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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.

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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.

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The genetic loci influencing individual resistance to Mycobacterium avium subsp. paratuberculosis (MAP) infection are still largely unknown. In the current study, we searched for genetic loci associated with resistance to MAP infection by evaluating the performance of monocyte-derived macrophages (MDMs) isolated from the peripheral blood of 75 healthy Holsteins cows and infected ex vivo with MAP. Bacterial load (log colony-forming units, log CFUs) within MDMs was quantified at 2 h and 7 days p. i. using a BACTEC MGIT 960 instrument. In addition, the expression levels of some genes with important roles in the innate immune response including epiregulin (EREG), complement component C3 (C3), galectin-9 (Gal9), and nitric oxide (NO-) were measured in the supernatant of the infected cells. DNA from peripheral blood samples of the animals included in the study was isolated and genotyped with the EuroG MD bead Chip (44,779 single nucleotide-polymorphisms, SNPs). Linear mixed models were used to calculate the heritability (h2 ) estimates for each indicator of MDM performance, MAP load within MDMs and EREG, C3, Gal9, and NO-expression. After performing a genome-wide association study, the only phenotypes that showed SNPs with a significant association were the bacterial load within MDMs at 2 h (h2 = 0. 87) and 7 days (h2 = 0.83) p.i. A total of 6 SNPs, 5 candidate genes, and one microRNA on the Bos taurus chromosomes BTA2, BTA17, BTA18, and BTA21 were associated with MAP load at 2 h p.i. Overlap was seen in two SNPs associated with the log CFUs at 2 h and 7 d p.i. The identified SNPs had negative regression coefficients, and were, therefore, associated with a low bacterial load within MDMs. Some of the identified SNPs were located within QTLs previously associated with longevity, reproductive, and udder health traits. Some of the identified candidate genes; Oxysterol Binding Protein Like 6, Cysteine and Serine Rich Nuclear Protein 3, and the Coiled-Coil Domain Containing 92 regulate cellular cholesterol trafficking and efflux, apoptosis, and interferon production, respectively. Taken together, our results define a heritable and distinct immunogenetic profile in MAP-infected macrophages designed to limit bacterial load early after infection.

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An in silico genomic-transcriptomic combined approach allowed the identification of a polymorphism (cis-eQTL-rs41976219) in the Bos taurus genome associated with the CTSG mRNA expression in bovine blood samples, which suggests that individual genetic variation might modulate the CTSG transcriptional response. In the current study, a sandwich ELISA is used to measure the CTSG protein levels in supernatants of monocyte-derived macrophages (MDMs) isolated from cows with the AA (n = 5) and AC (n = 11) genotypes for the rs41976219 and infected ex vivo with MAP. Cows with the AC genotype have significantly higher CTSG protein levels (1.85 ng/mL) in the supernatants of enriched CD14+-MDMs after 2 h of infection when compared with infected CD14+-MDMs from cows with the AA genotype (1.68 ng/mL). Statistically significant differences in the intracellular MAP load at 7 d p.i. are observed between animals with the AA (2.16 log CFUs) and AC (1.44 log CFUs) genotypes. Finally, the association between the rs41976219 allelic variants and resistance to PTB is tested in a larger cattle population (n = 943) classified according to the presence (n = 442) or absence (n = 501) of PTB-associated lesions. The presence of the two minor alleles in the rs41976219 (CC) is more frequent among healthy cows than in cows with PTB-associated lesions in gut tissues (2.2% vs. 1.4%, OR = 0.61). In agreement with this, the CTSG levels in plasma samples of cows without lesions in gut tissues and with the CC (n = 8) genotype are significantly higher than in the plasmas of cows with the AA + AC (n = 36) genotypes.

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A major challenge in tuberculosis is identifying correlates of a protective immune response. The Mycobacterial Growth Inhibition Assay (MGIA) is a functional assay providing an integrated measure of the host immune response to mycobacteria. However, its feasibility is limited by reliance on biosafety level 3 facilities, and its performance has not been widely evaluated in TB-endemic settings. Here, we compared two mycobacterial strains (M. tuberculosis H37Rv versus attenuated M. bovis BCG) in the performance of whole-blood MGIA in 30 TB-exposed children (median age 2 years) in Chennai, India. The time-to-positivity in both assays was similar (5.7 days vs 6 days) and the mycobacterial growth of M. tuberculosis H37Rv and M. bovis BCG were correlated (r = 0.64, p<0.0001). In Bland-Altman analysis, the bias was -0.54 days (95% limit of agreement -2.08, 0.99). Collectively, our results indicate that M. tuberculosis H37Rv can be substituted with the less virulent M. bovis BCG strain to improve feasibility of the MGIA assay, particularly in low-income settings.

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The only currently available approach to early efficacy testing of tuberculosis (TB) vaccine candidates is in vivo preclinical challenge models. These typically include mice, guinea pigs and non-human primates (NHPs), which must be exposed to virulent M.tb in a 'challenge' experiment following vaccination in order to evaluate protective efficacy. This procedure results in disease development and is classified as 'Moderate' in severity under EU legislation and UK ASPA licensure. Furthermore, experiments are relatively long and animals must be maintained in high containment level facilities, making them relatively costly. We describe an in vitro protocol for the direct mycobacterial growth inhibition assay (MGIA) for use in the macaque model of TB vaccine development with the aim of overcoming some of these limitations. Importantly, using an in vitro assay in place of in vivo M.tb challenge represents a significant refinement to the existing procedure for early vaccine efficacy testing. Peripheral blood mononuclear cell and autologous serum samples collected from vaccinated and unvaccinated control animals are co-cultured with mycobacteria in a 48-well plate format for 96 hours. Adherent monocytes are then lysed to release intracellular mycobacteria which is quantified using the BACTEC MGIT system and colony-forming units determined relative to an inoculum control and stock standard curve. We discuss related optimisation and characterisation experiments, and review evidence that the direct NHP MGIA provides a biologically relevant model of vaccine-induced protection. The potential end-users of the NHP MGIA are academic and industry organisations that conduct the assessment of TB vaccine candidates and associated protective immunity using the NHP model. This approach aims to provide a method for high-throughput down-selection of vaccine candidates going forward to in vivo efficacy testing, thus expediting the development of a more efficacious TB vaccine and offering potential refinement and reduction to the use of NHPs for this purpose.

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The lack of efficient and cost-effective diagnostic tools contributes to poor control of tuberculosis in endemic countries. Moreover, host biological processes influence susceptibility, and infection resolution. It is well known that comorbidities such as type 2 diabetes mellitus (DM2) affect the host immune response, making individuals more susceptible to Mycobacterium tuberculosis infection. Currently, there are no laboratory tools that can identify those subjects who have a higher risk of developing the disease. In this study, we used a whole blood mycobacterial growth inhibition assay to assess the immune response capacity to inhibit mycobacterial growth between healthy subjects and those living with DM2 with optimal and poor glycemic control. We also measured cytokine levels in the culture supernatant by cytokine bead arrays. We included 89 patients with DM2: 54 patients with optimal control (mean age 56.2 ± 11.75 years) and 35 patients with poor control (mean age 52.05 ± 9.94 years). We also included 44 healthy subjects as controls (mean age 42.12 ± 11.75 years). We compared the Δlog UFC (a value that represents the difference between mycobacterial growth in the control tube versus the subject's blood) between each group. Our results demonstrate that patients with DM2 had a lower capacity to inhibit M. tuberculosis growth (Δlog UFC DM2 subjects 0.9581 (-0.3897 to 2.495) vs Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p=0.013). Comparing subjects living with DM2 (optimal and poor glycemic control) vs healthy subjects, we found only significant differences between healthy subjects and patients poorly controlled (Δlog UFC optimal control group 0.876 (-0.3897 to 2.495); Δlog UFC poor control group 1.078 (0.068 to 2.33); Δlog UFC healthy subjects 0.7190 (-0.2678 to 2.098); p= 0.022). Therefore, glycemic control assessed by glycosylated hemoglobin values influences the capacity of the host to control the infection. Our results confirm that the whole blood mycobacterial growth inhibition assay has potential utility as an in vitro marker of M. tuberculosis immunological control in vivo in subjects living with DM2. This assay can be used to evaluate the immune response of each individual against M. tuberculosis, allowing clinicians to choose a more specific host-directed therapy.

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Background: Whole blood mycobacterial growth assays (WBMGA) quantify mycobacterial growth in fresh blood samples and may have potential for assessing tuberculosis vaccines and identifying individuals at risk of tuberculosis. We evaluated the evidence for the underlying assumption that in vitro WBMGA results can predict in vivo tuberculosis susceptibility. Methods: A systematic search was done for studies assessing associations between WBMGA results and tuberculosis susceptibility. Meta-analyses were performed for eligible studies by calculating population-weighted averages. Results: No studies directly assessed whether WBMGA results predicted tuberculosis susceptibility. 15 studies assessed associations between WBMGA results and proven correlates of tuberculosis susceptibility, which we divided in two categories. Firstly, WBMGA associations with factors believed to reduce tuberculosis susceptibility were statistically significant in all eight studies of: BCG vaccination; vitamin D supplementation; altitude; and HIV-negativity/therapy. Secondly, WBMGA associations with probable correlates of tuberculosis susceptibility were statistically significant in three studies of tuberculosis disease, in a parasitism study and in two of the five studies of latent tuberculosis infection. Meta-analyses for associations between WBMGA results and BCG vaccination, tuberculosis infection, tuberculosis disease and HIV infection revealed consistent effects. There was considerable methodological heterogeneity. Conclusions: The study results generally showed significant associations between WBMGA results and correlates of tuberculosis susceptibility. However, no study directly assessed whether WBMGA results predicted actual susceptibility to tuberculosis infection or disease. We recommend optimization and standardization of WBMGA methodology and prospective studies to determine whether WBMGA predict susceptibility to tuberculosis disease.

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BACKGROUND: Children are particularly susceptible to tuberculosis. However, most children exposed to Mycobacterium tuberculosis are able to control the pathogen without evidence of infection. Correlates of human protective immunity against tuberculosis infection are lacking, and their identification would aid vaccine design. METHODS: We recruited pairs of asymptomatic children with discordant tuberculin skin test status but the same sleeping proximity to the same adult with sputum smear-positive tuberculosis in a matched case-control study in The Gambia. Participants were classified as either Highly TB-Exposed Uninfected or Highly TB-Exposed Infected children. Serial luminescence measurements using an in vitro functional auto-luminescent Bacillus Calmette-Guérin (BCG) whole blood assay quantified the dynamics of host control of mycobacterial growth. Assay supernatants were analysed with a multiplex cytokine assay to measure associated inflammatory responses. FINDINGS: 29 pairs of matched Highly TB-Exposed Uninfected and Highly TB-Exposed Infected children aged 5 to 15 years old were enroled. Samples from Highly TB-Exposed Uninfected children had higher levels of mycobacterial luminescence at 96 hours than Highly TB-Exposed Infected children. Highly TB-Exposed Uninfected children also produced less BCG-specific interferon-γ than Highly TB-Exposed Infected children at 24 hours and at 96 hours. INTERPRETATION: Highly TB-Exposed Uninfected children showed less control of mycobacterial growth compared to Highly TB-Exposed Infected children in a functional assay, whilst cytokine responses mirrored infection status. FUNDING: Clinical Research Training Fellowship funded under UK Medical Research Council/Department for International Development Concordat agreement and part of EDCTP2 programme supported by European Union (MR/K023446/1). Also MRC Program Grants (MR/K007602/1, MR/K011944/1, MC_UP_A900/1122).

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Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays. Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model. Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001). Conclusion: The BCG-GFP-LuxFO assay requires 225 µL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis.

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A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development.

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Tuberculosis (TB) remains a leading global cause of morbidity and mortality and an effective new vaccine is urgently needed. A major barrier to the rational development of novel TB vaccines is the lack of a validated immune correlate or biomarker of protection. Mycobacterial Growth Inhibition Assays (MGIAs) provide an unbiased measure of ability to control mycobacterial growth in vitro, and may represent a functional correlate of protection. However, the biological relevance of any potential correlate can only be assessed by determining the association with in vivo protection from either a controlled mycobacterial infection or natural development of TB disease. Our data demonstrate that the direct MGIA using peripheral blood mononuclear cells (PBMC) is measuring a biologically relevant response that correlates with protection from in vivo human BCG infection across two independent cohorts. This is the first report of an MGIA correlating with in vivo protection in the species-of-interest, humans, and furthermore on a per-individual as well as per-group basis. Control of mycobacterial growth in the MGIA is associated with a range of immune parameters measured post-BCG infection in vivo including the IFN-γ ELISpot response, frequency of PPD-specific IFN-γ or TNF-α producing CD4+ T cells and frequency of specific sub-populations of polyfunctional CD4+ T cells. Distinct transcriptomic profiles are associated with good vs. poor mycobacterial control in the MGIA, with good controllers showing enrichment for gene sets associated with antigen processing/presentation and the IL-23 pathway, and poor controllers showing enrichment for hypoxia-related pathways. This study represents an important step toward biologically validating the direct PBMC MGIA for use in TB vaccine development and furthermore demonstrates the utility of this assay in determining relevant immune mechanisms and pathways of protection.

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BACKGROUND: In order to eliminate tuberculosis (TB), an effective vaccine is urgently needed to prevent infection with Mycobacterium tuberculosis. A key obstacle for the development of novel TB vaccines is the lack of surrogate markers for immune protection against M. tuberculosis. METHODS: We investigated growth rates of M. tuberculosis in the mycobacterial growth inhibition assay (MGIA) as a marker for mycobacterial growth control of human bronchoalveolar lavage (BALC) and peripheral blood mononuclear cells (PBMC) before and after vaccination with Mycobacterium bovis Bacille Calmette-Guérin (BCG) of healthy adult volunteers. RESULTS: Vaccination induced a positive response (p < 0.001) to purified protein derivate (PPD) in 58.8% of the individuals in an interferon-γ release assay-ELISpot. Intraindividual evaluation of the MGIA growth rates before and after M. bovis BCG-vaccination revealed no significant difference in time to culture positivity before and after vaccination in BALC (p = 0.604) and PBMC (p = 0.199). The magnitude of the PPD-response induced by M. bovis BCG-vaccination did not correlate with growth control in BALC and PBMC (correlation = 0.468, 95% CI: -0.016 to 0.775). CONCLUSION: In conclusion, M. bovis BCG-vaccination-induced mycobacterial-specific cytokine immune response does not result in functional immune control against M. tuberculosis in the MGIA.

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Intravesical bacillus Calmette-Guérin (BCG) is widely used for high-risk, non-muscle-invasive bladder cancer. This report describes four cases that illustrate the spectrum of BCG-induced complications, varying from granulomatous prostatitis to sepsis. There is considerable debate regarding whether inflammation or infection is the predominant mechanism in the pathogenesis of BCG disease. In two patients with a systemic illness, the symptoms first resolved after adding prednisone, indicating a principal role for inflammation in systemic disease. In vitro testing of T-cell responses and a mycobacterial growth inhibition assay were performed for these patients with systemic disease. The patient with mild symptoms showed more effective in vitro growth reduction of BCG, while the patient with sepsis and organ involvement had high T-cell responses but ineffective killing. While these findings are preliminary, it is believed that immunological assays, as described in this report, may provide a better insight into the pathogenesis of BCG disease in individual patients, justifying further research.

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Human tuberculosis remains a significant cause of mortality and morbidity throughout the world. The global economic impact of bovine TB is considerable. An effective vaccine would be the most cost-effective way to control both epidemics, particularly in emerging economies. TB vaccine research would benefit from the identification of an immune correlate of protection with which vaccines could be gated at both preclinical and clinical levels. In-vitro mycobacterial growth inhibition assays (MGIA) are functional assays that include most aspects of the complex host immune response to mycobacteria, and they may serve as functional immune correlates for vaccine development. We applied to cattle an MGIA that was developed for use with human and murine samples. Several technical difficulties were encountered while transferring it to the cattle model. However, our data demonstrate that the assay was not discriminatory in cattle and further work is needed before using it for bovine TB vaccine development.

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The development of a functional biomarker assay in the tuberculosis (TB) field would be widely recognized as a major advance in efforts to develop and to test novel TB vaccine candidates efficiently. We present preliminary studies using mycobacterial growth inhibition assays (MGIAs) to detect Mycobacterium bovis BCG vaccine responses across species, and we extend this work to determine whether a standardized MGIA can be applied in characterizing new TB vaccines. The comparative MGIA studies reviewed here aimed to evaluate robustness, reproducibility, and ability to reflect in vivo responses. In doing so, they have laid the foundation for the development of a MGIA that can be standardized and potentially qualified. A major challenge ahead lies in better understanding the relationships between in vivo protection, in vitro growth inhibition, and the immune mechanisms involved. The final outcome would be a MGIA that could be used with confidence in TB vaccine trials. We summarize data arising from this project, present a strategy to meet the goals of developing a functional assay for TB vaccine testing, and describe some of the challenges encountered in performing and transferring such assays.

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Tuberculosis (TB) continues to pose a serious global health threat, and the current vaccine, BCG, has variable efficacy. However, the development of a more effective vaccine is severely hampered by the lack of an immune correlate of protection. Candidate vaccines are currently evaluated using preclinical animal models, but experiments are long and costly and it is unclear whether the outcomes are predictive of efficacy in humans. Unlike measurements of single immunological parameters, mycobacterial growth inhibition assays (MGIAs) represent an unbiased functional approach which takes into account a range of immune mechanisms and their complex interactions. Such a controlled system offers the potential to evaluate vaccine efficacy and study mediators of protective immunity against Mycobacterium tuberculosis (M.tb). This review discusses the underlying principles and relative merits and limitations of the different published MGIAs, their demonstrated abilities to measure mycobacterial growth inhibition and vaccine efficacy, and what has been learned about the immune mechanisms involved.

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