A novel murine cytomegalovirus vaccine vector protects against Mycobacterium tuberculosis

Abstract

Tuberculosis remains a global health problem so that a more effective vaccine than bacillus Calmette-Guérin is urgently needed. Cytomegaloviruses persist lifelong in vivo and induce powerful immune and increasing ("inflationary") responses, making them attractive vaccine vectors. We have used an m1-m16-deleted recombinant murine CMV (MCMV) expressing Mycobacterium tuberculosis Ag 85A to show that infection of mice with this recombinant significantly reduces the mycobacterial load after challenge with M. tuberculosis, whereas control empty virus has a lesser effect. Both viruses induce immune responses to H-2(d)-restricted epitopes of MCMV pp89 and M18 Ags characteristic of infection with other MCMVs. A low frequency of 85A-specific memory cells could be revealed by in vivo or in vitro boosting or after challenge with M. tuberculosis. Kinetic analysis of M. tuberculosis growth in the lungs of CMV-infected mice shows early inhibition of M. tuberculosis growth abolished by treatment with NK-depleting anti-asialo ganglio-N-tetraosylceramide Ab. Microarray analysis of the lungs of naive and CMV-infected mice shows increased IL-21 mRNA in infected mice, whereas in vitro NK assays indicate increased levels of NK activity. These data indicate that activation of NK cells by MCMV provides early nonspecific protection against M. tuberculosis, potentiated by a weak 85A-specific T cell response, and they reinforce the view that the innate immune system plays an important role in both natural and vaccine-induced protection against M. tuberculosis.

FIGURE 1.

Protection against M. tuberculosis challenge. BALB/c mice were infected with 2 × 10⁶ PFU eMCMV or MCMV85A i.p. or i.v. and 4 wk later challenged with M. tuberculosis. Mice challenged at 8, 12, and 24 wk postinfection were immunized i.p. Controls were naive or immunized with 2 × 10⁹ virus particles Ad85A i.n. or 2 × 10⁵ CFU BCG s.c. Lung M. tuberculosis CFU were enumerated 5 wk after challenge. Symbols show individual mice, and the horizontal line indicates the mean. Data were analyzed by a one-way ANOVA with a Tukey posttest.

FIGURE 2.

Kinetics of pp89 and M18 responses. BALB/c mice were infected with 2 × 10⁶ PFU eMCMV or MCMV85A i.p. Lung, liver, and spleen cells were isolated at the indicated times and after stimulation for 6 h with pp89 (A) or M18 (B) peptides, and the proportion of CD8 IFN-γ–producing cells was determined by surface and intracellular immunofluorescence staining and flow cytometric analysis. Results are expressed as the means ± SD of three to four mice per group.

FIGURE 3.

Ag 85A–specific immune responses. (A) Mice were infected with eMCMV or MCMV85A and 4 wk later boosted with 2 × 10⁹ virus particles Ad85A i.m. Six days later, lung, spleen, and liver cells were isolated and stimulated for 6 h with pooled 85A peptides. (B) Spleen cells from eMCMV- or MCMV85A-infected mice were stimulated in vitro with a pool of 15-mer peptides covering the sequence of Ag 85A, expanded with IL-2, rested, and restimulated with 85A peptides before assaying by intracellular staining and flow cytometry for Ag-specific IFN-γ–producing cells. (C) Lung or spleen cells from MCMV85A-infected mice boosted with Ad85A were stimulated with individual 85A peptides covering the dominant H-2^d CD4 and CD8 epitopes. The frequencies of IFN-γ– or IL-2–producing cells were determined by flow cytometry on CD8- and CD4-gated cells. Results are expressed as the means ± SD of three or four mice per group, representative of two independent experiments. Data were analyzed by a one-way ANOVA with a Tukey posttest.

FIGURE 4.

Mechanisms of M. tuberculosis protection. (A) Mice were infected with 2 × 10⁶ PFU eMCMV or MCMV85A i.p. with uninfected (naive) mice as controls. Five weeks postinfection all the mice were challenged with M. tuberculosis and sacrificed at days 7 and 15 for enumeration of lung CFU. (B) Five weeks postinfection with eMCMV or MCMV85A, infected and control uninfected (naive) mice were challenged with M. tuberculosis i.n. and after 1 wk sacrificed for enumeration of lung M. tuberculosis CFU. The day before and on the day of M. tuberculosis challenge, mice were given anti-ASGM1 Ab i.p. Flow cytometry panels show depletion by anti-ASGM1 Ab as assessed by DX5 and NKp46 staining of lung cells. Representative CFU data from one of two experiments with four to five mice per group are shown. (C) Mice were infected and challenged as in (A). Lung cells were isolated 14 d after the M. tuberculosis challenge and stimulated for 6 h with pooled 85A peptides or pp89. The frequencies of IFN-γ–producing cells were determined by flow cytometry on CD8- and CD4-gated cells. Results are expressed as the means ± SD of four mice per group. Data were analyzed by a one-way ANOVA with a Tukey posttest.

FIGURE 5.

Effect of IL-21 on M. tuberculosis protection. (A) Mice infected with 2 × 10⁶ PFU eMCMV or uninfected controls were challenged with M. tuberculosis. Two days before and on the day of challenge, half of each group was treated with 75 μg IL-21RFc i.p. Mice were sacrificed 7 d later and M. tuberculosis CFU were enumerated. Human IgG1 was used as a control and had no effect (data not shown). (B) Naive mice were challenged with M. tuberculosis. On the days of challenge 100 ng recombinant mouse IL-21 or 100 ng CCL22 as control was administered i.n. Mice were sacrificed 7 d later and M. tuberculosis CFU were enumerated. (C) Spleen cells from five eMCMV and five uninfected mice were assayed on YAC-1 targets for NK activity. Data from 100:1 spleen cell to YAC-1 ratio are shown. Horizontal lines show the mean of each group. Data were analyzed by a one-way ANOVA with a Tukey posttest.