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Comprehensive Analysis of West Nile Virus-Specific T Cell Responses in Humans Author(s): Marion C. Lanteri, John W. Heitman, Rachel E. Owen, Thomas Busch, Nelly Gefter, Nancy Kiely, Hany T. Kamel, Leslie H. Tobler, Michael P. Busch and Philip J. Norris Source: The Journal of Infectious Diseases, Vol. 197, No. 9 (May 1, 2008), pp. 1296-1306Published by: Oxford University PressStable URL: http://www.jstor.org/stable/40254176Accessed: 12-05-2015 20:23 UTC

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MAJOR ARTICLE

Comprehensive Analysis of West Nile Virus-Specific T Cell Responses in Humans Marion C. Lanteri,1 John W. Heitman,1 Rachel E. Owen," Thomas Busch,1 Nelly Getter,1 Nancy Kiely,4 Hany T. Kamel,4 Leslie H. Tobler,1 Michael P. Busch," and Philip J. Norris1*3

1Blood Systems Research Institute and Departments of laboratory Medicine and 3Medicine, University of California, San Francisco, California;

and 4Blood 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 pheno- type 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-y (IFN-y) 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, positive- polarity RNA virus of the Flaviviridae family [ 1 ]. Its ge- nome encodes for capsid (C), envelope (E), premem- brane (prM), and membrane (M) proteins and for 7 nonstructural (NS) proteins that likely contribute to vi- ral 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

Received 6 July 2007; accepted 9 November 2007; electronically published 28 March 2008.

Potential conflicts of interest: none reported. Presented in part: 54th Annual Meeting of the American Society of Tropical

Medicine and Hygiene, Washington, DC, 11-15 December 2005. Financial support: Centers for Disease Control and Prevention (grant R01

C1 00021 4-01).

Reprints or correspondence: Dr. Philip J. Norris, Blood Systems Research

Institute, 270 Masonic Ave., San Francisco, CA 94118 (pnorris@bloodsystems.org).

The Journal of Infectious Diseases 2008; 197:1296-306 © 2008 by the Infectious Diseases Society of America. All rights reserved. 0022-1 899/2008/1 9709-001 2$1 5.00 D0I: 10.1086/586898

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 neurologi- cal symptoms [4]. No spécifie therapy or vaccine has been approved for use in humans, leaving only support- ive treatment for WNV infection [5].

Considerable progress has been made in understand-

ing 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 Ragl»"/M<"" mice (i.e., those with B cell deficiency) [8] that succumb to WNV infection in the absence of passive immuno-

therapy [ 1 1, 12]. In the macaque model, WNV clearance from the blood occurs 5-11 days after inoculation, whereas anti-WNV IgM is not detectable until 9-10

days after inoculation, with neutralizing antibodies ap- pearing an additional 1-2 days later [13]. Finally, in the natural history of WNV infection in humans, clearance

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Table 1. Characteristics of blood donors who tested positive for West Nile virus (WNV) RNA.

NOTE. Data are positive ( + ) or negative (-) results of tests, unless otherwise indicated. ND, not done; NR, not reactive; R, reactive; TMA, transcriptional mediated amplification.

a Travel to dengue endemic area within the past 5 years (DEN) or vaccination with yellow fever vaccine in 1975 (YF). b Day of collection of the first sample from which peripheral blood mononuclear cells were recovered. c Highest number reported on the index or follow-up questionnaires. Constitutional symptoms (e.g., West Nile fever) predominated, and neurological symptoms

were rare and mild, with no meningoencephalitis or acute flaccid paralysis. d Seroconversion occurred on day 12 after the index donation. e Results of plaque-reduction neutralization tests for WNV and St. Louis encephalitis virus were negative at day 1 0, with only WNV titers increasing to 1 :51 20

at day 16.

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-y (IFN-y) secretion by y 8 and a/3 T

cells decreases the WNV load in mouse brains, and adoptive transfer of y8 or a/3 T cells reduces the susceptibility of mice to lethal WNV infection [ 15-17]. CD8+ T cells contribute to WNV

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Figure 1. Magnitude and diversity of responses to West Nile virus (WNV) peptides. A, Study subjects' response to each individual peptide. Subjects were considered to have responded if they recognized a peptide at any 1 of the following 3 time points: 15 days, 1 month, and 2 months after the index donation. B, Peak positive responses, determined by interferon-y enzyme-linked immunosorbent spot analysis after overnight incubation of peripheral blood mononuclear cells (PBMCs) with individual WNV peptides. The highest response to a given peptide from the 3 time points tested was averaged among all donors with a response. Error bars, standard errors. SFC, spot-forming cell.

clearance, as CD8+ T cell- deficient mice show increased mor- tality, with surviving mice exhibiting persistent viremia [18]. WNV can also be detected in the central nervous system of C57BL/6 mice lacking either IFN-y [19] or perform granules [20]. Major histocompatibility complex (MHC) class I upregu- lation is mediated by IFN-y- dependent and nuclear factor- kB- dependent pathways, pointing to a role for T cell-secreted cyto- kines in combating WNV infection [21].

Although T cells likely play a role in controlling WNV rep- lication, a comprehensive characterization of human T cell responses to WNV has not been previously reported. Studies of other flaviviruses suggest that NS proteins are preferen- tially recognized by T cells [22]. Studies of humans immu- nized 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 identi- fied a number of HLA class I B7-restricted 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 phe- notype and functional markers of T cells specific for immu- nodominant 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 RNA-positive 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 differ-

ent 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 per-

centage 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 X 106 cells were

frozen in media that contained 90% FBS (HyClone) and 10%

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Table 2. Characteristics of West Nile virus (WNV) peptides frequently recognized by WNV-positive persons and findings of the Basic Local Alignment Search Tool (BLAST).

NOTE. DEN, dengue virus; JEV, Japanese encephalitis virus; SLE, St. Louis encephalitis virus. a Bold font indicates 3 epitopes that span each of the following pairs of overlapping peptides: 38 and 39 from E, 264 and 265 from NS3, and 299 and 300 from

NS4b. b Data are magnitudes of positive responses to each peptide. The highest response from each of 3 time points was used to calculate the mean response for

each donor. Bold italic font indicates the highest responses (mean magnitude, >100 spot-forming cells (SFCs] per 106 peripheral blood mononuclear cells [PBMCs]).

c Bold italic font indicates the region(s) of the BLAST-matched flavivirus peptide sequence that differs from that of the WNV peptide sequence.

dimethyl sulfoxide (DMSO [Fisher BioReagents] ) and were stored in liquid nitrogen.

WNV transcription-mediated amplification (TMA), load, and antibody testing. WNV TMA testing was performed at Blood Systems Laboratories (BSL; Tempe, AZ), using reagents manufactured by Gen-Probe. WNV loads were measured at the National Genetics Institute (Los Angeles, CA). WNV IgM and

IgG testing was performed using ELISA kits (Focus Diagnostics) in accordance with the manufacturer's instructions. Plaque re- duction neutralization testing was performed at the Centers for Disease Control and Prevention (Fort Collins, CO) [26].

WNV peptides. A set of 452 peptides (16-18 amino acids

long, overlapping by 10 amino acids) spanning the entire ex-

pressed genome of NY99-flamingo382-99 strain of WNV (Gen- Bank accession number, AF 196835) was obtained through the National Institutes of Health Biodefense and Emerging Infec- tions Research Repository (Manassas, VA). Peptides were recon- stituted in water with ̂10% DMSO.

Enzyme-linked immunosorbent spot (ELISPOT) assays. Peptides were divided into 93 pools of 8-10 peptides, with each

peptide present in 2 pools [27]. The pattern of responses to the

pools was indicative of candidate peptides, which were individ-

ually retested in duplicate. PBMCs were plated in 96-well poly- vinylidene plates (Millipore) precoated with 2 /xg/mL of anti-

IFN-7 antibody (mAbl-DlK [Mabtech]) in PBS (UCSF Cell Culture Facility). Cells were added at a concentration of 100,000 cells/well in 100 /llL of RPMI (UCSF Cell Culture Facility) plus 10% human AB serum (Sigma), 11 mmol/L of glucose, 2 mmol/L of L-glutamine, 23 mmol/L of NaHCO3P, and 50 U of

penicillin-streptomycin (i.e., R10H). The final concentration of each peptide was 5 jutg/mL. Duplicate negative control wells, which contained cells plus medium, and a positive control well, which contained 0.5 /xg/mL of phytohemagglutinin (PHA;

Sigma), were run on each plate. Plates were then incubated for 20 h at 37°C and 5% CO2 and washed 6 times with PBS, after which they were incubated at 25°C for 60 min with biotinylated anti-IFN- y antibody (Mabtech). Plates were washed 6 times and incubated with streptavidin-alkaline phosphatase (Mabtech) for 60 min at 25°C. Plates were washed before addition of 5-bromo-

4-chloro-3-indolylphosphate and nitroblue tetrazolium sub- strates (Bio-Rad). Spots were counted using an automated ELISPOT plate reader (Cellular Technology). Results were ex-

pressed as spot-forming cells (SFCs) per 106 PBMCs after sub- traction of background values. Responses in 5 WNV-negative healthy control subjects ranged from 0 to 30 SFCs/106 PBMCs, with >80% of wells showing no spots. Responses were counted as positive if they were 3 times above the background value of the

plate and consisted of ̂50 SFCs/106 PBMCs.

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Figure 2. HLA restriction analysis of peripheral blood mononuclear cells (PBMCs) from donors who had the highest magnitude response. PBMCs were tested for presentation of the most frequently recognized peptides, by means of allogeneic B cell lines with partially matched HLA alleles (bold font; displayed on the left side of the charts). Peptide 30 Wand peptide 38 (B)were tested with PBMCs from donor 2350. Peptide 299 fCjwas tested with PBMCs from donor 2250. Charts show raw data for peptide-pulsed (black bars) and non-peptide-pulsed (open barsjB cell lines. Responses were counted if they were 2*1.5 times the background level. SFC, spot-forming cell.

Amino acid sequence comparison. Homology to antigens potentially able to induce cross-reactive responses to WNV pep- tides was investigated using the protein-specific Basic Local

Alignment Search Tool (BLASTP) from the National Center for

Biotechnology Information (NCBI) [28]. We used the "nr" pep- tide sequence databases, including all nonredundant GenBank CDS translations, RefSeq Proteins, PDB, SwissProt, PIR, and PRF databases.

Peptide stimulation and flow cytometry. One million PBMCs were stimulated in 250 jllL of R10H with 200 ng/mL of

staphylococcal enterotoxin B (SEB; Sigma) as a positive control or with 10 jULg/mL of individual peptides at 37°C and 5% CO2 for 2 h before the addition of 10 jutg/mL of brefeldin A (MP Biomedicals). Cells were incubated overnight at 37°C and 5% CO2 and then washed and stained. Except where noted, reagents were purchased from BD Biosciences/Pharmingen. For the phenotypic panel, cells were stained with EMA, anti-CD4-Alexa 700, anti-CD8-PE, anti-

CD45RA-PECy7, and anti-CD27-FITC at 4°C in the dark for 10 min and under bright light for 10 min. Cells were fixed with me- dium A (Caltag-Invitrogen), permeabilized with medium B

(Caltag), and stained with anti-CD3-Pacific Blue and anti-IFN-y-

APC. For the cytotoxic panel, cells were stained with anti-CD4- Alexa 700 and anti-CD8-PECy7 at 4°C for 10 min in the dark and for 10 min under bright light. A dump channel with EMA, CD 14, CD 16, and CD 19 was used to exclude dead cells, monocytes/mac- rophages, NK cells, and B cells. Cells were then fixed, permeabilized, and stained with anti-CD3-Pacific Blue, anti-IFN-y-APC, anti-

perforin-FITC, and anti-granzyme B-PECy5.5 (Caltag). Data were

acquired on a 3-laser BD LSRII instrument and were analyzed using Flowjo software (Tree Star).

HLA typing and restriction. DNA was extracted from PBMCs by use of QIAamp DNA Mini kits (Qiagen) in accordance with the manufacturers instructions. HLA class I/II typing was per- formed at BSL for 1 8 subjects with the highest-level responses, using a sequence-specific primer PCR [29]. HLA restriction analysis of

responses from subjects with high-magnitude T cell responses was

performed by ELISPOT, in which 5 ju,g/mL of peptides were prein- cubated for 16 h with B cell lines partially HLA matched to respond- ers. After 2 washes, 50,000 loaded B cells were incubated with

100,000 PBMCs in duplicate wells. Positive controls included PB- MCs plus peptide or PHA, and the background level was calculated

using PBMCs with unloaded B cells.

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RESULTS

Breadth and magnitude of T cell responses to acute WNV infection. Comprehensive WNV-specific T cell responses were measured by ELISPOT for each of the 35 WNV-positive participants at 3 time points: 15 days, 1 month, and 2 months after the index donation. There was a wide distribution of re- sponses: 27 of 35 WNV-positive individuals recognized 1-9 pep- tides at 55 1 of the 3 time points after the index donation, with the majority of peptides recognized at only 1 time point. The most frequently recognized regions of the WNV genome were those encoding the proteins C, M, E, NS3, NS4a, and NS4b (figure \A and table 2). Overall, 8% of the peptides expressed in the WNV genome were recognized by the T cells of at least 1 participant. The percentage of peptides recognized within individual pro- teins was variable, with the greatest breadth of recognition in C (20% of peptides), M (19%), E (12%), NS4a (17%), and NS4b (18%). Peptide 30 from M was highly targeted and was recog- nized by >20% of individuals (table 2). Other peptides targeted by >9% of the donors were peptide 30 from M, peptides 38 and 39 from E, peptides 217, 264, and 265 from NS3, and peptides 299 and 300 from NS4b. Among the frequently targeted pep- tides, 3 epitopes spanned each of the following pairs of overlap- ping peptides: 38 and 39 from E, 264 and 265 from NS3, and 299 and 300 from NS4b (table 2). None of the frequently recognized WNV peptides elicited responses in uninfected control subjects. A BLASTP search on the NCBI Web site revealed matching amino acid sequences only between WNV and other flaviviruses (table 2).

Among the 27 donors with T cell responses, the average summed magnitude of positive responses within a given donor against individual peptides ranged from 50 to 4210 SFCs/106 PBMCs (median, 130 SFCs/106 PBMCs). The highest magnitude responses (mean magnitude, >100 SFCs/106 PBMCs) were elic- ited by peptides within M, E, NS3, NS4a, and NS4b (table 2). Peptides 30, 38 and 39, 264 and 265, and 299 and 300 appeared to be immunodominant epitopes, because they elicited high- magnitude responses and were among the most frequently rec- ognized peptides (figure 1 and table 2). The breadth of WNV epitopes recognized did not differ between symptomatic and asymptomatic subjects (mean, 2.0 ± 2.6 vs. 2.2 ± 1.88 pep- tides; P = .76), nor did the magnitude of positive responses (326 vs. 425 SFCs/106 PBMCs; P = .59).

Late immune response to WNV. The durability of T cell responses to WNV was tested 1 year after the index donation in samples from 20 donors, which included the 5 donors with the highest early responses. None of the samples had ̂50 SFCs/106 PBMCs to peptide pools at 1 year. If the ELISPOT assay cutoff was adjusted to just above the value for negative control wells, responses to the immunodominant peptides could still be de- tected using peptide pools in 10% of previously responsive sub- jects (data not shown). These data demonstrated only very low-

Table 3. HLA class 1 binding motifs for West Nile virus peptides 30 and 299.

NOTE. Data are based on predictions made by use of SYFPEITHI software [31].

a Bold font indicates preferred anchor residues, and un- derlining indicates preferred auxiliary anchor residues.

b Scores are calculated by ranking amino acid anchor positions. Ideal anchors are given 10 points, unusual an- chors 6-8 points, auxiliary anchors 4-6 points, and pre- ferred residues 1-4 points. Amino acids with a negative effect on binding are scored between -1 and -3.

frequency persistence of WNV-specific T cells 1 year after infection.

HLA restriction. Responses to peptides 30, 38, and 299 were associated with particular HLA types. Seven of 10 do- nors expressing A*0101 recognized peptide 30, compared with 0 of 8 Al-negative donors. Four of 5 C*0303-positive or

Cw*0304-positive donors recognized peptide 38, compared with 1 of 13 HLA C3-negative donors. The Cw*0303 and Cw*0304 alleles were treated as equivalent, because the only coding difference between them is at codon 91, which is not

predicted to affect the HLA binding groove or T cell receptor contact sites [30]. Finally, 3 of 6 A*0201 -positive donors re-

sponded to peptide 299, and 0 of 12 HLA A2-negative donors

responded. These associations agreed with HLA restriction

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Figure 3. CD4+ versus CD8+ T cell mediation of high-magnitude West Nile virus (WNVHpecific responses. A, Results of flow cytometry to determine CD4+ and CD8+ populations of T lymphocytes. Gates were set on unstimulated (unstim) cells for interferon-y (IFN-y) secretion. CD4+ and CD8+ T cell populations were stained for IFN-y+ expression after overnight stimulation and incubation with brefeldin A. EMA, ethidium monoazide; FSC, forward scatter; SSC, side scatter. B, Rate of IFN-y+ secretion among CD4+ or CD8+ T cell populations after no stimulation, staphylococcal enterotoxin B (SEB) stimulation, WNV peptide stimulation. Gates for IFN-y secretion were set using an aliquot of unstimulated cells. Results are shown for responses >0.1% IFN--^positive cells, and a minimum of 100,000 lymphocyte-gated events were collected in each experiment.

experiments that involved B cell lines partially HLA matched to subjects 2250 or 2350, the subjects with the highest mag- nitude responses (figure 2A-C). Exploration of HLA-binding motifs by use of SYFPEITHI software [31] showed that the second most likely binding motif for peptide 30 was A*0101 and that the most likely binding motif for peptide 299 was A*0201 (table 3). The binding motif analysis supported the HLA restriction results: peptide 30 was presented by A*0101, peptide 38 by Cw*0303/0304, and peptide 299 by A*0201.

High-magnitude WNV-specific responses are mediated pri- marily by CD8+ T cells. WNV-specific T cells from donors with high-magnitude responses (i.e., >500 SFCs/106 PBMCs) were examined by flow cytometry (figure 3). PBMCs from 3

subjects (symptomatic donor 2250 and asymptomatic donors 2350 and 2450) were stimulated with their respective cognate WNV peptides or with SEB as a positive control. Cells were gated by forward scatter and side scatter to determine which cells were

lymphocytes. Lymphocytes were then separated into live CD4+ and CD8+ populations. IFN-y positivity was plotted against CD3 positivity to enumerate the fraction of cells specific for WNV (figure 3A). IFN-y-positive cells induced by peptide stim- ulation were predominantly CD8+ T cells, except in donor 2450, in whom low-frequency CD4+ T cell responses were also ob- served (figure 3B).

Phenotype of WNV-specific T cells. To further characterize the phenotype of WNV-specific T cells, PBMCs were stained before and after stimulation (figure 4). Cells were classified as naive (CD45RA+CD27+), effector (CD45RA+/-CD27-), or

memory (CD45RA-CD27+) [32] (figure 4A). Effector cell fre-

quencies in the CD8+ T cell subset were not significantly differ-

ent between WNV-infected and WNV-uninfected subjects (P = .32), whereas the frequency of effector CD4+T cells was lower among WNV-infected subjects (P = .006, by the t test)

(figure 4B). For donor 2250, 47% of WNV-specific CD8+ T cells 1 month after infection were effector cells, compared with 19% of unstimulated CD8+ T cells, whereas for donor 2350, effector cells comprised 55% and 49% of WNV-specific and unstimu- lated CD8+ T cells, respectively, with WNV-specific CD8+ T cells almost equally divided between effector and memory pop- ulations (figure AC). WNV-specific T cells also showed high ex-

pression of the cytolytic markers perforin and granzyme B, con- sistent with the effector phenotype of these cells (figure 5). Donors 2250 and 2350 exhibited higher median levels of WNV-

specific CD8+ T cells that expressed cytolytic markers, compared with unstimulated (i.e., non-WNV-specific) cells (85% vs. 23%; P<.001).

DISCUSSION

In this study, we performed the first comprehensive analysis of the breadth and specificity of WNV-specific T cell responses in humans. We report WNV-specific T cell responses in the major- ity of WNV-infected individuals. Responses were preferentially directed against C, M, E, NS3, and NS4b, with 12%-20% of

peptides from these regions recognized by at least 1 subject in our cohort. By 1 year after the index donation, the diversity of

responses had waned, with only peptides associated with a

strong early response still able to elicit responses directly ex vivo.

Analysis of subjects with the highest magnitude responses re- vealed most responses to be CD8+ T cell mediated, with re-

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Figure 4. Phenotype of West Nile virus (WNV)-specific T cells. A, Gating strategy to define naive T cells (CD45RA+CD27+), memory T cells (CD45RA-CD27+), and effector T cells (CD45RA+/CD27~). Gates for interferon-y (IFN-y) secretion were set on unstimulated cells. Live lymphocytes were gated into CD4+ and CD8+ populations. CD45RA+ and CD27+ gates were set on the IFN-^negative (IFN-y~) and IFN-^positive (IFN-y+) CD3+ T cell populations. Fluorescence-minus-one tubes were used for CD45RA and CD27 stains. EMA, ethidium monoazide; FSC, forward scatter; SSC, side scatter. B, Phenotypes of T cells in 10 WNV^ control subjects and 23 WNV+ subjects 1 month after the index donation. C, Phenotypes of T cells after no stimulation (unstim; i.e., IFN-y~), staphylococcal enterotoxin B (SEB) stimulation, and antigen-specific stimulation (i.e., IFN-y+) in 2 donors with the

highest magnitude CD8+ T cell responses 1 month after the index donation. At least 100,000 lymphocyte-gated events were collected, and at least 100 events in the IFN-y+ gate were collected.

sponding cells having an effector phenotype and expressing per- forin and granzyme B.

The combined magnitude of positive responses to WNV (me- dian, 130 SFCs/106 PBMCs [range, 50-4210 SFCs/106 PBMCs]) was less than that observed in human immunodeficiency virus (HIV) infection (median, 4245 SFCs/106 PBMCs [range, 280- 25,860 SFCs/106 PBMCs]) [33] but comparable to that observed in Epstein-Barr virus (EBV) infection (median, 136 SFCs/106 PBMCs [range, 27-1344 SFCs/106 PBMCs]) [34], cytomegalo- virus (CMV) infection (median, 973 SFCs/106 PBMCs for IE1

protein recognition [range, 0-3900 SFCs/106 PBMCs]) [35], and HCV infection (range, 25-285 SFCs/106 PBMCs) [36] . Eight of 35 subjects did not have a WNV-specific T cell response of >50 SFCs/106PBMCs, implying that systemic infection can oc-

cur with weak induction of T cell activity. Of the 3 subjects with the strongest WNV-specific T cell responses, subject 2250 (who had symptomatic infection) had traveled to a dengue-endemic area in the past 5 years and had preexisting IgG antibody cross- reactive with WNV, and subject 2450 (who had asymptomatic infection) had been vaccinated against yellow fever. These data raise the possibility that prior flavivirus exposure increased the

magnitude of WNV-specific T cell responses during primary in- fection but did not necessarily prevent symptoms.

WNV E protein is known to be a major target for neutralizing antibodies [37, 38]. IFN-y T cell responses to E peptides were

reported after immunization of humans with a live attenuated recombinant WNV vaccine, using WNV M and E in a yellow fever virus backbone [39]. Our study confirmed the importance

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Figure 5. Cytolytic effector molecule expression by West Nile virus (WNV)-specific T cells. Gates were set on unstimulated cells for interferon-y (IFN-y) secretion and fluorescence-minus-one (FMO) tubes for perform and granzyme B stains. A Unstimulated, staphylococcal enterotoxin B (SEB)-stimulated, and peptide-stimulated conditions for donor 2250 (smoothed lines) 1 month after the index donation. Peripheral blood mononuclear cells were stained for IFN-y plus perform and granzyme B after secretion after overnight stimulation and incubation with brefeldin A. Live lymphocytes were gated on IFN-y-negative (IFN-y-) and IFN-y-positive (IFN-y+) populations. B, Rates of perform and/or granzyme B expression among CD8+ IFN-y+ cells after no stimulation, stimulation with SEB, or stimulation with WNV-specific peptides in donors 2250 and 2350 1 month after the index donation. Gates for perform and granzyme B were set using FMO controls. A minimum of 100,000 lymphocyte-gated events were collected, and at least 100 events in the IFN-y+ gate were collected.

of WNV E protein as a major target of immune responses. Po- tential T cell epitopes in the E proteins of Murray Valley enceph- alitis virus, Japanese encephalitis virus, WNV, and dengue virus have been identified by computer prediction algorithms. One

epitope in the C- terminal region of the E glycoprotein (approx- imate location, amino acids 420-455) was predicted to induce immune responses in all flaviviruses analyzed [40] and was con- firmed in our study as peptide 95, which was 94% homologous with dengue virus and induced high-magnitude responses (table 2). Another bioinformatics approach to T cell epitope mapping identified 16 HLA B7-restricted epitopes in WNV [24]. None of these epitopes induced responses in our study, which included 2 HLA B7-positive donors who responded to other WNV pep- tides.

The nonstructural proteins NS3, NS4a, and NS4b have been described as immunodominant targets for class I MHC-re-

stricted responses in mice infected with Kunjin virus or WNV

[22]. Cytotoxic T lymphocytes (CTLs) that recognize prM, E, NS1, NS2a, and NS3 proteins were also identified in volunteers immunized against dengue virus, and some responses cross- reacted with WNV [23] . Human T cell responses against dengue virus C, prM, M, E, NSI, NS2a, NS3, NS4a, NS4b, and NS5

epitopes have been reported [23, 41-45]. Several of the WNV

peptides we reported are derived from proteins that are homol-

ogous to immunogenic flavivirus proteins and have similar amino acid sequences. Of the 8 most frequently recognized pep- tides, 3 were from NS3, and 2 from NS4b (figure \A).

We were able to characterize the phenotype of T cells respond- ing to WNV in the 2 subjects with the highest frequency of CD8+ T cell responses (figures 4 and 5). The results showed that WNV-

specific CTLs express the cytolytic markers perforin and gran- zyme B and were roughly equally divided between memory and

1304 • JID 2008:197 (1 May) • Lanterietal.

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effector cell phenotypes 1 month after the index donation. Al-

though further studies would be required to confirm these find-

ings in more individuals, WNV would appear to induce CTLs in the middle of the spectrum of maturity, compared with chronic viral infections inducing early (e.g., EBV or HCV), intermediate

(HIV), or late (CMV) memory differentiated populations in the months following the acute phase of infection [27, 32, 46]. The

early high-frequency responses we observed were only very weakly detected 1 year after infection, implying that the WNV-

specific memory cells persisted at very low frequencies. Potential limitations of this study include the peptides used to

stimulate WNV-specific responses and the specimen handling and storage process. The WNV peptides used were 16-18 amino acids long, overlapping by 10 amino acids. Although longer than

optimal CD8+ T cell epitopes, these peptide lengths have been shown to induce reasonably strong CD8+ and CD4+ T cell re-

sponses, representing a cost-effective compromise in measuring responses to the whole expressed viral genome [47, 48]. Few fixed mutations have been described in currently circulating US

strains, compared with the reference WNV strain used as the

template for peptide synthesis [49]. Though WNV is not highly variable, it is possible that sequence variation decreased the abil-

ity to observe responses in the current study. Finally, blood was collected at regional blood centers and shipped overnight to the

laboratory, delaying cell processing by up to 24 h. This may also have reduced the magnitude of T cell responses we observed,

resulting in underestimation of the true magnitude of WNV-

specific responses [50]. In addition to defining frequently recognized regions of

WNV, HLA restriction was performed for 3 immunodominant

epitopes (figure 2). These epitopes were presented by the rela-

tively common HLA alleles A*0101, A*0201, and Cw*0303/04,

implying that these may be useful epitopes to include in vaccine candidates. Notably, despite a high diversity of HLA class I alleles

among the WNV-positive individuals, 54% of the subjects and 70% of individuals with responses targeted at least 1 peptide within the subset of 13 frequently recognized peptides (table 2). Our results show that a limited number of peptides can induce

WNV-specific T cell immune responses in a genetically diverse

population. Our data suggest that immunodominant epitopes were present in M, E, NS3, and NS4b proteins. M and E proteins, already known to be targets for neutralizing antibodies and a focus of vaccine development [51 ], are also targeted by CTLs in natural WNV infection. Natural infection also induced T cell

responses to C, NS3, NS4a, and NS4b proteins, and these regions of the virus should be further studied as new or complementary candidates for WNV vaccine development.

Acknowledgments

We thank the National Institutes of Health Biodefense and Emerging In- fections Research Resources Repository for kindly providing peptide arrays spanning all West Nile virus proteins; Drs. Robert Endres and Jar-How Lee

for helpful discussions on HLA-C molecular structure; and Dr. Robert Lan- ciotti for performing West Nile virus plaque reduction neutralization tests.

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