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

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

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

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

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

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

© 2013 Matsumiya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedA 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.This work was funded by the Wellcome Trust. MM has a Wellcome Trust PhD studentship and HM is a Wellcome Trust Senior Fello

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

Innate immune responses to tuberculosis vaccines

Tuberculosis, caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem. Drug resistance and high rates of HIV infection have fuelled the pandemic and, although a vaccine exists, its ability to protect from pulmonary tuberculosis varies between 0 and 80%. Bacille Calmette Guerin (BCG) has been administered to billions worldwide yet its protective mechanisms remain unknown, as do the reasons for its failure to protect in many parts of the world. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel candidate vaccine designed to boost immune responses to BCG and improve protection. An aim of this thesis has been to characterise the innate immune response to an MVA85A boosting vaccination in both UK adults and South African infants. In the former, volunteers develop a strong innate response following vaccination however this does not always translate into a robust adaptive response to antigen 85A (Ag85A), which is determined in part by Treg expansion and the nuclear protein HMGB1 signaling through the TLR1-2-6 axis. By contrast, not all South Africa infants mount a strong innate immune response to MVA85A yet this response is correlated with the magnitude of the adaptive response. The immune response to BCG in both populations is also characterised and an association found between increased production of IL-17, IL-22 and IFN-γ in response to BCG stimulation and control of mycobacterial growth. The results presented here further the knowledge on the links between innate and adaptive responses to vaccination with BCG and MVA85A and the variation in mechanisms involved in different populations.</p

Innate immune responses to tuberculosis vaccines

Tuberculosis, caused by infection with Mycobacterium tuberculosis (M.tb), remains a global health problem. Drug resistance and high rates of HIV infection have fuelled the pandemic and, although a vaccine exists, its ability to protect from pulmonary tuberculosis varies between 0 and 80%. Bacille Calmette Guerin (BCG) has been administered to billions worldwide yet its protective mechanisms remain unknown, as do the reasons for its failure to protect in many parts of the world. Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) is a novel candidate vaccine designed to boost immune responses to BCG and improve protection. An aim of this thesis has been to characterise the innate immune response to an MVA85A boosting vaccination in both UK adults and South African infants. In the former, volunteers develop a strong innate response following vaccination however this does not always translate into a robust adaptive response to antigen 85A (Ag85A), which is determined in part by Treg expansion and the nuclear protein HMGB1 signaling through the TLR1-2-6 axis. By contrast, not all South Africa infants mount a strong innate immune response to MVA85A yet this response is correlated with the magnitude of the adaptive response. The immune response to BCG in both populations is also characterised and an association found between increased production of IL-17, IL-22 and IFN-γ in response to BCG stimulation and control of mycobacterial growth. The results presented here further the knowledge on the links between innate and adaptive responses to vaccination with BCG and MVA85A and the variation in mechanisms involved in different populations.This thesis is not currently available in ORA

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