Immunogenicity and protective efficacy of a multi-antigenic adenovirus-based vaccine candidate against Mycobacterium tuberculosis

Abstract

Introduction: The inadequate efficacy of the Bacillus Calmette-Guérin (BCG) vaccine against adult pulmonary tuberculosis (TB) necessitates the development of new and effective vaccines. Human adenovirus serotype 5 (Ad5), which induces T-cell response, is a widely used viral vector. In this study, we aimed to evaluate the efficacy of a multi-antigenic recombinant Ad5 vectored vaccine and determine the optimal immunization route for enhanced immune response against Mycobacterium tuberculosis.

Methods: We constructed a multi-antigenic recombinant Ad5 vectored vaccine expressing four antigens (Ag85B-ESAT6-MPT64-Rv2660c) of M. tuberculosis (rAd-TB4), immunized with rAd-TB4 (5 × 10⁷ infectious virus units/mouse) twice at an interval of 4 weeks starting at 10 weeks after BCG priming, and evaluated its boosting efficacy in a BCG-primed mouse model, and determined the optimal immunization route.

Results: Compared with the BCG-only (2 × 10⁵ colony forming units/mouse), subcutaneous injection of rAd-TB4 (1 × 10⁷ infectious virus units/mL; two doses) elicited a T-cell response and cytokine production in lung lymphocytes and splenocytes. rAd-TB4 immunization significantly reduced bacterial loads and inflamed lung areas compared to BCG immunization (p < 0.01) and protected against the H37Rv challenge performed at 17 weeks of BCG priming. RNA sequencing of the whole blood of rAd-TB4-vaccinated mice collected pre- and, 1 and 4 weeks post-infection, identified differentially expressed genes associated with immune and inflammatory responses, especially those in the Wnt signaling pathway.

Conclusion: Our results indicate that rAd-TB4 immunization enhances the immune response to the vaccine boosting antigens in BCG-primed mice, making it a potential adult pulmonary TB vaccine candidate.

Keywords: BCG vaccine; adenovirus vector; immune response; immunization; mouse model; multi-antigenic vaccine; pulmonary tuberculosis.

FIGURE 1

Evaluation of rAd-TB4 immunogenicity as a Bacille Calmette–Guérin (BCG) booster vaccine. (A) Schematic of the study design. C57BL/6 mice were subcutaneously vaccinated with BCG (2 × 10⁵ CFU/mouse) on day 0. The mice were immunized with rAd-TB4 (1 × 10⁷ IFU/mouse) twice at 10 and 14 weeks, and then at 15 weeks, five mice in each group were sacrificed to collect their lungs, spleens, and sera. Immunogenicity was evaluated using an enzyme-linked immunosorbent spot (ELISpot) assay and immunoglobulin G (IgG) titer measurement. (B–D) Interferon-γ (IFN-γ) secretion in (B) lung lymphocytes and (C) splenocytes detected using the ELISpot assay following 36 h of incubation of single cells with purified protein derivative (PPD), Ag85B, Rv2660c, and ESAT-6 peptide (100 ng/mL). The BCG group is presented as a gray bar and the rAd-TB4 group was presented as a black bar. (D) Antigen-specific total IgG titer in serum samples measured using enzyme-linked immunosorbent assay (ELISA). Data show the mean ± standard deviation from triplicate wells in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 obtained using ordinary two-way ANOVA.

FIGURE 2

Antigen-specific multifunctional T-cells in the lung lymphocytes of rAd-TB4- and BCG-only immunized mice. One week after the final immunization (see Figure 1A), the mice in BCG-only and BCG-primed-rAd-TB4 immunized groups (n = 5 in each) were sacrificed, and their lungs were collected for intracellular cytokine staining assays to evaluate multifunctional T-cells. Lung lymphocytes from each group were stimulated with PPD, Ag85B, Rv2660c, and ESAT-6 peptides (100 ng/mL) for 5 h at 37°C in the presence of GolgiPlug and GolgiStop. Data are shown as the intensity of (A) CD4⁺ or (B) CD8⁺ T-cells expressing one, two, or three specific cytokines upon (i) PPD stimulation, (ii) Ag85B stimulation, (iii) Rv2660c stimulation, (iv) ESAT-6 stimulation in each group. Data show the mean ± standard deviation from triplicate wells in each group; *p < 0.05, **p < 0.01, ***p < 0.001 obtained using ordinary two-way ANOVA.

FIGURE 3

Immunogenicity of rAd-TB4 injected using different routes in BCG-primed mice. The mice were divided into BCG-only and BCG-primed- rAd-TB4 immunized groups. BCG/rAd-TB4 group was further divided into two groups depending on the route of rAd-TB4 administration—subcutaneous (SC) or intramuscular (IM). (A) IFN-γ secreted by single cells of mice immunized via the two routes detected using the ELISpot assay following 36 h of incubation with the Ag85B peptide (100 ng/mL). (B,C) Cytokines (%) and CD4⁺ or CD8⁺ T-cells in (B) lung lymphocytes and (C) splenocytes of the respective groups stimulated with Ag85B peptide for 5 h at 37°C in the presence of GolgiPlug and GolgiStop. Data are shown as the intensity of CD4⁺ (left) or CD8⁺ (right) T cells expressing one, two, or three cytokines in each group. Statistical analysis was performed using the unpaired Student’s t-test; **p < 0.01, ***p < 0.001, compared with the BCG group. Data show the mean ± standard deviation from triplicate wells in each group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 obtained using ordinary two-way ANOVA.

FIGURE 4

Comparison of inflammatory cytokine production in lung cells following immunization using different routes. Mice in each group were immunized via the SC or IM route. One week after the final immunization, the mice were euthanized, and lung lymphocytes were harvested. Cultured supernatants of lung lymphocytes were collected for cytokine level measurement after stimulation with the Ag85B peptide (100 ng/mL) for 36 h at 37°C. Production levels of nine cytokines (A) IL-2, (B) TNF-α, (C) IFN-γ, (D) GM-CSF, (E) IL-12p40, (F) IL-12p70, (G) IL-6, (H) MCP-1, and (I) IL-17A were examined using a bead-based multiplex cytokine assay. Statistical analysis was performed using the unpaired Student’s t-test; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the BCG group.

FIGURE 5

Protective efficacy of rAd-TB4 against H37Rv aerosol challenge. (A) Schematic of the immunization schedule and subsequent evaluation. The SC-vaccinated mice were challenged with H37Rv via the aerosol route (n = 5 or n = 3 per group). (B) After 8 weeks, the mice sera were collected and measured Ag85B specific total IgG, IgG1, and IgG2c. (C) Lung bacterial loads 8 weeks after H37RVchallenge. (D) The percentage of inflamed area of the lung. (C,D) Conducted twice. (E) Representative H&E staining images from the lungs of Experiment 1. Statistical analysis was performed using analysis of variance, followed by Dunnett’s multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001. Data are means ± SEM.

FIGURE 6

Protective efficacy of rAd-TB4 against clinical isolated strain HN878. (A) Schematic of the immunization schedule and subsequent evaluation. The SC-vaccinated mice were challenged with HN878 via the aerosol route (n = 6). (B) Lung and spleen bacterial loads 8 weeks after HN878 challenge. (C) Representative H&E staining images of infected lungs. (D) Lung lymphocytes and (E) splenocytes of infected mice were stimulated with Ag85B peptide for 5 h at 37°C. Frequency of Naïve T cell (CD44^– CD62⁺), Tcm (CD44⁺ CD62^–), Teff (CD44⁺ CD62^– CD17^–), Tem (CD44⁺ CD62^– CD17⁺) were presented along with the median. Data show the mean ± standard deviation from triplicate wells in each group; *p < 0.05, **p < 0.01, ****p < 0.0001 obtained using ordinary two-way ANOVA.

FIGURE 7

Transcriptional profiles and hierarchical clustering analysis of differentially expressed genes (DEGs). (A) Principal component analysis (PCA) of 18 samples using single normalized mRNA expression levels of three replicate samples in each group. (B) Bar graph showing significantly [fold change, 2; normalized data (log₂), 2; and p-value, 0.05] up- or down-regulated genes between rAd-TB4 vs. BCG groups. Hierarchically clustered heat map calculated with Euclidean distance to confirm gene expression profiles in (C) respective groups and (D) rAd-TB4 group compared with BCG group at each time point: pre-infection, 1 week post-infection (p.i.1week), and 4 weeks post-infection (p.i.4weeks).

FIGURE 8

Functional annotation analysis of the DEGs at each time point. Functional annotation analysis of the DEGs [fold change: 2, normalized data (log₂): 4, p-value: 0.05] identified between rAd-TB4 vs. BCG based on gene ontology (GO) was performed using the DAVID 6.7 database at (A) pre-infection, (B) 1 week post-infection (p.i.1week), and (C) 4 weeks post-infection (p.i.4weeks).