Immunization of mice with a recombinant adenovirus vaccine inhibits the early growth of Mycobacterium tuberculosis after infection

Abstract

Background: In pulmonary Mycobacterium tuberculosis (Mtb) infection, immune responses are delayed compared to other respiratory infections, so that antigen-specific cells are not detected in the lungs earlier than day 14. Even after parenteral immunization with Bacille Calmette Guerin (BCG) or a subunit vaccine, the immune response after Mtb challenge is only slightly accelerated and the kinetics of pulmonary Mtb growth do not differ between naïve and immunized animals up to day 14.

Methods and findings: Mice were immunized intranasally with a recombinant adenovirus expressing mycobacterial antigen 85A (Ad85A), challenged by aerosol with Mtb and the kinetics of Mtb growth in the lungs measured. Intranasal immunization with Ad85A inhibits Mtb growth in the early phase of infection, up to day 8. Protection is sustained for at least 7 months and correlates with the presence of antigen-specific activated effector CD8 T cells in the lungs. Antigen 85A-specific T cells respond to antigen presenting cells from the lungs of mice immunized with Ad85A 23 weeks previously, demonstrating the persistence of antigen in the lungs.

Conclusions/significance: Intranasal immunization with Ad85A can inhibit early growth of Mtb because it establishes a lung antigen depot and maintains an activated lung-resident lymphocyte population. We propose that an optimal immunization strategy for tuberculosis should aim to induce both lung and systemic immunity, targeting the early and late phases of Mtb growth.

Figure 1. Early control of mycobacterial growth by intranasal administration of Ad85A.

BALB/c mice were immunized with BCG or primed with BCG and 10 weeks later boosted with Ad85A i.n. (A) or immunized only with Ad85A i.n. (B) Naïve mice were used as controls. Mice were challenged with Mtb by aerosol 4 weeks after the last immunization and sacrificed at days 1, 3, 8, 14, 21 and 28. Lung and spleen CFU were enumerated. Results are expressed as the mean counts of 5–7 mice per group. * p<0.05 Ad85A or B-Ad85A immunized mice versus Naïve or BCG, **p<0.05 for B-Ad85A versus Naïve, BCG versus B-Ad85A, BCG versus Naïve. Similar results were obtained in a repeat experiment. Standard deviations are small, so that the error bars are within the symbols when not visible. (C) Inhibition of Mtb growth in macrophages by lung cells in vitro. Murine peritoneal macrophages were infected with Mtb and co-cultured with lung cells from naïve mice or mice immunized with Ad85A i.n. 3 weeks previously. Inhibition of mycobacterial growth was determined after 3 days. Results are expressed as the mean % inhibition +/− SEM of four experiments with four mice per group.

Figure 2. Intranasal administration of Ad85A increases protection at 4 and 24 weeks.

(A) Time line of the experiment. BALB/c mice were immunized with BCG, or primed with BCG and 10 weeks later boosted intranasally with Ad85A (B-Ad85A i.n.), or immunized only with Ad85A i.n. Naïve mice were used as challenge controls. Mice were challenged with Mtb by aerosol 4 (B) or 24 (C) weeks after the boost. Deposition in the lungs was measured 24 h after challenge and was ∼200 CFU/lung. Mice were sacrificed 6 weeks later and lung and spleen CFU enumerated. Symbols show CFU counts of individual mice and the horizontal line indicates the mean. * p<0.05 versus Naïve, **p<0.05 versus BCG. Similar results were obtained in two other experiments where the lungs and spleens were harvested at 4 weeks post Mtb challenge.

Figure 3. Cytokine responses of lung T cells to antigen 85A.

Mice were immunized with Ad85A i.n. (Ad85A i.n.) or primed with BCG and boosted 8 weeks later with Ad85A intranasally (B-Ad85A i.n.). Lung cells were isolated at the indicated times after the last immunization and stimulated with pooled 85A peptides for 6 hours. The frequencies of IFNγ, IL-2 and TNFα producing cells were determined by flow cytometry on CD8 (A) and CD4 (B) gated cells. Pie charts indicate the proportions of single (light grey), dual (dark grey) and triple (black) CD8 producers of IFNγ, TNFα and IL-2 in the lungs of mice 4 (C) and 23 (D) weeks after immunization. Results are expressed as the mean +/− SEM of three/four mice per group, representative of two independent experiments.

Figure 4. Phenotype of antigen-specific cells following intranasal immunization with Ad85A.

Mice were immunized as described in the legend to Fig. 2 and lung cells stained for CD8, IFNγ, CD62L and CD127 (A) or CD27 (B). All results are from Ad85A i.n. mice at 4 and 23 weeks. Similar data were obtained from B-Ad85A i.n. mice. Results are expressed as the mean +/− SEM from three or four mice except for blood where the PBMCs from three/four mice were pooled. Similar results were obtained from two independent experiments. (C) Persistence of antigen 85A in the lungs. Cells from the lungs of mice immunized 23 weeks previously with Ad85A i.n. (open histogram) or naïve control mice (shaded histogram) were used as APCs and co-cultured with CFSE-labelled 85A-specific splenic T cells. Cells were recovered at day 5 and stained with H-2L^d 85A peptide _70–78aa (MPVGGQSSF) tetramer. The histogram shows the CFSE profile of 85A tetramer positive CD8 cells. The result of one representative experiment of three is shown.