Backtranslation of human RNA biosignatures of tuberculosis disease risk into the preclinical pipeline is condition dependent.

It is not clear whether human progression to active tuberculosis disease (TB) risk signatures are viable endpoint criteria for evaluations of treatments in clinical or preclinical development. TB is the deadliest infectious disease globally and more efficacious vaccines are needed to reduce this mortality. However, the immune correlates of protection for either preventing infection with Mycobacterium tuberculosis or preventing TB disease have yet to be completely defined, making the advancement of candidate vaccines through the pipeline slow, costly, and fraught with risk. Human-derived correlate of risk (COR) gene signatures, which identify an individual's risk to progressing to active TB disease, provide an opportunity for evaluating new therapies for TB with clear and defined endpoints. Though prospective clinical trials with longitudinal sampling are prohibitively expensive, characterization of COR gene signatures is practical with preclinical models. Using a 3Rs (Replacement, Reduction and Refinement) approach we reanalyzed heterogeneous publicly available transcriptional datasets to determine whether a specific set of COR signatures are viable endpoints in the preclinical pipeline. We selected RISK6, Sweeney3 and BATF2 human-derived blood-based RNA biosignatures because they require relatively few genes to assign a score and have been carefully evaluated across several clinical cohorts. Excitingly, these data provide proof-of-concept that human COR signatures seem to have high fidelity across several tissue types in the preclinical TB model pipeline and show best performance when the model most closely reflected human infection or disease conditions. Human-derived COR signatures offer an opportunity for high-throughput preclinical endpoint criteria of vaccine and drug therapy evaluations. One Sentence Summary: Human-derived biosignatures of tuberculosis disease progression were evaluated for their predictive fidelity across preclinical species and derived tissues using available public data sets.

Demonstrating the utility of the ex vivo murine mycobacterial growth inhibition assay (MGIA) for high-throughput screening of tuberculosis vaccine candidates against multiple Mycobacterium tuberculosis complex strains.

Human tuberculosis (TB) is caused by various members of the Mycobacterium tuberculosis (Mtb) complex. Differences in host response to infection have been reported, illustrative of a need to evaluate efficacy of novel vaccine candidates against multiple strains in preclinical studies. We previously showed that the murine lung and spleen direct mycobacterial growth inhibition assay (MGIA) can be used to assess control of ex vivo mycobacterial growth by host cells. The number of mice required for the assay is significantly lower than in vivo studies, facilitating testing of multiple strains and/or the incorporation of other cellular analyses. Here, we provide proof-of-concept that the murine MGIA can be applied to evaluate vaccine-induced protection against multiple Mtb clinical isolates. Using an ancient and modern strain of the Mtb complex, we demonstrate that ex vivo bacillus Calmette-Guérin (BCG)-mediated mycobacterial growth inhibition recapitulates protection observed in the lung and spleen following in vivo infection of mice. Further, we provide the first report of cellular and transcriptional correlates of BCG-induced growth inhibition in the lung MGIA. The ex vivo MGIA represents a promising platform to gain early insight into vaccine performance against a collection of Mtb strains and improve preclinical evaluation of TB vaccine candidates.

Development and application of the direct mycobacterial growth inhibition assay: a systematic review.

Introduction: First described by Wallis et al. in 2001 for the assessment of TB drugs, the direct mycobacterial growth inhibition assay (MGIA) offers a tractable ex vivo tool measuring the combined influences of host immunity, strain virulence and intervention effects. Over the past 13 years, we have led efforts to adapt the direct MGIA for the assessment of TB vaccines including optimisation, harmonisation and validation of BCG vaccine-induced responses as a benchmark, as well as assay transfer to institutes worldwide. Methods: We have performed a systematic review on the primary published literature describing the development and applications of the direct MGIA from 2001 to June 2023 in accordance with the PRISMA reporting guidelines. Results: We describe 63 studies in which the direct MGIA has been applied across species for the evaluation of TB drugs and novel TB vaccine candidates, the study of clinical cohorts including those with comorbidities, and to further understanding of potential immune correlates of protection from TB. We provide a comprehensive update on progress of the assay since its conception and critically evaluate current findings and evidence supporting its utility, highlighting priorities for future directions. Discussion: While further standardisation and validation work is required, significant advancements have been made in the past two decades. The direct MGIA provides a potentially valuable tool for the early evaluation of TB drug and vaccine candidates, clinical cohorts, and immune mechanisms of mycobacterial control. Systematic review registration: https://www.crd.york.ac.uk/prospero/, identifier CRD42023423491.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Adaption of the ex vivo mycobacterial growth inhibition assay for use with murine lung cells.

In the absence of a correlate(s) of protection against human tuberculosis and a validated animal model of the disease, tools to facilitate vaccine development must be identified. We present an optimised ex vivo mycobacterial growth inhibition assay (MGIA) to assess the ability of host cells within the lung to inhibit mycobacterial growth, including Bacille Calmette-Guérin (BCG) and Mycobacterium tuberculosis (MTB) Erdman. Growth of BCG was reduced by 0.39, 0.96 and 0.73 log10 CFU following subcutaneous (s.c.) BCG, intranasal (i.n.) BCG, or BCG s.c. + mucosal boost, respectively, versus naïve mice. Comparatively, a 0.49 (s.c.), 0.60 (i.n.) and 0.81 (s.c. + mucosal boost) log10 reduction in MTB CFU was found. A BCG growth inhibitor, 2-thiophenecarboxylic acid hydrazide (TCH), was used to prevent quantification of residual BCG from i.n. immunisation and allow accurate MTB quantification. Using TCH, a further 0.58 log10 reduction in MTB CFU was revealed in the i.n. group. In combination with existing methods, the ex vivo lung MGIA may represent an important tool for analysis of vaccine efficacy and the immune mechanisms associated with vaccination in the organ primarily affected by MTB disease.

How can we improve priority-setting for investments in health research? A case study of tuberculosis.

BACKGROUND: Although enhanced priority-setting for investments in health research for development is essential to tackling inequalities in global health, there is a lack of consensus on an optimal priority-setting process. In light of the current surge in tuberculosis (TB) research investment, we use TB as a case study. METHODS: We investigated two critical aspects of a research prioritisation process, namely the criteria that should be used to rank alternative research options and which stakeholders should be involved in priority-setting. We conducted semi-structured interviews with 24 key informants purposively selected from four distinct groups - academia, funding bodies, international policy or technical agencies, and national disease control programmes. Interview transcripts were analysed verbatim using a framework approach. We also performed a systematic analysis of seven diverse TB research prioritisation processes. RESULTS: There was consensus that well-defined and transparent criteria for assessing research options need to be agreed at the outset of any prioritisation process. It was recommended that criteria should select for research that is likely to have the greatest public health impact in affected countries rather than research that mainly fills scientific knowledge gaps. Some interviewees expressed strong views about the need - and reluctance - to make politically challenging decisions that place some research areas at a lower priority for funding. The importance of taking input from stakeholders from countries with high disease burden was emphasised; such stakeholders were notably absent from the majority of prioritisation processes we analysed. CONCLUSIONS: This study indicated two critical areas for improvement of research prioritisation processes such that inequalities in health are better addressed - the need to deprioritise some research areas to generate a specific and meaningful list for investment, and greater involvement of experts working in high disease-burden countries.

RUTI Vaccination Enhances Inhibition of Mycobacterial Growth ex vivo and Induces a Shift of Monocyte Phenotype in Mice.

Tuberculosis (TB) is a major global health problem and there is a dire need for an improved treatment. A strategy to combine vaccination with drug treatment, termed therapeutic vaccination, is expected to provide benefit in shortening treatment duration and augmenting treatment success rate. RUTI candidate vaccine has been specifically developed as a therapeutic vaccine for TB. The vaccine is shown to reduce bacillary load when administered after chemotherapy in murine and guinea pig models, and is also immunogenic when given to healthy adults and individuals with latent TB. In the absence of a validated correlate of vaccine-induced protection for TB vaccine testing, mycobacterial growth inhibition assay (MGIA) has been developed as a comprehensive tool to evaluate vaccine potency ex vivo. In this study, we investigated the potential of RUTI vaccine to control mycobacterial growth ex vivo and demonstrated the capacity of MGIA to aid the identification of essential immune mechanism. We found an association between the peak response of vaccine-induced growth inhibition and a shift in monocyte phenotype following RUTI vaccination in healthy mice. The vaccination significantly increased the frequency of non-classical Ly6C- monocytes in the spleen after two doses of RUTI. Furthermore, mRNA expressions of Ly6C--related transcripts (Nr4a1, Itgax, Pparg, Bcl2) were upregulated at the peak vaccine response. This is the first time the impact of RUTI has been assessed on monocyte phenotype. Given that non-classical Ly6C- monocytes are considered to play an anti-inflammatory role, our findings in conjunction with previous studies have demonstrated that RUTI could induce a balanced immune response, promoting an effective cell-mediated response whilst at the same time limiting excessive inflammation. On the other hand, the impact of RUTI on non-classical monocytes could also reflect its impact on trained innate immunity which warrants further investigation. In summary, we have demonstrated a novel mechanism of action of the RUTI vaccine, which suggests the importance of a balanced M1/M2 monocyte function in controlling mycobacterial infection. The MGIA could be used as a screening tool for therapeutic TB vaccine candidates and may aid the development of therapeutic vaccination regimens for TB in the near future.

HIV, HCMV and mycobacterial antibody levels: a cross-sectional study in a rural Ugandan cohort.

OBJECTIVES: A growing evidence base implicates human cytomegalovirus (HCMV) as a risk factor for TB disease. We investigated total IgG and mycobacteria-specific antibodies in a cross-sectional study nested within a rural Ugandan General Population Cohort (GPC), in relation to HIV infection and the magnitude of HCMV IgG response. METHODS: Sera from 2189 individuals (including 27 sputum-positive TB cases) were analysed for antibodies against mycobacteria (Ag85A, PPD, LAM, ESAT6/CFP10) and HCMV, tetanus toxoid (TT) and total IgG. RESULTS: Anti-mycobacterial antibodies increased with age until approximately 20 years, when they plateaued. Higher HCMV exposure (measured by IgG) was associated with lower levels of some anti-mycobacterial antibodies, but no increase in total IgG. HIV infection was associated with a decrease in all anti-mycobacterial antibodies measured and with an increase in total IgG. CONCLUSIONS: The increase in anti-mycobacterial antibodies with age suggests increasing exposure to non-tuberculous mycobacteria (NTM), and to M.tb itself. HIV infection is associated with decreased levels of all mycobacterial antibodies studied here, and high levels of HCMV IgG are associated with decreased levels of some mycobacterial antibodies. These findings point towards the importance of humoral immune responses in HIV/TB co-infection and highlight a possible role of HCMV as a risk factor for TB disease.

Human cytomegalovirus epidemiology and relationship to tuberculosis and cardiovascular disease risk factors in a rural Ugandan cohort.

Human cytomegalovirus (HCMV) infection has been associated with increased mortality, specifically cardiovascular disease (CVD), in high-income countries (HICs). There is a paucity of data in low- and middle-income countries (LMICs) where HCMV seropositivity is higher. Serum samples from 2,174 Ugandan individuals were investigated for HCMV antibodies and data linked to demographic information, co-infections and a variety of CVD measurements. HCMV seropositivity was 83% by one year of age, increasing to 95% by five years. Female sex, HIV positivity and active pulmonary tuberculosis (TB) were associated with an increase in HCMV IgG levels in adjusted analyses. There was no evidence of any associations with risk factors for CVD after adjusting for age and sex. HCMV infection is ubiquitous in this rural Ugandan cohort from a young age. The association between TB disease and high HCMV IgG levels merits further research. Known CVD risk factors do not appear to be associated with higher HCMV antibody levels in this Ugandan cohort.

Decorin antagonizes the angiogenic network: concurrent inhibition of Met, hypoxia inducible factor 1α, vascular endothelial growth factor A, and induction of thrombospondin-1 and TIMP3.

Decorin, a small leucine-rich proteoglycan, inhibits tumor growth by antagonizing multiple receptor tyrosine kinases including EGFR and Met. Here, we investigated decorin during normoxic angiogenic signaling. An angiogenic PCR array revealed a profound decorin-evoked transcriptional inhibition of pro-angiogenic genes, such as HIF1A. Decorin evoked a reduction of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor A (VEGFA) in MDA-231 breast carcinoma cells expressing constitutively-active HIF-1α. Suppression of Met with decorin or siRNA evoked a similar reduction of VEGFA by attenuating downstream ß-catenin signaling. These data establish a noncanonical role for ß-catenin in regulating VEGFA expression. We found that exogenous decorin induced expression of thrombospondin-1 and TIMP3, two powerful angiostatic agents. In contrast, decorin suppressed both the expression and enzymatic activity of matrix metalloprotease (MMP)-9 and MMP-2, two pro-angiogenic proteases. Our data establish a novel duality for decorin as a suppressor of tumor angiogenesis under normoxia by simultaneously down-regulating potent pro-angiogenic factors and inducing endogenous anti-angiogenic agents.