Cytomegalovirus infection is a risk factor for tuberculosis disease in infants.

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman's rho, P = 6 × 10-8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02-4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell-associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot-positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and -negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV- signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.

Author Correction: The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

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Tools for Assessing the Protective Efficacy of TB Vaccines in Humans: in vitro Mycobacterial Growth Inhibition Predicts Outcome of in vivo Mycobacterial Infection.

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.

Regulation of mycobacterial infection by macrophage Gch1 and tetrahydrobiopterin.

Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.

Human Hookworm Infection Enhances Mycobacterial Growth Inhibition and Associates With Reduced Risk of Tuberculosis Infection.

Soil-transmitted helminths and Mycobacterium tuberculosis frequently coincide geographically and it is hypothesized that gastrointestinal helminth infection may exacerbate tuberculosis (TB) disease by suppression of Th1 and Th17 responses. However, few studies have focused on latent TB infection (LTBI), which predominates globally. We performed a large observational study of healthy adults migrating from Nepal to the UK (n = 645). Individuals were screened for LTBI and gastrointestinal parasite infections. A significant negative association between hookworm and LTBI-positivity was seen (OR = 0.221; p = 0.039). Hookworm infection treatment did not affect LTBI conversions. Blood from individuals with hookworm had a significantly greater ability to control virulent mycobacterial growth in vitro than from those without, which was lost following hookworm treatment. There was a significant negative relationship between mycobacterial growth and eosinophil counts. Eosinophil-associated differential gene expression characterized the whole blood transcriptome of hookworm infection and correlated with improved mycobacterial control. These data provide a potential alternative explanation for the reduced prevalence of LTBI among individuals with hookworm infection, and possibly an anti-mycobacterial role for helminth-induced eosinophils.

The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

The current vaccine against tuberculosis, live attenuated Mycobacterium bovis BCG, has variable efficacy, but development of an effective alternative is severely hampered by the lack of an immune correlate of protection. There has been a recent resurgence of interest in functional in vitro mycobacterial growth inhibition assays (MGIAs), which provide a measure of a range of different immune mechanisms and their interactions. We identified a positive correlation between mean corpuscular haemoglobin and in vitro growth of BCG in whole blood from healthy UK human volunteers. Mycobacterial growth in peripheral blood mononuclear cells (PBMC) from both humans and macaques was increased following the experimental addition of haemoglobin (Hb) or ferric iron, and reduced following addition of the iron chelator deferoxamine (DFO). Expression of Hb genes correlated positively with mycobacterial growth in whole blood from UK/Asian adults and, to a lesser extent, in PBMC from South African infants. Taken together our data indicate an association between Hb/iron levels and BCG growth in vitro, which may in part explain differences in findings between whole blood and PBMC MGIAs and should be considered when using such assays.

T-cell activation is an immune correlate of risk in BCG vaccinated infants.

Vaccines to protect against tuberculosis (TB) are urgently needed. We performed a case-control analysis to identify immune correlates of TB disease risk in Bacille Calmette-Guerin (BCG) immunized infants from the MVA85A efficacy trial. Among 53 TB case infants and 205 matched controls, the frequency of activated HLA-DR(+) CD4(+) T cells associates with increased TB disease risk (OR=1.828, 95% CI=1.25-2.68, P=0.002, FDR=0.04, conditional logistic regression). In an independent study of Mycobacterium tuberculosis-infected adolescents, activated HLA-DR(+) CD4(+) T cells also associate with increased TB disease risk (OR=1.387, 95% CI=1.068-1.801, P=0.014, conditional logistic regression). In infants, BCG-specific T cells secreting IFN-γ associate with reduced risk of TB (OR=0.502, 95% CI=0.29-0.86, P=0.013, FDR=0.14). The causes and impact of T-cell activation on disease risk should be considered when designing and testing TB vaccine candidates for these populations.

Gene expression and cytokine profile correlate with mycobacterial growth in a human BCG challenge model.

BACKGROUND: Bacillus Calmette-Guerin (BCG) vaccine is the most widely administered vaccine in the world, yet its mechanism of action remains unclear. We hypothesize that certain immune pathways are associated with reduced mycobacterial growth following BCG challenge in human volunteers. METHODS: We used samples from a mycobacterial challenge in which previously BCG-vaccinated or BCG-naive adults in the United Kingdom were challenged intradermally with a standard dose of BCG. Any remaining BCG was quantified in a skin biopsy specimen obtained 2 weeks after challenge and used as a measure of BCG growth and functional antimycobacterial immunity. We measured the immune response over the 2-week challenge, using DNA microarrays and flow cytometry, and correlated this with mycobacterial growth. RESULTS: The magnitude of the immune response to BCG is greater in previously vaccinated volunteers, and this correlates with reduced mycobacterial growth but increased scarring at the vaccination site. In particular, the interferon γ and interleukin 17 pathways are strongly induced in previously vaccinated volunteers and correlate with reduced mycobacterial growth in this population. CONCLUSION: This study identifies pathways associated with control of mycobacterial growth in vivo in human volunteers and supports the use of BCG challenge as a tool for evaluating vaccine efficacy and identifying mechanisms of antimycobacterial immunity.

Inflammatory and myeloid-associated gene expression before and one day after infant vaccination with MVA85A correlates with induction of a T cell response.

BACKGROUND: Tuberculosis (TB) remains a global health problem, with vaccination likely to be a necessary part of a successful control strategy. Results of the first Phase 2b efficacy trial of a candidate vaccine, MVA85A, evaluated in BCG-vaccinated infants were published last year. Although no improvement in efficacy above BCG alone was seen, cryopreserved samples from this trial provide an opportunity to study the immune response to vaccination in this population. METHODS: We investigated blood samples taken before vaccination (baseline) and one and 28 days post-vaccination with MVA85A or placebo (Candin). The IFN-γ ELISpot assay was performed at baseline and on day 28 to quantify the adaptive response to Ag85A peptides. Gene expression analysis was performed at all three timepoints to identify early gene signatures predictive of the magnitude of the subsequent adaptive T cell response using the significance analysis of microarrays (SAM) statistical package and gene set enrichment analysis. RESULTS: One day post-MVA85A, there is an induction of inflammatory pathways compared to placebo samples. Modules associated with myeloid cells and inflammation pre- and one day post-MVA85A correlate with a higher IFN-γ ELISpot response post-vaccination. By contrast, previous work done in UK adults shows early inflammation in this population is not associated with a strong T cell response but that induction of regulatory pathways inversely correlates with the magnitude of the T cell response. This may be indicative of important mechanistic differences in how T cell responses develop in these two populations following vaccination with MVA85A. CONCLUSION: The results suggest the capacity of MVA85A to induce a strong innate response is key to the initiation of an adaptive immune response in South African infants but induction of regulatory pathways may be more important in UK adults. Understanding differences in immune response to vaccination between populations is likely to be an important aspect of developing successful vaccines and vaccination strategies. TRIAL REGISTRATION: ClinicalTrials.gov number NCT00953927.

Process of assay selection and optimization for the study of case and control samples from a phase IIb efficacy trial of a candidate tuberculosis vaccine, MVA85A.

The first phase IIb safety and efficacy trial of a new tuberculosis vaccine since that for BCG was completed in October 2012. BCG-vaccinated South African infants were randomized to receive modified vaccinia virus Ankara, expressing the Mycobacterium tuberculosis antigen 85A (MVA85A), or placebo. MVA85A did not significantly boost the protective effect of BCG. Cryopreserved samples provide a unique opportunity for investigating the correlates of the risk of tuberculosis disease in this population. Due to the limited amount of sample available from each infant, preliminary work was necessary to determine which assays and conditions give the most useful information. Peripheral blood mononuclear cells (PBMC) were stimulated with antigen 85A (Ag85A) and purified protein derivative from M. tuberculosis in an ex vivo gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) and a Ki67 proliferation assay. The effects of a 2-h or overnight rest of thawed PBMC on ELISpot responses and cell populations were determined. Both the ELISpot and Ki67 assays detected differences between the MVA85A and placebo groups, and the results correlated well. The cell numbers and ELISpot responses decreased significantly after an overnight rest, and surface flow cytometry showed a significant loss of CD4(+) and CD8(+) T cells. Of the infants tested, 50% had a positive ELISpot response to a single pool of flu, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) (FEC) peptides. This pilot work has been essential in determining the assays and conditions to be used in the correlate study. Moving forward, PBMC will be rested for 2 h before assay setup. The ELISpot assay, performed in duplicate, will be selected over the Ki67 assay, and further work is needed to evaluate the effect of high FEC responses on vaccine-induced immunity and susceptibility to tuberculosis disease.

Roles for Treg expansion and HMGB1 signaling through the TLR1-2-6 axis in determining the magnitude of the antigen-specific immune response to MVA85A.

A better understanding of the relationships between vaccine, immunogenicity and protection from disease would greatly facilitate vaccine development. Modified vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel tuberculosis vaccine candidate designed to enhance responses induced by BCG. Antigen-specific interferon-γ (IFN-γ) production is greatly enhanced by MVA85A, however the variability between healthy individuals is extensive. In this study we have sought to characterize the early changes in gene expression in humans following vaccination with MVA85A and relate these to long-term immunogenicity. Two days post-vaccination, MVA85A induces a strong interferon and inflammatory response. Separating volunteers into high and low responders on the basis of T cell responses to 85A peptides measured during the trial, an expansion of circulating CD4+ CD25+ Foxp3+ cells is seen in low but not high responders. Additionally, high levels of Toll-like Receptor (TLR) 1 on day of vaccination are associated with an increased response to antigen 85A. In a classification model, combined expression levels of TLR1, TICAM2 and CD14 on day of vaccination and CTLA4 and IL2Rα two days post-vaccination can classify high and low responders with over 80% accuracy. Furthermore, administering MVA85A in mice with anti-TLR2 antibodies may abrogate high responses, and neutralising antibodies to TLRs 1, 2 or 6 or HMGB1 decrease CXCL2 production during in vitro stimulation with MVA85A. HMGB1 is released into the supernatant following atimulation with MVA85A and we propose this signal may be the trigger activating the TLR pathway. This study suggests an important role for an endogenous ligand in innate sensing of MVA and demonstrates the importance of pattern recognition receptors and regulatory T cell responses in determining the magnitude of the antigen specific immune response to vaccination with MVA85A in humans.

Mycobacterial growth inhibition in murine splenocytes as a surrogate for protection against Mycobacterium tuberculosis (M. tb).

Development of an improved vaccine against tuberculosis (TB) is hindered by the lack of a surrogate of protection. Efficacy of new TB vaccines in humans can only be evaluated by expensive and time consuming efficacy trials within TB endemic areas. It is critical that vaccines with the greatest potential to protect are selected for these trials. Mycobacterial growth inhibition assays (MGIAs) have been developed with the hope that these in-vitro functional assays will correlate with protection, which could aid in the selection of the best vaccine candidates. The present study describes the use of the BACTEC system to perform MGIAs in mice. We demonstrate reproducible mycobacterial growth inhibition in splenocytes from BCG immunised mice compared with unimmunised mice (P < 0.023), which corresponded with in-vivo efficacy against Mycobacterium tuberculosis (M. tb) challenge. Microarray data showed extensive differential gene expression in splenocyte responses to ex-vivo BCG stimulation between unimmunised and BCG-immunised mice. TH1 responses, including IFN-γ, nitric oxide synthase (NOS2) and Interleukin -17 (IL-17) expression were enhanced in BCG immunised mice, indicating a possible mechanism for mycobacterial growth inhibition. Further investigation into whether the BACTEC MGIA can be used as a surrogate of protection in humans and preclinical animal models is now warranted.

Safety and immunogenicity of an FP9-vectored candidate tuberculosis vaccine (FP85A), alone and with candidate vaccine MVA85A in BCG-vaccinated healthy adults: a phase I clinical trial.

The safety and immunogenicity of a new candidate tuberculosis (TB) vaccine, FP85A was evaluated alone and in heterologous prime-boost regimes with another candidate TB vaccine, MVA85A. This was an open label, non-controlled, non-randomized Phase I clinical trial. Healthy previously BCG-vaccinated adult subjects were enrolled sequentially into three groups and vaccinated with FP85A alone, or both FP85A and MVA85A, with a four week interval between vaccinations. Passive and active data on adverse events were collected. Immunogenicity was evaluated by Enzyme Linked Immunospot (ELISpot), flow cytometry and Enzyme Linked Immunosorbent assay (ELISA). Most adverse events were mild and there were no vaccine-related serious adverse events. FP85A vaccination did not enhance antigen 85A-specific cellular immunity. When MVA85A vaccination was preceded by FP85A vaccination, cellular immune responses were lower compared with when MVA85A vaccination was the first immunisation. MVA85A vaccination, but not FP85A vaccination, induced anti-MVA IgG antibodies. Both MVA85A and FP85A vaccinations induced anti-FP9 IgG antibodies. In conclusion, FP85A vaccination was well tolerated but did not induce antigen-specific cellular immune responses. We hypothesize that FP85A induced anti-FP9 IgG antibodies with cross-reactivity for MVA85A, which may have mediated inhibition of the immune response to subsequent MVA85A. ClinicalTrials.gov identification number: NCT00653770.