Vaccines for Leprosy and Tuberculosis: Opportunities for Shared Research, Development, and Application

Abstract

Tuberculosis (TB) and leprosy still represent significant public health challenges, especially in low- and lower middle-income countries. Both poverty-related mycobacterial diseases require better tools to improve disease control. For leprosy, there has been an increased emphasis on developing tools for improved detection of infection and early diagnosis of disease. For TB, there has been a similar emphasis on such diagnostic tests, while increased research efforts have also focused on the development of new vaccines. Bacille Calmette-Guérin (BCG), the only available TB vaccine, provides insufficient and inconsistent protection to pulmonary TB in adults. The impact of BCG on leprosy, however, is significant, and the introduction of new TB vaccines that might replace BCG could, therefore, have serious impact also on leprosy. Given the similarities in antigenic makeup between the pathogens Mycobacterium tuberculosis (Mtb) and M. leprae, it is well possible, however, that new TB vaccines could cross-protect against leprosy. New TB subunit vaccines currently evaluated in human phase I and II studies indeed often contain antigens with homologs in M. leprae. In this review, we discuss pre-clinical studies and clinical trials of subunit or whole mycobacterial vaccines for TB and leprosy and reflect on the development of vaccines that could provide protection against both diseases. Furthermore, we provide the first preclinical evidence of such cross-protection by Mtb antigen 85B (Ag85B)-early secretory antigenic target (ESAT6) fusion recombinant proteins in in vivo mouse models of Mtb and M. leprae infection. We propose that preclinical integration and harmonization of TB and leprosy research should be considered and included in global strategies with respect to cross-protective vaccine research and development.

Keywords: Mycobacterium leprae; Mycobacterium tuberculosis; antigen 85B; early secretory antigenic target; hybrid recombinant protein; leprosy; tuberculosis; vaccines.

Figure 1

Leprosy and tuberculosis (TB) vaccine pipelines. Schematic representation of leprosy (upper segment) and TB (lower segment) candidate vaccines in clinical trials. Source: adapted from Ref. (59), TBVI/Aeras September 2017 and https://clinicaltrials.gov/. The primary endpoints are indicated for each trial with the exception of TB/FLU-04L for which primary outcome is yet not registered. POI, prevention of infection; POR, prevention of recurrence; POD, prevention of disease.

Figure 2

IFN-γ secretion after Ag85-ESAT immunization. C57BL/6j and HLA-A2tg mice B6.Cg-Tg (117) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed under specific pathogen-free conditions. Recombinant proteins were overexpressed in E. coli BL21 (DE3) and purified to remove any traces of endotoxin as described in Ref. (116, 118). For the production of the antigen 85B (Ag85B)-early secretory antigenic target (ESAT6), hybrid recombinant hybrid protein, the Ag85B and ESAT6 genes were fused together by PCR with a linker coding for the amino acids NVA. C57BL/6j mice [(A); 13–14 animals per group] and HLA-A2tg mice [(B); 5 animals per group] were immunized three times subcutaneously with Mycobacterium tuberculosis (Mtb) Ag85-ESAT or Mycobacterium leprae Ag85-ESAT recombinant protein (25 µg) adjuvanted with GLA-SE [glucopyranosyl lipid adjuvant-stable emulsion (23) kindly provided by Infectious Disease Research Institute; Seattle, WA, USA; TLR4 agonist; 20 µg]; or CpG (ODN1826 5′-TCC ATG ACG TTC CTG ACG TT -3′; InvivoGen, San Diego, CA, USA; TLR9 agonist; 50 µg) (119). Splenocytes were harvested 4 weeks after final injections and restimulated in vitro with Mtb or M. leprae Ag85-ESAT hybrid recombinant proteins or the single Ag85B and ESAT6 recombinant proteins (all 10 µg/ml). IFN-γ secretion was analyzed by ELISA after 5 days. All mice were analyzed separately. Data shown indicate the mean and SE value of five mice per group.

Figure 3

Quantification of serum antibodies. Following immunization of C57BL/6j (A) and HLA-A2 tg (B) mice with adjuvant alone, Mycobacterium tuberculosis (Mtb) Ag85-ESAT or Mycobacterium leprae Ag85-ESAT recombinant protein in GLA-SE (A) or CpG (B), antibody titers (OD₄₅₀) against Mtb Ag85-ESAT, M. leprae Ag85-ESAT, or Mtb/M. leprae Antigen 85B (Ag85B) and early secretory antigenic target (ESAT6) were determined by ELISA as described in Ref. (120). As a control coating with BSA (0.4% in PBS) was used. Sera from immunized mice were collected from cardiac blood 3 weeks after final immunization Serum dilutions are shown on the x-axis. Test groups included 3–5 mice. All mice were analyzed separately. Results are shown for one representative animal.

Figure 4

Determination of bacterial burden. C57BL/6j mice were injected with 10⁴ live Mycobacterium leprae (121) (viability: 11,000; in 40 µl PBS) in each hind foot pads 4 weeks after the final protein immunization. 7 months after M. leprae challenge, mouse footpads were harvested, and M. leprae were enumerated by RLEP PCR (122). HLA-A2tg mice were infected with live Mycobacterium tuberculosis (Mtb) strain H37Rv 6 weeks after the final protein immunization and 10 weeks after Bacille Calmette–Guérin (BCG) immunization (119). All animals included in the experiments were observed daily in order to ensure ethics requirements and to monitor any adverse effects possibly related to the vaccination or infection. (A) Bacteria were determined by the RLEP PCR from footpads from M. leprae infected C57BL/6j mice that had been immunized with GLA-SE adjuvant alone (−), M. leprae Ag85-ESAT/GLA-SE, Mtb Ag85-ESAT/GLA-SE, or heat killed M. leprae (HKML; 2 × 10⁸ in 40 µl; viability: 6,400) as indicated on the x-axis. Each symbol represents one mouse. Calculated bacterial loads are expressed as RLEP counts on the y-axis. Horizontal lines indicate median values with interquartile range. (B) CFUs were determined in lung homogenates from Mtb-infected unimmunized (−) or Mtb-infected HLA-A2 tg mice that were immunized with BCG1331 (10⁶ CFU), M. leprae Ag85-ESAT or Mtb Ag85-ESAT as indicated under the x-axis. Each symbol represents one mouse. Bacterial loads are expressed as log10 bacterial counts. Horizontal lines indicate median values with interquartile range. CFU of test and control groups were compared to the controls using the Mann–Whitney test and a p < 0.01 was considered significant. * marks differences that remained significant after multiple test correction using Kruskal–Wallis testing with Dunn’s post-test.