1 Scientific RepoRts | 6:36355 | DOI: 10.1038/srep36355 www.nature.com/scientificreports Adenovirally-Induced polyfunctional t Cells Do Not Necessarily Recognize the Infected target: Lessons from a phase I trial of the AERAS-402 Vaccine Melissa Nyendak 1 , Gwendolyn M. swarbrick 2,3 , Amanda Duncan 2 , Meghan Cansler 2 , ervina Winata Huf 3 , David Hokey 4 , tom evans 4 , Lewellys Barker 4 , Gretta Blatner 4 , Jerald Sadof 5 , Macaya Douoguih 5 , Maria Grazia pau 5 , Deborah A. Lewinsohn 2 & David M. Lewinsohn 1,2,3 the development of a vaccine for Mycobacterium tuberculosis (Mtb) has been impeded by the absence of correlates of protective immunity. one correlate would be the ability of cells induced by vaccination to recognize the Mtb-infected cell. AERAS-402 is a replication-defcient serotype 35 adenovirus containing DNA expressing a fusion protein of Mtb antigens 85A, 85B and TB10.4. We undertook a phase I double- blind, randomized placebo controlled trial of vaccination with AERAS-402 following BCG. Analysis of the vaccine-induced immune response revealed strong antigen-specifc polyfunctional CD4 + and CD8 + T cell responses. However, analysis of the vaccine-induced CD8 + t cells revealed that in many instances these cells did not recognize the Mtb-infected cell. Our fndings highlight the measurement of vaccine- induced, polyfunctional T cells may not refect the extent or degree to which these cells are capable of identifying the Mtb-infected cell and correspondingly, the value of detailed experimental medicine studies early in vaccine development. To eradicate tuberculosis (TB), a multifaceted approach is needed, including the development of a robust and durable vaccine 1,2 . Whereas serologic correlates of protective immunity have been established for many vaccine preventable illnesses, correlates for protective immunity for TB have remained elusive 3 . Containment of Mtb infection requires the induction and maintenance of a robust T1 immune response 2,4–6 and evidence from pre-clinical animal 7 and human 8 vaccination studies suggest the breadth of the vaccine-induced cytokine response (IFN-γ and TNF-α, IL-2) is associated with efcacy 9 . Collectively, these T cells have been termed polyfunctional 10 . Recent results from the frst Phase IIb vaccine study using MVA-Ag85A in human infants has highlighted the possibility that the induction of polyfunctional CD4 + T cell immunity, while important, may not be sufcient 11 to confer protection. While human Mtb specifc CD4 + and CD8 + T cells are similar in the cytolytic and pro-infammatory capacity 12,13 , CD8 + T cells are capable of discerning Mtb-infected cells, particularly those that are HLA-II negative. Human Mtb-specifc CD8 + T cells are further distinguished by both their preferential recognition of heavily infected cells and restriction by HLA-B 14,15 . Additionally, it is increasingly evident that CD8 + T cells have an important and complex role in Mtb containment and immunity 14,16–20 . Specifcally, we note that CD8 + T cells are uniquely capable of discerning the Mtb-infected cell, and that a role for these cells in the long-term progression of myco- bacterial growth has been demonstrated in the mouse and non-human primate models. For most vaccination studies, the assessment of vaccine-induced CD8 + T cells has relied upon the measurement of antigen-specifc polyfunctional cells, typically using peptide pools. However, as these measurements have been considered as a 1 Department of Medicine, Oregon Health and Science University, Portland, Oregon, USA. 2 Department of Pediatrics, Oregon Health and Science University, Portland, Oregon, USA. 3 Department of Medicine, Portland VA Medical center, Portland, Oregon, USA. 4 Aeras, Rockville, Maryland, USA. 5 Janssen infectious Diseases and Vaccines (formerly crucell), Leiden, the netherlands. correspondence and requests for materials should be addressed to M.n. (email: nyendakm@ohsu.edu) or D.M.L. (email: lewinsod@ohsu.edu) Received: 29 April 2016 Accepted: 13 October 2016 Published: 02 November 2016 opeN