rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B

Abstract

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy.

FIG 1

Frequency of cytokine-producing CD4⁺ T cells in response to M. leprae antigens. Mice were sham immunized or immunized with BCG or rBCG30-ARMF-II Tice (rBCG30) at week 0. At week 7, half of the mice immunized with BCG or rBCG30 received a booster vaccination with r30Mtb protein in adjuvant. At week 8, splenocytes were isolated from all mice and stimulated in vitro with MLCwA, HKML, r30ML protein, or r30ML peptide for 6 h and 24 h. GolgiPlug (containing brefeldin A) was included for the final 3 h of stimulation prior to intracellular cytokine staining, followed by analysis for cytokine-producing T cells by multiparameter flow cytometry. The frequencies of live CD3⁺ CD4⁺ T cells producing each of the seven different possible combinations of IFN-γ, IL-2, and TNF (indicated by the plus and minus signs underneath the graphs), as well as the summation of all cytokine-producing T cells (“All”), are shown. Background numbers of cells producing cytokines without antigen stimulation were subtracted from the data. Values are means and standard errors for 4 mice. The experiment was repeated once with similar results, and the data from both experiments with respect to r30ML-specific cytokine-producing CD4⁺ T cells are summarized in Fig. 3.

FIG 2

Frequency of cytokine-producing CD8⁺ T cells in response to M. leprae antigens. Mice were immunized, and T cells were analyzed by multiparameter flow cytometry as described in the Fig. 1 legend. The frequencies of live CD3⁺ CD8⁺ T cells producing each of the seven different possible combinations of IFN-γ, IL-2, and TNF after 24 h of in vitro stimulation (indicated by the plus and minus signs underneath the graphs) as well as the summation of all cytokine-producing T cells (“All”) are shown. Background numbers of cells producing cytokines without antigen stimulation were subtracted from the data. Values are means and standard errors for 4 mice.

FIG 3

Priming with rBCG30 and boosting with r30Mtb protein in adjuvant significantly enhances r30ML-specific cytokine-producing CD4⁺ T cells and IFN-γ secretion. Mice were immunized as described in Fig. 1. At 8 weeks, splenocytes were isolated and stimulated in vitro with r30ML protein and r30ML peptide for 6 h prior to intracellular cytokine staining (A and B), for 24 h prior to intracellular cytokine staining (C and D), or for 3 days prior to analyzing the culture medium for IFN-γ by ELISA (E and F). Two independent experiments were performed: experiment I (A, C, and E) and experiment II (B, D, and F). For intracellular cytokine staining (A to D), the summation of all CD4⁺ T cells producing cytokine (IFN-γ, IL-2, and/or TNF) is shown and background numbers of cells producing cytokines without antigen stimulation were subtracted from the data. Values are means and standard errors for 4 mice. Statistical comparisons are indicated by lines above the bars with P values calculated by one-way ANOVA and multiple comparisons performed by a Tukey-Kramer post hoc analysis.

FIG 4

Protective efficacy. Mice were sham immunized (Sham) or immunized with BCG or rBCG30 at week 0 and challenged at week 10 with 5,000 live M. leprae bacteria in each hind footpad. In experiment II, additional mice were sham immunized with IFA (Sham IFA) or immunized with purified r30Mtb in IFA or r30ML in IFA at week 7 and also challenged at week 10. Also in experiment II, additional mice were immunized with BCG or rBCG30 at week 0 and additionally boosted with r30Mtb or r30ML in IFA as indicated at week 7 and challenged at week 10. Seven months after challenge, the animals were euthanized and the number of AFB was enumerated in their hind footpads. Data are the log AFB/footpad; for graphing purposes, mice with 0 AFB per 60 oil immersion fields were assigned a value of 1 equivalent to log 4 in experiment I and log 3.7 in experiment II. (A) Experiment I. *, P < 0.0001 by ANOVA and P = 0.0004 by Kruskal-Wallis rank sum test; **, P < 0.0001 by ANOVA and P = 0.002 by Kruskal-Wallis rank sum test; ***, P < 0.04 by ANOVA and P = 0.04 by Kruskal-Wallis rank sum test. (B) Experiment II. Data where AFB counts were 0 are shown by open circles. †, P < 0.001 versus Sham by ANOVA and P = 0.002 versus Sham by nonparametric analysis; γ, P < 0.02 by ANOVA and P = 0.02 by nonparametric analysis; γγ, P < 0.02 by ANOVA and P = 0.04 by nonparametric analysis; §, P = 0.03 by nonparametric analysis.