Protective immune responses to a recombinant adenovirus type 35 tuberculosis vaccine in two mouse strains: CD4 and CD8 T-cell epitope mapping and role of gamma interferon

Abstract

There is an urgent need for an efficacious vaccine against tuberculosis (TB). Cellular immune responses are key to an effective protective response against TB. Recombinant adenovirus (rAd) vectors are especially suited to the induction of strong T-cell immunity and thus represent promising vaccine vehicles for the prevention of TB. We have previously reported on rAd vector serotype 35, the serotype of choice due to low preexisting immunity worldwide, which expresses a unique fusion protein of Mycobacterium tuberculosis antigens Ag85A, Ag85B, and TB10.4 (Ad35-TBS). Here, we demonstrate that Ad35-TBS confers protection against M. tuberculosis when administered to mice through either an intranasal or an intramuscular route. Histological evaluation of lung tissue corroborated the protection and, in addition, demonstrated differences between two mouse strains, with diffuse inflammation in BALB/c mice and distinct granuloma formation in C57BL/6 mice. Epitope mapping analysis in these mouse strains showed that the major T-cell epitopes are conserved in the artificial fusion protein, while three novel CD8 peptides were discovered. Using a defined set of T-cell epitopes, we reveal differences between the two mouse strains in the type of protective immune response, demonstrating that different antigen-specific gamma interferon (IFN-gamma)-producing T cells can provide protection against M. tuberculosis challenge. While in BALB/c (H-2(d)) mice, a dominant CD8 T-cell response was detected, in C57BL/6 (H-2(b)) mice, more balanced CD4/CD8 T-cell responses were observed, with a more pronounced CD4 response in the lungs. These results unify conflicting reports on the relative importance of CD4 versus CD8 T-cell responses in protection and emphasize the key role of IFN-gamma.

FIG. 1.

Immunogenicity of Ad35-TBS. Groups of C57BL/6 mice were immunized i.m. with 10⁷ to 10¹⁰ VP of Ad35-TBS (five mice per group) or 10¹⁰ VP of Ad35-E (control) (three mice per group). Two weeks after immunization, the antigen-specific T-cell response was determined using ICS upon an in vitro stimulation of splenocytes with the peptide pools covering the whole sequence of Ag85A or TB10.4. The percentages of antigen-specific IFN-γ-positive CD8 and CD4 cells are shown for Ag85A (A and C) and TB10.4 (B and D). Bars represent geometric means.

FIG. 2.

Identification of an Ag85-specific T-cell epitope as an example of the epitope mapping procedure. Groups of BALB/c mice were immunized with 10⁹ VP of Ad35-TBS (five mice per group) or Ad35-E (three mice per group) (control). Two weeks after immunization, the T-cell immune response was determined using ICS upon an in vitro stimulation of splenocytes with the small 15-mer peptide pools (A). Possibly reactive peptides were identified as the intersection of positive R and C pools (B) and used for in vitro stimulation in the follow-up experiment (C). From the identified positive peptide, overlapping 9-mer peptides were generated, covering the whole sequence, and used for in vitro stimulation in the follow-up experiment (D). The identification of the positive 9-mer peptide enables the generation of a specific pentamer, which makes it possible to monitor the development of an immune response in the blood in time (E). Black bars represent results obtained with the pooled splenocytes of Ad35-TBS-immunized mice, while white bars indicate the background levels measured with splenocytes from Ad35-E (control)-immunized mice. Bars in E represent standard errors of the means. T, total pool.

FIG. 3.

Histological evaluation. The cumulative inflammation score (A and C) and the percentage of lung inflammation (B and D) were determined for BALB/c (A and B) and C57BL/6 (C and D) mice 6 weeks after challenge. Bars represent means. ****, P < 0.0001; ***, P < 0.0005; **, P < 0.005; *, P < 0.05.

FIG. 4.

Histopathology of the lungs. The representative microscopic images of lungs from an untreated mouse (control) (A and E), a nonvaccinated challenged mouse (naïve) (B and F), an Ad35-TBS i.n. vaccinated challenged mouse (C and G), and an Ad35-TBS i.m. vaccinated challenged mouse (D and H) are shown for BALB/c (A to D) and C57BL/6 (E to H) mouse lines. Inserts show a part of the lung at a higher magnification (×20).

FIG. 5.

Immune status of animals at the time of challenge. Six weeks after the immunization with 10⁹ VP of Ad35-TBS, the antigen-specific CD8 and CD4 T-cell responses were determined using ELISPOT upon an in vitro stimulation of lung cells (A and B) and splenocytes (C and D) of BALB/c (A and C) and C57BL/6 (B and D) mice. The results obtained upon i.n. immunization are shown in upper left panels, and those obtained upon i.m. immunization are shown in lower left panels. The corresponding protection, expressed as the ratio of CFU of vaccinated to CFU of naïve mice, is shown in the right panels. Bars in the left panels represent geometric means, and bars in the right panels show 95% confidence intervals of the mean values (diamonds). Dashed lines indicate the background levels in the ELISPOT assay. SFU, spot-forming units.