Comprehensive Analysis of West Nile Virus–Specific T Cell Responses in Humans

Marion C. Lanteri 1 John W. Heitman 1 Rachel E. Owen 1 Thomas Busch 1 Nelly Gefter 1 Nancy Kiely 2 Hany T. Kamel 2 Leslie H. Tobler 1 Michael P. Busch 1 Philip J. Norris () 0 1 0 Medicine, University of California , San Francisco, California 1 Blood Systems Research Institute and Departments of 2 Blood Systems , Scottsdale, Arizona Background. Cellular responses have been shown to play a role in immune control and clearance of West Nile virus (WNV) in murine models. However, little is known about the immunogenic regions of the virus or the phenotype of responding T cells in human infection. Methods. Frozen peripheral blood mononuclear cells (PBMCs) from 35 WNV-infected blood donors were screened for virus-specific T cell responses by an interferon- (IFN- ) enzyme-linked immunosorbent spot assay that used 452 overlapping peptides spanning all WNV proteins. More-detailed phenotypic studies were performed on subjects with high-magnitude T cell responses. Results. In individuals with identified responses, the total number of recognized WNV peptides ranged from 1 to 9 (median, 2 peptides), and the overall magnitude of responses ranged from 50 to 4210 spot-forming cells (SFCs) per 106 PBMCs (median, 130 SFCs/106 PBMCs). A subset of 8 frequently recognized peptides from the regions of the genome encoding membrane, envelope, and nonstructural 3 and 4b proteins was identified. Phenotypic study of the highest magnitude WNV-specific T cell responses revealed that most were mediated by CD8 cells that expressed perforin and/or granzyme B. Conclusions. These findings are the first to define the breadth and characteristics of the human T cell response to WNV and have implications for candidate vaccine design and evaluation. - West Nile virus (WNV) is a single-stranded, positivepolarity RNA virus of the Flaviviridae family [1]. Its genome encodes for capsid (C), envelope (E), premembrane (prM), and membrane (M) proteins and for 7 nonstructural (NS) proteins that likely contribute to viral replication [2]. Since the introduction of WNV to the United States in 1999, the virus has become endemic nationwide. Humans are typically infected as incidental hosts via mosquito bites. Seasonal outbreaks in the US population have been responsible for 26,000 cases of disease and 1038 deaths. Many more underlying human infections occurred, as WNV infection is asymptomatic in 80% of cases [3]. In the 20% of infected individuals who are symptomatic, WNV fever predominates, with only 1% of infected persons presenting with neurological symptoms [4]. No specific therapy or vaccine has been approved for use in humans, leaving only supportive treatment for WNV infection [5]. Considerable progress has been made in understanding immunity to flavivirus infection, particularly in the murine model. Innate immune responses [6, 7] as well as humoral [8, 9] and cellular immune responses have been implicated in the control of WNV infection. A protective role for T and B lymphocytes against WNV has been demonstrated by transfer experiments involving mice with severe combined immunodeficiency (i.e., those with T cell and B cell deficiency) [10] and Rag1tm/Mom mice (i.e., those with B cell deficiency) [8] that succumb to WNV infection in the absence of passive immunotherapy [11, 12]. In the macaque model, WNV clearance from the blood occurs 511 days after inoculation, whereas anti-WNV IgM is not detectable until 9 10 days after inoculation, with neutralizing antibodies appearing an additional 12 days later [13]. Finally, in the natural history of WNV infection in humans, clearance NR 1 5 2 4 1 5 5 3 0 5 0 0 12 2 4 9 0 8 0 0 3 4 5 0 7 8 8 0 2 0 5 1 5 2 4 Index donation First follow-up sample Second follow-up sample Exposure historya TMA IgM IgG of viremia occurs before detection of anti-WNV IgM [14]. Thus, WNV-specific antibodies, although known to play a critical role in initial control of viral replication and clearance of viremia, likely are not the only arm of the immune system responsible for the clearance of WNV from the body. T cells possibly play a role in viral clearance and in protection from disease. Interferon- (IFN- ) secretion by and T cells decreases the WNV load in mouse brains, and adoptive transfer of or T cells reduces the susceptibility of mice to lethal WNV infection [1517]. CD8 T cells contribute to WNV clearance, as CD8 T cell deficient mice show increased mortality, with surviving mice exhibiting persistent viremia [18]. WNV can also be detected in the central nervous system of C57BL/6 mice lacking either IFN- [19] or perforin granules [20]. Major histocompatibility complex (MHC) class I upregulation is mediated by IFN- dependent and nuclear factor- B dependent pathways, pointing to a role for T cellsecreted cytokines in combating WNV infection [21]. Although T cells likely play a role in controlling WNV replication, a comprehensive characterization of human T cell responses to WNV has not been previously reported. Studies of other flaviviruses suggest that NS proteins are preferentially recognized by T cells [22]. Studies of humans immunized against dengue virus also support NS proteins as the prime region of T cell immunogenicity [23]. A study that used bioinformatic approaches to T cell epitope mapping identified a number of HLA class I B7restricted epitopes in WNV [24]. The current study was designed to determine the breadth and specificity of WNV-specific T cell responses across the entire genome expressed in WNV [25]. The phenotype and functional markers of T cells specific for immunodominant epitopes were also characterized. SUBJECTS, MATERIALS, AND METHODS Study subjects. Of 800,000 United Blood Services donors whose blood was screened for WNV in 2005, samples from 45 were confirmed to be positive for WNV RNA. Thirty-five donors with a WNV RNApositive blood specimen were enrolled in this study after provision of informed consent, as required by the institutional review board of the University of California, San Francisco (UCSF). Questionnaires that covered 12 possible WNV-related symptoms were administered at study enrollment and 2 weeks later. Donors were considered symptomatic if they reported 4 symptoms on either questionnaire (table 1). Samples were collected in EDTA and shipped overnight to the Blood Systems Research Institute (San Francisco). Ten uninfected control subjects (5 laboratory workers and 5 blood donors) were included and were not statistically different from study subjects with respect to mean age (37.6 years [range, 29 53 years] vs. 49 years [range, 26 73 years]; P .05) and percentage of females (40% vs. 71%; P .05). Isolation of peripheral blood mononuclear cells (PBMCs). PBMCs were isolated on a Ficoll-Paque Plus density gradient (GE Healthcare Bio-Sciences). Aliquots of 10 106 cells were frozen in media that contained 90% FBS (HyClone) and 10% KGAWMDSTKATRYLVK KEAWLNSTKATRYLVK BLAST findings Matching peptide sequencec Matching flavivirus Homology (...truncated)