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.

Vaccines for a sustainable planet.

The health of the planet is one objective of the United Nations' Sustainable Development Goals. Vaccines can affect not only human health but also planet health by reducing poverty, preserving microbial diversity, reducing antimicrobial resistance, and preventing an increase in pandemics that is fueled partly by climate change.

Inflammation and immune activation are associated with risk of Mycobacterium tuberculosis infection in BCG-vaccinated infants.

Tuberculosis vaccine development is hindered by the lack of validated immune correlates of protection. Exploring immune correlates of risk of disease and/or infection in prospective samples can inform this field. We investigate whether previously identified immune correlates of risk of TB disease also associate with increased risk of M.tb infection in BCG-vaccinated South African infants, who became infected with M.tb during 2-3 years of follow-up. M.tb infection is defined by conversion to positive reactivity in the QuantiFERON test. We demonstrate that inflammation and immune activation are associated with risk of M.tb infection. Ag85A-specific IgG is elevated in infants that were subsequently infected with M.tb, and this is coupled with upregulated gene expression of immunoglobulin-associated genes and type-I interferon. Plasma levels of IFN-[Formula: see text]2, TNF-[Formula: see text], CXCL10 (IP-10) and complement C2 are also higher in infants that were subsequently infected with M.tb.

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.

Understanding the interaction between cytomegalovirus and tuberculosis in children: The way forward.

Over 1 million children develop tuberculosis (TB) each year, with a quarter dying. Multiple factors impact the risk of a child being exposed to Mycobacterium tuberculosis (Mtb), the risk of progressing to TB disease, and the risk of dying. However, an emerging body of evidence suggests that coinfection with cytomegalovirus (CMV), a ubiquitous herpes virus, impacts the host response to Mtb, potentially influencing the probability of disease progression, type of TB disease, performance of TB diagnostics, and disease outcome. It is also likely that infection with Mtb impacts CMV pathogenesis. Our current understanding of the burden of these 2 diseases in children, their immunological interactions, and the clinical consequence of coinfection is incomplete. It is also unclear how potential interventions might affect disease progression and outcome for TB or CMV. This article reviews the epidemiological, clinical, and immunological literature on CMV and TB in children and explores how the 2 pathogens interact, while also considering the impact of HIV on this relationship. It outlines areas of research uncertainty and makes practical suggestions as to potential studies that might address these gaps. Current research is hampered by inconsistent definitions, study designs, and laboratory practices, and more consistency and collaboration between researchers would lead to greater clarity. The ambitious targets outlined in the World Health Organization End TB Strategy will only be met through a better understanding of all aspects of child TB, including the substantial impact of coinfections.

The in vitro direct mycobacterial growth inhibition assay (MGIA) for the early evaluation of TB vaccine candidates and assessment of protective immunity: a protocol for non-human primate cells.

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.

Characterization of the Infant Immune System and the Influence and Immunogenicity of BCG Vaccination in Infant and Adult Rhesus Macaques.

In many countries where tuberculosis (TB) is endemic, the Bacillus Calmette-Guérin (BCG) vaccine is given as close to birth as possible to protect infants and children from severe forms of TB. However, BCG has variable efficacy and is not as effective against adult pulmonary TB. At present, most animal models used to study novel TB vaccine candidates rely on the use of adult animals. Human studies show that the infant immune system is different to that of an adult. Understanding how the phenotypic profile and functional ability of the immature host immune system compares to that of a mature adult, together with the subsequent BCG immune response, is critical to ensuring that new TB vaccines are tested in the most appropriate models. BCG-specific immune responses were detected in macaques vaccinated within a week of birth from six weeks after immunization indicating that neonatal macaques are able to generate a functional cellular response to the vaccine. However, the responses measured were significantly lower than those typically observed following BCG vaccination in adult rhesus macaques and infant profiles were skewed towards the activation and attraction of macrophages and monocytes and the synthesis in addition to release of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α. The frequency of specific immune cell populations changed significantly through the first three years of life as the infants developed into young adult macaques. Notably, the CD4:CD8 ratio significantly declined as the macaques aged due to a significant decrease in the proportion of CD4+ T-cells relative to a significant increase in CD8+ T-cells. Also, the frequency of both CD4+ and CD8+ T-cells expressing the memory marker CD95, and memory subset populations including effector memory, central memory and stem cell memory, increased significantly as animals matured. Infant macaques, vaccinated with BCG within a week of birth, possessed a significantly higher frequency of CD14+ classical monocytes and granulocytes which remained different throughout the first three years of life compared to unvaccinated age matched animals. These findings, along with the increase in monokines following vaccination in infants, may provide an insight into the mechanism by which vaccination with BCG is able to provide non-specific immunity against non-mycobacterial organisms.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

Automatic real-time analysis and interpretation of arterial blood gas sample for Point-of-care testing: Clinical validation.

BACKGROUND: Point-of-care arterial blood gas (ABG) is a blood measurement test and a useful diagnostic tool that assists with treatment and therefore improves clinical outcomes. However, numerically reported test results make rapid interpretation difficult or open to interpretation. The arterial blood gas algorithm (ABG-a) is a new digital diagnostics solution that can provide clinicians with real-time interpretation of preliminary data on safety features, oxygenation, acid-base disturbances and renal profile. The main aim of this study was to clinically validate the algorithm against senior experienced clinicians, for acid-base interpretation, in a clinical context. METHODS: We conducted a prospective international multicentre observational cross-sectional study. 346 sample sets and 64 inpatients eligible for ABG met strict sampling criteria. Agreement was evaluated using Cohen's kappa index, diagnostic accuracy was evaluated with sensitivity, specificity, efficiency or global accuracy and positive predictive values (PPV) and negative predictive values (NPV) for the prevalence in the study population. RESULTS: The concordance rates between the interpretations of the clinicians and the ABG-a for acid-base disorders were an observed global agreement of 84,3% with a Cohen's kappa coefficient 0.81; 95% CI 0.77 to 0.86; p < 0.001. For detecting accuracy normal acid-base status the algorithm has a sensitivity of 90.0% (95% CI 79.9 to 95.3), a specificity 97.2% (95% CI 94.5 to 98.6) and a global accuracy of 95.9% (95% CI 93.3 to 97.6). For the four simple acid-base disorders, respiratory alkalosis: sensitivity of 91.2 (77.0 to 97.0), a specificity 100.0 (98.8 to 100.0) and global accuracy of 99.1 (97.5 to 99.7); respiratory acidosis: sensitivity of 61.1 (38.6 to 79.7), a specificity of 100.0 (98.8 to 100.0) and global accuracy of 98.0 (95.9 to 99.0); metabolic acidosis: sensitivity of 75.8 (59.0 to 87.2), a specificity of 99.7 (98.2 to 99.9) and a global accuracy of 97.4 (95.1 to 98.6); metabolic alkalosis sensitivity of 72.2 (56.0 to 84.2), a specificity of 95.5 (92.5 to 97.3) and a global accuracy of 93.0 (88.8 to 95.3); the four complex acid-base disorders, respiratory and metabolic alkalosis, respiratory and metabolic acidosis, respiratory alkalosis and metabolic acidosis, respiratory acidosis and metabolic alkalosis, the sensitivity, specificity and global accuracy was also high. For normal acid-base status the algorithm has PPV 87.1 (95% CI 76.6 to 93.3) %, and NPV 97.9 (95% CI 95.4 to 99.0) for a prevalence of 17.4 (95% CI 13.8 to 21.8). For the four-simple acid-base disorders and the four complex acid-base disorders the PPV and NPV were also statistically significant. CONCLUSIONS: The ABG-a showed very high agreement and diagnostic accuracy with experienced senior clinicians in the acid-base disorders in a clinical context. The method also provides refinement and deep complex analysis at the point-of-care that a clinician could have at the bedside on a day-to-day basis. The ABG-a method could also have the potential to reduce human errors by checking for imminent life-threatening situations, analysing the internal consistency of the results, the oxygenation and renal status of the patient.

A non-human primate in vitro functional assay for the early evaluation of TB vaccine candidates.

We present a non-human primate mycobacterial growth inhibition assay (MGIA) using in vitro blood or cell co-culture with the aim of refining and expediting early tuberculosis vaccine testing. We have taken steps to optimise the assay using cryopreserved peripheral blood mononuclear cells, transfer it to end-user institutes, and assess technical and biological validity. Increasing cell concentration or mycobacterial input and co-culturing in static 48-well plates compared with rotating tubes improved intra-assay repeatability and sensitivity. Standardisation and harmonisation efforts resulted in high consistency agreements, with repeatability and intermediate precision <10% coefficient of variation (CV) and inter-site reproducibility <20% CV; although some systematic differences were observed. As proof-of-concept, we demonstrated ability to detect a BCG vaccine-induced improvement in growth inhibition in macaque samples, and a correlation between MGIA outcome and measures of protection from in vivo disease development following challenge with either intradermal BCG or aerosol/endobronchial Mycobacterium tuberculosis (M.tb) at a group and individual animal level.

Are people with an intellectual disability at increased risk of attachment difficulties? A critical review.

Attachment difficulties are associated with a range of adverse outcomes in mental health, and people with intellectual disabilities (IDs) may be at greater risk of experiencing difficulties in their attachment relationships. This review critically evaluated recent research measuring the prevalence of attachment difficulties in people with ID. Eight studies met the inclusion criteria, and a higher prevalence of insecure and disorganized attachment classifications, and symptoms of attachment disorder, was found across a number of subgroups of people with diagnoses of ID. However, the validity and reliability of measures of attachment have not been empirically established in this population, and control groups were not always appropriate. These findings indicate the need to (1) develop reliable and standardized assessments of attachment for people with ID and (2) evaluate the efficacy of attachment-based interventions in relation to reducing psychological distress, mental health problems and expression of behaviours experienced by others as challenging.

Serum From Melioidosis Survivors Diminished Intracellular Burkholderia pseudomallei Growth in Macrophages: A Brief Research Report.

Melioidosis is a neglected tropical disease with high mortality rate. It is caused by the Gram-negative, CDC category B select agent Burkholderia pseudomallei (B. ps) that is intrinsically resistant to first-line antibiotics. An antibody-based vaccine is likely to be the most effective control measure. Previous studies have demonstrated significant mechanistic roles of antibodies in protection against death in animal models, but data from human melioidosis is scarce. Herein, we used in-vitro antibody-dependent cellular phagocytosis and growth inhibition assays to assess the mechanism of protective antibodies in patients with acute melioidosis. We found that serum from patients who survived the disease enable more live B. ps to be engulfed by THP-1 derived macrophages (median 1.7 × 103 CFU/ml, IQR 1.1 × 103-2.5 × 103 CFU/ml) than serum from patients who did not survive (median 1.2 × 103 CFU/ml, IQR 0.7 × 103-1.8 × 103, p = 0.02). In addition, the intracellular growth rate of B. ps pre-opsonized with serum from survivors (median 7.89, IQR 5.58-10.85) was diminished when compared with those with serum from non-survivors (median 10.88, IQR 5.42-14.88, p = 0.04). However, the difference of intracellular bacterial growth rate failed to reach statistical significance when using purified IgG antibodies (p = 0.09). These results provide new insights into a mechanistic role of serum in protection against death in human melioidosis for antibody-based vaccine development.

Glibenclamide alters interleukin-8 and interleukin-1ß of primary human monocytes from diabetes patients against Mycobacterium tuberculosis infection.

Type 2 diabetes mellitus (T2DM) is an important risk factor for development of tuberculosis (TB). Our previous study showed glibenclamide, an anti-diabetic drug used to control blood glucose concentration, reduced interleukin (IL)-8 secretion from primary human monocytes challenged with M. tuberculosis (Mtb). In mice infected with Mtb, IL-1ß is essential for host resistance through the enhancement of cyclooxygenase that limits excessive Type I interferon (IFN) production and fosters Mtb containment. We hypothesize that glibenclamide may also interfere with monocyte mediated immune responses against Mtb and alter the balance between IL-1ß and IFNα-mediated immunity. Purified monocytes from non-diabetic and diabetic individuals were infected with Mtb or M. bovis BCG. We demonstrate that monocytes from diabetes patients who were being treated with glibenclamide showed reduced IL-1ß and IL-8 secretion when exposed to Mtb. Additionally, these responses also occurred when monocytes from non-diabetic individuals were pre-treated with glibenclamide in vitro. Moreover, this pre-treatment enhanced IFNa1 expression but was not involved with prostaglandin E2 (PGE2) expression in response to Mtb infection. Taken together, our data show that glibenclamide might exacerbate susceptibility of diabetes patients to Mtb infection by reducing IL-1ß and IL-8 production by monocytes.

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.

Human Immune Responses to Melioidosis and Cross-Reactivity to Low-Virulence Burkholderia Species, Thailand1.

Melioidosis is a neglected tropical disease with an estimated annual mortality rate of 89,000 in 45 countries across tropical regions. The causative agent is Burkholderia pseudomallei, a gram-negative soil-dwelling bacterium. In Thailand, B. pseudomallei can be found across multiple regions, along with the low-virulence B. thailandensis and the recently discovered B. thailandensis variant (BTCV), which expresses B. pseudomallei-like capsular polysaccharide. Comprehensive studies of human immune responses to B. thailandensis variants and cross-reactivity to B. pseudomallei are not complete. We evaluated human immune responses to B. pseudomallei, B. thailandensis, and BTCV in melioidosis patients and healthy persons in B. pseudomallei-endemic areas using a range of humoral and cellular immune assays. We found immune cross-reactivity to be strong for both humoral and cellular immunity among B. pseudomallei, B. thailandensis, and BTCV. Our findings suggest that environmental exposure to low-virulence strains may build cellular immunity to B. pseudomallei.

A systematic review of the impact of psychosocial factors on immunity: Implications for enhancing BCG response against tuberculosis.

BACKGROUND: Tuberculosis (TB) remains an urgent global public health priority, causing 1.5 million deaths worldwide in 2018. There is evidence that psychosocial factors modulate immune function; however, how this may influence TB risk or BCG vaccine response, and whether this pathway can be modified through social protection, has not been investigated. This paper aims to: a) systematically review evidence of how psychosocial factors influence the expression of biomarkers of immunity, and b) apply this general evidence to propose plausible TB-specific pathways for future study. METHODS: Papers reporting on the impact of psychosocial stressors on immune biomarkers in relation to infectious disease risk were identified through a search of the databases MEDLINE, PsycINFO, Global Health and PsycEXTRA alongside reference list and citation searching of key papers. Data extraction and critical appraisal were carried out using a standardised form. The findings were tabulated and synthesised narratively by infectious disease category, and used to propose plausible mechanisms for how psychosocial exposures might influence immune outcomes relevant to TB and BCG response. RESULTS: 27,026 citations were identified, of which 51 met the inclusion criteria. The literature provides evidence of a relationship between psychosocial factors and immune biomarkers. While the direction and strength of associations is heterogenous, some overarching patterns emerged: adverse psychosocial factors (e.g. stress) were generally associated with compromised vaccine response and higher antibody titres to herpesviruses, and vice versa for positive psychosocial factors (e.g. social support). CONCLUSIONS: The evidence identifies pathways linking psychosocial factors and immune response: co-viral infection and immune suppression, both of which are potentially relevant to TB and BCG response. However, the heterogeneity in the strength and nature of the impact of psychosocial factors on immune function, and lack of research on the implications of this relationship for TB, underscore the need for TB-specific research.

Cytomegalovirus Antibody Responses Associated With Increased Risk of Tuberculosis Disease in Ugandan Adults.

BACKGROUND: Recent evidence highlights human cytomegalovirus (HCMV) and immune activation as risk factors for tuberculosis disease. It is not known whether other herpesviruses are also implicated, nor whether a dose-response relationship exists between tuberculosis risk and herpes coinfection. METHODS: This nested case-control study used stored serum samples from 25 persons with tuberculosis up to 10 years before tuberculosis diagnosis and between 3 and 6 matched controls without tuberculosis from a rural Ugandan cohort. Samples were investigated for Epstein-Barr virus, herpes simplex virus, and HCMV-specific immunoglobulin G (IgG), serum markers of inflammation, and mycobacterial antibody levels. RESULTS: Humoral response to HCMV, but not Epstein-Barr or herpes simplex virus, was associated with increased risk of active tuberculosis disease up to 10 years before diagnosis. Individuals with medium HCMV IgG were 2.8 times more likely to have tuberculosis (P = .055), and those with high HCMV IgG 3.4 times more likely to have tuberculosis (P = .007). Mycobacterial antibody levels were not associated with differences in odds of tuberculosis disease. Interferon-induced protein 10 was independently associated with increased odds of tuberculosis (odds ratio, 4.2; P = .009). CONCLUSIONS: These data provide evidence of a dose response between magnitude of HCMV IgG with risk of tuberculosis disease. An inflammatory environment, characterized by serum interferon-induced protein 10 and interleukin 1α, is independently associated with increased risk of tuberculosis disease.