2; Delta) or B (B.1.1.529; Omicron) variant. Five days post-challenge, mice were euthanized, and lung samples were collected and processed for lung virus titration. Data is shown as log₁₀ TCID₅₀/g and detection limit is 0.5 log₁₀ TCID₅₀/g. Each symbol represents an individual animal, and the error is shown as SD. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001. C–F Monitoring numbers of IL-10- or IL-4-secreting cells in the spleen of immunized mice. Splenocytes from Study #2 were revived and analyzed for N- (C, E) and M-specific (D, F) IL-10- (C, D) or IL-4- (E, F) secreting cells by ELISpot assay using antigen-specific peptides. Numbers of IL-10- or IL-4-secreting cells per 10⁶ splenocytes are presented. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test. *, significant at p ≤ 0.05; **, significant at p ≤ 0.01; ***, significant at p ≤ 0.001; and ****, significant at p ≤ 0.0001.