Immunogenicity and protection against Mycobacterium avium with a heterologous RNA prime and protein boost vaccine regimen

Abstract

Prophylactic efficacy of two different delivery platforms for vaccination against Mycobacterium avium (M. avium) were tested in this study; a subunit and an RNA-based vaccine. The vaccine antigen, ID91, includes four mycobacterial antigens: Rv3619, Rv2389, Rv3478, and Rv1886. We have shown that ID91+GLA-SE is effective against a clinical NTM isolate, M. avium 2-151 smt. Here, we extend these results and show that a heterologous prime/boost strategy with a repRNA-ID91 (replicon RNA) followed by protein ID91+GLA-SE boost is superior to the subunit protein vaccine given as a homologous prime/boost regimen. The repRNA-ID91/ID91+GLA-SE heterologous regimen elicited a higher polyfunctional CD4⁺ T_H1 immune response when compared to the homologous protein prime/boost regimen. More significantly, among all the vaccine regimens tested only repRNA-ID91/ID91+GLA-SE induced IFN-γ and TNF-secreting CD8⁺ T cells. Furthermore, the repRNA-ID91/ID91+GLA-SE vaccine strategy elicited high systemic proinflammatory cytokine responses and induced strong ID91 and an Ag85B-specific humoral antibody response a pre- and post-challenge with M. avium 2-151 smt. Finally, while all prophylactic prime/boost vaccine regimens elicited a degree of protection in beige mice, the heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen provided greater pulmonary protection than the homologous protein prime/boost regimen. These data indicate that a prophylactic heterologous repRNA-ID91/ID91+GLA-SE vaccine regimen augments immunogenicity and confers protection against M. avium.

Figure 1.. Overview of vaccine strategies and experimental design.

(A) Schematic of ID91 replicon RNA (repRNA) formulated in a Lipid InOrganic Nanoparticle (LION^™) delivery system. (B) Prime/boost vaccine strategies received by each group. (C) Timeline of immunizations and M. avium 2-151 smt aerosol challenge. Experimental samples were collected at two time-points: D35 (one week post boost immunization) and D96 (six weeks post M. avium challenge).

Figure 2.. Heterologous RNA prime/protein boost vaccine strategy drives a robust systemic proinflammatory response both post-boost and post- M. avium challenge.

Circulating proinflammatory cytokine levels were measured in serum samples collected one week post boost immunization and six weeks post M. avium challenge. (A-H) Concentration of IFN-γ, TNF-α, IL-1β, IL-2, IL-6, KC/GRO, IL-10, and IL-5 (measured in pg/mL) at both time points. Bars show mean ± SEM, dots represent individual mice, n = 3–4/group. Asterisks indicate statistical significance compared to the saline alone group, where **p<0.01, ***p < 0.001, and ****p < 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test.

Figure 3.. Heterologous RNA prime/protein boost vaccine strategy induces ID91 and Ag85B antigen-specific humoral immune responses post-boost and post M. avium challenge.

Serum samples were collected one week post boost immunization and six weeks post M. avium challenge and were evaluated for: (A-C) ID91 antigen specific Total IgG (A), IgG1 (B), and IgG2c (C) responses; (D-F) Ag85B-specific Total IgG (D), IgG1 (E), and IgG2c (F) responses. Log₁₀ endpoint titer (EPT) is shown (LOD = limit of detection). Bars show mean ± SEM, dots represent individual mice, n = 3–4/group. Asterisks indicate statistical significance compared to saline alone cohort, where **p < 0.01, ***p < 0.001, and ****p < 0.0001 using two-way ANOVA with Dunnett’s multiple comparisons test.

Figure 4.. Heterologous RNA prime/protein boost vaccine strategy elicits robust ID91-specific CD4⁺ T_H1 immune responses post-boost.

Splenocytes were cultured from all six groups of mice one week post boost immunization and stimulated with ID91 or ID91 components (Rv1886, Rv3619, Rv3478, Rv2389) ex vivo and evaluated for CD4⁺ T cell responses by intracellular cytokine staining flow cytometry including: (A) Percent frequency of CD4⁺CD44⁺ ID91-specific single-cytokine-producing cells; (B) ID91-specific CD4⁺CD44⁺ polyfunctional T_H1 cytokine-producing cells; (C-F) Percent frequency of CD4⁺CD44⁺ Rv1886 (C), Rv3619 (D), Rv3478 (E), and Rv2389 (F) specific single-cytokine-producing cells. Bars show mean ± SEM, dots represent individual mice, n = 4/group. Asterisks indicate statistical significance, where **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test.

Figure 5.. Heterologous RNA prime/protein boost vaccine strategy is the most effective regimen for CD8⁺ T cell responses post-boost.

Splenocytes were cultured one week post boost immunization and stimulated with ID91 ex vivo and evaluated for CD8⁺ T cell responses by intracellular cytokine staining flow cytometry including: (A) Percent frequency of CD8⁺CD44⁺ ID91-specific single-cytokine-producing cells; (B) ID91-specific CD8⁺CD44⁺ polyfunctional cytokine-producing cells. Bars show mean ± SEM, dots represent individual mice, n = 4/group. Asterisks indicate statistical significance, where **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test.

Figure 6.. All prime/boost vaccine strategies provide prophylactic pulmonary protection against M. avium in beige mice.

Beige mice were infected with M. avium 2-151 smt by aerosol route four weeks post final immunization. (A-C) Bacterial burden was assessed by colony forming unit (CFU) in lung (A), spleen (B), and liver (C) organ homogenates six weeks post challenge. CFU means were compared between each group using one-way ANOVA with Dunnett’s multiple comparisons test. Black line and error bars show mean ± SEM, dots represent individual mice, n = 6–7/group. Asterisks indicate statistical significance, where *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 7.. All prophylactic prime/boost vaccine strategies decrease lung immunopathology in Beige mice post challenge with M. avium.

(A-F) Representative images of accessory lung lobes from beige mice immunized with saline (A), BCG (B)v, repRNA-ID91 (C), ID91+GLA-SE (D), repRNA-ID91 prime/ID91+GLA-SE boost (E), or ID91+GLA-SE prime/repRNA-ID91 boost (F) after H&E staining for pulmonary lesions (dark purple). (G) Bars show mean ± SEM percent lesion area, dots represent individual mice, n = 7/group. Asterisks indicate statistical significance, where ****p < 0.0001 using one-way ANOVA with Dunnett’s multiple comparisons test.