Proteome-Wide Screening Reveals Immunodominance in the CD8 T Cell Response against Classical Swine Fever Virus with Antigen-Specificity Dependent on MHC Class I Haplotype Expression Giulia Franzoni 1,2 , Nitin V. Kurkure 1,3 , Sabine E. Essler 4 , Miriam Pedrera 1 , Helen E. Everett 1 , Kikki B. Bodman-Smith 2 , Helen R. Crooke 1 , Simon P. Graham 1 * 1 Virology Department, Animal Health and Veterinary Laboratories Agency, Addlestone, United Kingdom, 2 Department of Microbial & Cellular Sciences, University of Surrey, Guildford, United Kingdom, 3 Nagpur Veterinary College, Maharashtra Animal & Fishery Sciences University, Nagpur, India, 4 Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria Abstract Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-c responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-c expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-c induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core 241–255 PESRKKLEKALLAWA and NS3 1902–1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2 996–1003 YEPRDSYF, NS2 1223–1230 STVTGIFL and NS5A 3070– 3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-a in addition to IFN-c. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS2 1223–1230 STVTGIFL and NS3 1902–1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV. Citation: Franzoni G, Kurkure NV, Essler SE, Pedrera M, Everett HE, et al. (2013) Proteome-Wide Screening Reveals Immunodominance in the CD8 T Cell Response against Classical Swine Fever Virus with Antigen-Specificity Dependent on MHC Class I Haplotype Expression. PLoS ONE 8(12): e84246. doi:10.1371/ journal.pone.0084246 Editor: Gourapura J. Renukaradhya, The Ohio State University, United States of America Received August 23, 2013; Accepted November 13, 2013; Published December 23, 2013 Copyright: ß 2013 Franzoni 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 study was supported by project (SE0796) from the Department for Environment, Food and Rural Affairs of the United Kingdom. Dr. Nitin V. Kurkure’s contribution to the study was facilitated by a DBT CREST Award from the Ministry of Science & Technology, Government of India. Dr. Miriam Pedrera’s contribution was supported by a Research Grant for Foreign Universities or Research Centres 2011 from the Alfonso Martin Escudero Foundation, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. AHVLA has no commercial connection with the manufacturer of the vaccine used in this study, Riemser Arzneimittel AG, Germany. Competing Interests: The sequences of the antigenic peptides identified in this study are the subject of a provisional patent application GB1315130.3, entitled ‘Peptides’, filed at the UK Intellectual Property Office, 24th August 2013. The authors confirm that this provisional patent application does not alter their adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: Simon.Graham@ahvla.gsi.gov.uk Introduction Classical swine fever (CSF) is a severe and often lethal viral disease of domestic pigs and wild boars. The aetiological agent is classical swine fever virus (CSFV), a small, enveloped, positive- sense, single-stranded RNA virus belonging to the pestivirus genus of the Flaviviridae family [1,2]. The disease is endemic in South East Asia, parts of Central and South America and the Russian Federation. Despite the stringent controls adopted in the EU, the virus continues to be an epizootic threat with recent outbreaks in Lithuania (2009 and 2011) and Latvia (2012) [3]. CSF is amenable to control by vaccination and live attenuated C-strain vaccines are highly efficacious. However, the inability to differentiate vaccinat- ed animals from those infected with CSFV limits their utility as a control tool in outbreak settings in the EU [4]. Control of CSF outbreaks via a stamping-out policy is expensive, because large numbers of animals have to be culled including those slaughtered pre-emptively. Public resistance against such drastic measures is also growing. As a consequence, there is increased pressure to develop and adopt alternative strategies, like marker vaccines, to aid the control of CSF outbreaks [4]. C-strain vaccine induced IFN-c responses have been correlated to rapid protection against the disease [5] and CSFV-specific IFN- c secreting CD8 T cells are detected in the blood early after PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e84246