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 Magali Matsumiya * , Elena Stylianou, Kristin Griffiths, Zoe Lang, Joel Meyer, Stephanie A. Harris, Rosalind Rowland, Angela M. Minassian, Ansar A. Pathan ¤ , Helen Fletcher, Helen McShane The Jenner Institute, University of Oxford, Oxford, United Kingdom Abstract 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-Ȗ (IFN- Ȗ) 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+ CDβ5+ Foxpγ+ 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, TICAMβ and CD14 on day of vaccination and CTLA4 and ILβRα two days post-vaccination can classify high and low responders with over 80% accuracy. Furthermore, administering MVA85A in mice with anti-TLRβ antibodies may abrogate high responses, and neutralising antibodies to TLRs 1, β or 6 or HMGB1 decrease CXCLβ 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. Citation: Matsumiya M, Stylianou E, Griffiths K, Lang Z, Meyer J, et al. (β01γ) Roles for Treg Expansion and HMGB1 Signaling through the TLR1-β-6 Axis in Determining the Magnitude of the Antigen-Specific Immune Response to MVA85A. PLoS ONE 8(7): e679ββ. doi:10.1γ71/journal.pone.00679ββ Editor: Riccardo Manganelli, University of Padova, Medical School, Italy Received April 16, β01γ; Accepted May ββ, β01γ; Published July γ, β01γ Copyright: © β01γ 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 credited. Funding: This work was funded by the Wellcome Trust. MM has a Wellcome Trust PhD studentship and HM is a Wellcome Trust Senior Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have read the journal's policy and have the following conflicts: AAP and HM are named inventors in a patent filing related to MVA85A and are shareholders in a joint venture, OETC, formed for the future development of this vaccine. This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials. There are no other conflicts of interest. * E-mail: magali.matsumiya@msdtc.ox.ac.uk ¤ Current Address: Centre for Infection, Immunity and Disease Mechanisms, School of Health Sciences and Social Care, Brunel University, Middlesex, United Kingdom Introduction Tuberculosis (TB) remains a major global health issue, with an estimated 8.7 million cases and 1.4 million deaths in β011 [1]. BCG, the only licensed vaccine against TB, shows only partial, variable efficacy against pulmonary TB [β–4]. Twelve candidate vaccines are currently in clinical trials [5] and results of the first efficacy trial of a new vaccine against Mycobacterium tuberculosis (M.tb), in which Modified Vaccinia virus Ankara expressing antigen 85A (MVA85A) was given to BCG-vaccinated South African infants, have recently been reported [6]. Although MVA85A did not confer improved protection to TB compared to BCG alone in this setting, further analysis of samples collected will provide a valuable opportunity to investigate the immune basis of protection against TB. Efforts to produce T cell inducing vaccines against diseases such as TB, HIV and malaria, have made use of viral vectors as antigen delivery systems to enhance the immune response to the antigen of interest. Vaccine candidates are selected on the basis of safety, efficacy in preclinical animal disease models and the ability of the vaccine to induce the secretion of PLOS ONE | www.plosone.org 1 July β01γ | Volume 8 | Issue 7 | e679ββ