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

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

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

Building Volunteer Engagement in the Tennessee Extension Master Gardener Program from the Ground Up

This study was designed to assess key volunteer attitudes and perceptions about local and state Extension Master Gardener (EMG) volunteer programs. EMG volunteers in Tennessee completed a total of 759 surveys between January 22 and March 10, 2020. Survey responses showed there was a strong positive perception of local EMG groups and the connection with the University of Tennessee system overall. When respondents were segmented, answers to key questions showed illuminating differences between volunteers with positive and neutral or negative views of the program. The variations in answers show areas of potential emphasis to support healthy local group culture and broader efficacy of the EMG program

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

Assessment of the reproducibility and inter-site transferability of the murine direct splenocyte mycobacterial growth inhibition assay (MGIA)

Tuberculosis (TB) vaccine candidates must be tested for safety and efficacy using preclinical challenge models prior to advancement to human trials, because of the lack of a validated immune correlate or biomarker of protection. New, unbiased tools are urgently needed to expedite the selection of vaccine candidates at an early stage of development and reduce the number of animals experimentally infected with virulent Mycobacterium tuberculosis (M.tb). In recent years, there has been a concerted effort to develop standardised functional ex vivo mycobacterial growth inhibition assays (MGIAs) as a potential surrogate read-out of vaccine efficacy. We have previously described a direct MGIA for use with mouse splenocytes. In the current study, we set out to systematically compare co-culture conditions for the murine direct splenocyte MGIA with respect to both intra-assay repeatability and inter-site reproducibility. Common sample sets were shared between laboratory sites and reproducibility and sensitivity to detect a BCG-vaccine induced response were assessed. Co-culturing 5×106 splenocytes in 48-well plates resulted in improved reproducibility and superior sensitivity to detect a vaccine response compared with standing or rotating sealed 2ml screw-cap tubes. As the difference between naïve and BCG vaccinated mice was not consistently detected across both sample sets at both sites, we sought to further improve assay sensitivity by altering the multiplicity of infection (MOI). Cell viability at the end of the co-culture period was improved when splenocyte input number was reduced, with the highest viability for the condition of 3×106 splenocytes in 48-well plates. This cell input was also associated with the greatest sensitivity to detect a BCG vaccine-mediated MGIA response using an M.tb inoculum. Based on our findings, we recommend optimal co-culture conditions in a move towards aligning direct MGIA protocols and generating a cross-species consensus for early evaluation of TB vaccine candidates and biomarker studies

How to model the impact of vaccines for policymaking when the characteristics are uncertain: a case study in Thailand prior to the vaccine rollout during the COVID-19 pandemic

Thailand faced a dilemma of which groups to prioritise with a limited first tranche of COVID-19 vaccinations in early 2021, at a time when there was low incidence and low mortality in the country. A mathematical modelling analysis was performed to compare the potential short-term impact of allocating the available doses to either the high severity group (over 65-year-olds) or the high transmission group (aged 20-39). At the time of the analysis, there was uncertainty about the precise characteristics of the vaccines available, in terms of their potential impact on transmission and reductions to the severity of infection. As such, a range of vaccine characteristic scenarios, with differing levels of severity and transmission reductions were explored. Using the evidence available at the time regarding severity reduction of infection due to the vaccines, the model suggested that vaccinating high severity group should be the priority if reductions in deaths is the priority. Vaccinating this group was found to have a direct impact on reducing the number of deaths, while the incidence and hospitalisations remained unchanged. However, the model found that vaccinating the high transmission group with a vaccine with sufficiently high protection against infection (more than 70%) could provide enough herd effects to delay the expected epidemic peak, resulting in both case and death reductions in both target groups. The model explored a 12-month time horizon. These analyses helped to inform the vaccination strategy in Thailand throughout 2021 and can inform future modelling studies for policymaking when the characteristics of vaccines are uncertain

A theory-based framework for understanding the establishment, persistence, and diffusion of community-based conservation

Over decades, biodiversity conservation researchers and practitioners have developed theories and conceptual frameworks to inform the planning, implementation, and evaluation of community-based conservation (CBC). While a diversity of mechanisms for understanding and supporting CBC has helped tailor approaches to local needs and conditions, the absence of a unified lens to understand CBC has limited the capacity for integrating foundational theory into practice more systemically, and for learning across different projects, stakeholders, and institutions. We introduce a theory-based framework called “the CBC framework” that draws upon three foundational theories from sociology, economics, and political science to understand the establishment, persistence, and diffusion of CBC. Experience applying aspects of the framework within different conservation organizations demonstrates how this integrative approach can provide a gateway for practitioners to engage with social science theory to understand the status and context of CBC interventions and efforts. For practitioners, scientists, evaluators, and strategists, the framework can guide the design of CBC interventions and monitoring and evaluation systems to facilitate theory-based learning and enable evidence-informed decision-making. Approaches like the CBC framework that facilitate collaborative planning, evaluation, and research can help better integrate social science theory in conservation practice while increasing the capacity for conservation scientists, practitioners, and stakeholders to learn together and adaptively manage CBC to deliver positive results for both people and nature

Examining the stability of membrane proteins within SMALPs

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co2+ and Zn2+ resulted in an increased sensitivity