Mark A. Rodgers

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a major global health challenge, especially for people living with HIV (PLWH) not on antiretroviral therapy. We previously showed that intravenous Bacillus Calmette-Guérin (IV-BCG) provides robust protection in SIV+ Mauritian cynomolgus macaques (MCM). Here, we evaluate whether the immunogenicity and efficacy of IV-BCG in SIV+ MCM was impaired by eliminating BCG with drugs. SIV+ macaques were treated with an anti-BCG regimen of isoniazid, rifampicin, and ethambutol (HRE), starting either 1- or 3-weeks post-BCG vaccination. Five months post-vaccination, macaques were challenged with Mtb for 12 weeks. Protection conferred by IV-BCG was significant regardless of HRE timing. However, non-controllers, characterized by high viremia and reduced CD4+ T cells in the lungs, were significantly less protected than controllers. Thus, the robust efficacy of IV-BCG in SIV+ MCM is retained regardless of anti-BCG drug treatment, indicating that BCG does not need to survive long to provide protection. In contrast, SIV control was necessary for full protection induced by IV-BCG. Thus, our results support that viral control is critical for vaccine-elicited protection from TB in PLWH.

Tuberculosis (TB) relapse after appropriate drug treatment is poorly understood but critical to developing shorter treatment regimens. Using a cynomolgus macaque model of human TB, macaques with active TB disease were treated with a short course of isoniazid and rifampin and subsequently infected with SIV. Serial clinical, microbiologic, immunologic, and position emission and computed tomography (PET CT) assessments were performed to identify risk factors of relapse. Of the 12 animals, eight developed radiologically defined relapse, including four that had clinical and/or microbiologic signs. Greater gross pathology and bacterial burden were observed in relapse animals. PET CT characteristics before, during, and at the end of the treatment were similar among relapse and non-relapse animals. We show that complete sterilization or very low Mtb burden is protective against SIV-induced TB relapse but cannot be predicted by PET CT. Using barcoded M. tuberculosis , we found that Mtb dissemination during relapse originated from both lung and thoracic lymph nodes, underscoring the importance of lymph nodes as a reservoir. By matching barcoded Mtb and serial PET CT, we also demonstrate that not every site of persistent Mtb growth after drug treatment is capable of dissemination and relapse, underscoring the complex nature of drug treatment and relapse.

Tuberculosis (TB) relapse after appropriate drug treatment is poorly understood but critical to developing shorter treatment regimens. Using a cynomolgus macaque model of human TB, macaques with active TB disease were treated with a short course of isoniazid and rifampin and subsequently infected with SIV. Serial clinical, microbiologic, immunologic and position emission and computed tomography (PET CT) assessments were performed to identify risk factors of relapse. Of the 12 animals, eight developed radiologically defined relapse including four that had clinical and/or microbiologic signs. Greater gross pathology and bacterial burden were observed in relapse animals. PET CT characteristics before, during, and at the end of treatment were similar amongst relapse and non-relapse animals. We show that complete sterilization or very low Mtb burden is protective against SIV-induced TB relapse but cannot be predicted by PET CT. Using bar-coded M. tuberculosis , we found that Mtb dissemination during relapse originated from both lung and thoracic lymph nodes, underscoring the importance of lymph nodes as a reservoir. By matching bar-coded Mtb and serial PET CT, we also demonstrate that not every site of persistent Mtb growth after drug treatment is capable of dissemination and relapse, underscoring the complex nature of drug treatment and relapse.

Tuberculosis (TB) is a major health burden worldwide despite widespread intradermal (ID) BCG vaccination in newborns. We previously demonstrated that changing the BCG route and dose from 5 × 105 CFUs ID to 5 × 107 CFUs i.v. resulted in prevention of Mycobacterium tuberculosis (Mtb) infection and TB disease in highly susceptible nonhuman primates. Identifying immune mechanisms protection following i.v. BCG will facilitate development of more effective vaccines against TB. Here, we depleted lymphocyte subsets prior to and during Mtb challenge in i.v. BCG–vaccinated macaques to identify those necessary for protection. Depletion of adaptive CD4 T cells, but not adaptive CD8αβ T cells, resulted in loss of protection with increased Mtb burdens and dissemination, indicating that CD4 T cells are critical to i.v. BCG–mediated protection. Depletion of unconventional CD8α-expressing lymphocytes (NK cells, innate T cells, and CD4+CD8α+ double-positive T cells) abrogated protection in most i.v. BCG–immunized macaques, supporting further investigation into which of these cell subsets contribute to protection after vaccination.

Human challenge experiments could accelerate tuberculosis vaccine development. This requires a safe Mycobacterium tuberculosis (Mtb) strain that can both replicate in the host and be reliably cleared. Here we genetically engineered Mtb strains encoding up to three kill switches: two mycobacteriophage lysin operons negatively regulated by tetracycline and a degron domain–NadE fusion, which induces ClpC1-dependent degradation of the essential enzyme NadE, negatively regulated by trimethoprim. The triple-kill-switch (TKS) strain showed similar growth kinetics and antibiotic susceptibilities to wild-type Mtb under permissive conditions but was rapidly killed in vitro without trimethoprim and doxycycline. It established infection in mice receiving antibiotics but was rapidly cleared upon cessation of treatment, and no relapse was observed in infected severe combined immunodeficiency mice or Rag^−/− mice. The TKS strain had an escape mutation rate of less than 10⁻¹⁰ per genome per generation. These findings suggest that the TKS strain could be a safe, effective candidate for a human challenge model.

Improved vaccination strategies for tuberculosis are needed. Intravenous (i.v.) delivery of live attenuated Mycobacterium bovis BCG provides protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here we genetically engineered two strains, BCG-TetON-DL and BCG-TetOFF-DL, to either induce or inhibit expression of two phage lysin operons, respectively, upon tetracycline exposure. We show that lysin expression kills BCG in vitro, in infected macrophages, and following infection of immunocompetent (C57BL/6) and immunocompromised (SCID) mice. Modified BCG elicited similar immune responses and provided similar protection against Mtb challenge as wild-type BCG in mice. In macaques, cessation of tetracycline treatment reduced i.v.-administered BCG-TetOFF-DL numbers. Intravenous BCG-TetOFF-DL increased pulmonary CD4 T-cell responses compared with wild-type BCG-induced responses and provided robust protection against Mtb challenge. Sterilizing immunity occurred in 6 of 8 macaques compared with 2 of 8 wild-type BCG-immunized macaques. Thus, a ‘kill-switch’ BCG strain provides additional safety and robust protection against Mtb infection.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4- to 8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, ( n = 5; i.v.) at 3 and 17 days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group ( n = 5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy 8 weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the leading causes of death due to an infectious agent. Coinfection with HIV exacerbates M. tuberculosis infection outcomes in people living with HIV. Bacillus Calmette–Guérin (BCG), the only approved TB vaccine, is effective in infants, but its efficacy in adolescents and adults is limited. In this study, we investigated the immune responses elicited by BCG administered via i.v. or intradermal (i.d.) routes in SIV-infected Mauritian cynomolgus macaques (MCM) without the confounding effects of M. tuberculosis challenge. We assessed the impact of vaccination on T cell responses in the airway, blood, and tissues (lung, thoracic lymph nodes, and spleen), as well as the expression of cytokines, cytotoxic effectors, and key transcription factors. Our results showed that i.v. BCG induces a robust and sustained immune response, including tissue-resident memory T cells in lungs, polyfunctional CD4+ and CD8αβ+ T cells expressing multiple cytokines, and CD8αβ+ T cells and NK cells expressing cytotoxic effectors in airways. We also detected higher levels of mycobacteria-specific IgG and IgM in the airways of i.v. BCG-vaccinated MCM. Although i.v. BCG vaccination resulted in an influx of tissue-resident memory T cells in lungs of MCM with controlled SIV replication, MCM with high plasma SIV RNA (>105 copies/ml) typically displayed reduced T cell responses, suggesting that uncontrolled SIV or HIV replication would have a detrimental effect on i.v. BCG-induced protection against M. tuberculosis.

Children living with HIV have a higher risk of developing tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Gamma delta (γδ) T cells in the context of HIV/Mtb coinfection have been understudied in children, despite in vitro evidence suggesting γδ T cells assist with Mtb control. We investigated whether boosting a specific subset of γδ T cells, phosphoantigen-reactive Vγ9+Vδ2+ cells, could improve TB outcome using a nonhuman primate model of pediatric HIV/Mtb coinfection. Juvenile Mauritian cynomolgus macaques (MCM), equivalent to 4–8-year-old children, were infected intravenously (i.v.) with SIV. After 6 months, MCM were coinfected with a low dose of Mtb and then randomized to receive zoledronate (ZOL), a drug that increases phosphoantigen levels, (n=5; i.v.) at 3- and 17-days after Mtb accompanied by recombinant human IL-2 (s.c.) for 5 days following each ZOL injection. A similarly coinfected MCM group (n=5) was injected with saline as a control. Vγ9+Vδ2+ γδ T cell frequencies spiked in the blood, but not airways, of ZOL+IL-2-treated MCM following the first dose, however, were refractory to the second dose. At necropsy eight weeks after Mtb, ZOL+IL-2 treatment did not reduce pathology or bacterial burden. γδ T cell subset frequencies in granulomas did not differ between treatment groups. These data show that transiently boosting peripheral γδ T cells with ZOL+IL-2 soon after Mtb coinfection of SIV-infected MCM did not improve Mtb host defense.