Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL

et al. (2010) Identification of CD8+ T Cell Epitopes in the West Nile Virus Polyprotein by Reverse- Immunology Using NetCTL. PLoS ONE 5(9): e12697. doi:10.1371/journal.pone.0012697 + Identification of CD8 T Cell Epitopes in the West Nile Virus Polyprotein by Reverse-Immunology Using NetCTL Mette Voldby Larsen 0 Alina Lelic 0 Robin Parsons 0 Morten Nielsen 0 Ilka Hoof 0 Kasper Lamberth 0 Mark B. Loeb 0 Sren Buus 0 Jonathan Bramson 0 Ole Lund 0 Derya Unutmaz, New York University, United States of America 0 1 Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark , Lyngby , Denmark , 2 Department of Pathology and Molecular Medicine, Institute for Molecular Medicine and Health, McMaster University , Hamilton, Ontario , Canada , 3 Division of Experimental Immunology, Institute of Medical Microbiology and Immunology, The Panum Institute, University of Copenhagen , Copenhagen , Denmark Background: West Nile virus (WNV) is a growing threat to public health and a greater understanding of the immune response raised against WNV is important for the development of prophylactic and therapeutic strategies. Methodology/Principal Findings: In a reverse-immunology approach, we used bioinformatics methods to predict WNVspecific CD8+ T cell epitopes and selected a set of peptides that constitutes maximum coverage of 20 fully-sequenced WNV strains. We then tested these putative epitopes for cellular reactivity in a cohort of WNV-infected patients. We identified 26 new CD8+ T cell epitopes, which we propose are restricted by 11 different HLA class I alleles. Aiming for optimal coverage of human populations, we suggest that 11 of these new WNV epitopes would be sufficient to cover from 48% to 93% of ethnic populations in various areas of the World. Conclusions/Significance: The 26 identified CD8+ T cell epitopes contribute to our knowledge of the immune response against WNV infection and greatly extend the list of known WNV CD8+ T cell epitopes. A polytope incorporating these and other epitopes could possibly serve as the basis for a WNV vaccine. - Funding: This work was supported by the National Institutes of Health (NIH) (contracts HHSN266200400025C and HHSN266200400083C) and N01-AI-40066 to J.L.B. and M.B.L. as well as by a grant from the Danish Research Council for Technology and Production Sciences (project title Disease Gene Finding, Somatic Mutations, and Vaccine Design, principal funding recipient is Soeren Brunak). 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 declared that no competing interests exist. West Nile virus belongs to the family Flaviviridae, along with other human pathogens like Yellow fever virus and Dengue fever virus. It is an enveloped, spherical virus containing a single strand of RNA that is translated into a continuous polypeptide of approximately 3,400 amino acids. The polypeptide is post-translationally cleaved into ten distinct proteins including three structural proteins; capsid (C) protein, envelope (E) protein, and pre-membrane (prM) protein, and seven non-structural (NS) proteins; NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [1]. The virus is transmitted to humans by infected mosquitoes and causes West Nile fever in about 20% of infected people. The symptoms of West Nile fever are fever, headache, tiredness, and body aches that can last for a few days to several weeks. Less than one in 100 infected people will develop severe West Nile disease that may lead to fatal encephalitis [2]. The first incidents of WNV infection in the western hemisphere were detected in 1999 during an outbreak of encephalitis in New York City. Since then, the virus has spread across North America and is now a serious threat for public health in the United States, especially for immunocompromised recipients of transplanted organs [1]. Currently, no specific therapy is available for treatment and no vaccine has been approved for prevention of WNV infection in humans [3]. CD8+ Cytotoxic T Lymphocytes (CTLs) of the immune system have the capacity to eradicate virus-infected host cells. CTL activation is achieved when peptides originating from virus proteins are presented at the surface of infected cells in complex with Human Leukocyte Antigen (HLA) class I molecules. Several studies have shown that CTLs indeed play a role in the cellular antiviral response against WNV infection in mice and humans [47]. Although the important role of CTLs in combating WNV is well-established, only a limited number of WNV CD8+ T cell epitopes have so far been identified in humans. De Groot et al. applied a bioinformatics approach for predicting HLA-B*07 restricted WNV CD8+ T cell epitopes [8]: Out of 16 predicted epitopes, 12 were confirmed to bind HLA-B*07 in vitro, but the peptides ability to induce T-cell responses was not tested. Recent reports from our group and collaborators have described two different strategies for identifying CD8+ T cell epitopes in WNV. In the first case, a mass spectroscopy method developed by the Hildebrand laboratory successfully identified four HLA-A*0201 restricted WNV CD8+ T cell epitopes [9]. In a second study, we used a shotgun approach, employing overlapping peptides spanning the entire WNV polyprotein and identified additional epitopes restricted by HLA-A*01 and HLA-B*35, as well as several epitopes for which the HLA restriction was not ascertained [10]. In a study by Lanteri et al., overlapping peptides spanning all WNV proteins were likewise tested for their ability to induce T cell responses and led to the discovery of eight frequently recognised WNV peptides [5]. Three of the responses were associated with particular HLA class I types (A*0101, A*0201, and Cw*0303/ Cw*0304). In the current study, our objective is to extend the discovery of WNV CD8+ T cell epitopes to additional HLA class I alleles, while also considering the sequence variability of WNV proteins. Koo et al. have recently identified regions of the WNV polyprotein that are fully conserved across all analysed WNV sequences and examined whether these regions contain experimentally confirmed or predicted CD8+ T cell epitopes [11]. The variability of the WNV proteome is, however, unevenly distributed across the proteome with the structural proteins being most variably. At the amino acid level, the C protein has up to 23% differences across examined sequences, while the NS4b protein has the lowest diversity with at most 8% differences [11]. Accordingly, the majority of the conserved regions identified by Koo et al. were found in the non-structural proteins, while the C protein had none, and the two other structural proteins, prM and E, had the third and fourth least number of conserved regions [11]. It is likely that the structural proteins contain highly immunogenic epitopes that are missed when focusing solely on fully conserved (...truncated)