Acquired Immunity? in Seronegative Subjects

T Cell Immunity to Herpes Simplex Viruses in Seronegative Subjects: Silent Infection or Acquired Immunity?

This information is current as Christine M. Posavad, Anna Wald, Nancy Hosken, Meei Li of September 23, 2021. Huang, David M. Koelle, Rhoda L. Ashley and Lawrence Corey J Immunol 2003; 170:4380-4388; ; doi: 10.4049/jimmunol.170.8.4380

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

T Cell Immunity to Herpes Simplex Viruses in Seronegative Subjects: Silent Infection or Acquired Immunity?1

Christine M. Posavad,2*§ Anna Wald,*†‡ Nancy Hosken,¶ Meei Li Huang,* David M. Koelle,*†§ Rhoda L. Ashley,* and Lawrence Corey*†§

During the course of investigating T cell responses to HSV among volunteers entering trials of investigational genital herpes vaccines, 6 of the 24 immunocompetent subjects with no prior history of oral/labial or genital herpes possessed HSV-specific T cell immunity but, by multiple determinants of even the most sensitive serological assays, remained seronegative to HSV-1 and -2. Of these six immune seronegative (IS; HSV-seronegative with HSV-specific T cell responses) subjects, two had transient HSV-specific T cell responses, while four had CD4؉ and CD8؉ T cell responses directed at HSV that persisted for up to 4 years. CD4؉ T cell clones were isolated that recognized and had high binding affinities to epitopes in HSV-2 tegument proteins. All six IS subjects had potential sexual exposure to an HSV-2-infected sexual partner. Oral and genital mucosal secretions were sampled and tested for Downloaded from the presence of infectious HSV and HSV DNA. No evidence of HSV was detected in >1500 samples obtained from these IS subjects. The identification of persistent T cell responses to HSV in seronegative subjects is a novel finding in the herpesvirus field and suggests either undetected infection or acquired immunity in the absence of infection. Understanding the basis of these acquired immune responses may be critical in developing effective vaccines for genital herpes. The Journal of Immunology, 2003, 170: 4380–4388. http://www.jimmunol.org/ erpes simplex viruses type 1 (HSV-1) and 2 (HSV-2) During the process of testing the safety and immunogenicity of cause a variety of medically significant infections, espe- a candidate HSV-2 vaccine in subjects seronegative for HSV-1 and H cially in immunosuppressed subjects. HSV-1 and -2 are -2, we detected HSV-specific T cell responses in blood obtained ubiquitous pathogens, and hence, susceptibility to infection was before vaccine administration in six of 24 subjects tested. In this believed to be common, if not universal, in all populations. In study we detail the HSV-specific T cell and Ab responses in these prospective studies of couples discordant for HSV-2 where the six subjects with HSV-specific T cell responses. In addition, we HSV-seronegative partner was consistently exposed to infectious studied mucosal secretion patterns by daily sampling of these pa- secretions, the acquisition rate of HSV-1 and HSV-2 averaged tients for an extended time period. Ͻ7% yearly (1Ð3), suggesting that resistance to HSV infection by guest on September 23, 2021 may be acquired by persons with chronic exposure to the virus. Materials and Methods Acquired resistance to a human herpesvirus has not been docu- Subjects mented; however, there are precedents for acquired resistance in We enrolled 24 healthy subjects who were seronegative for HSV-1 and other human viral infections. This has been best documented in HSV-2 (HSV-seronegative) into a protocol testing the safety and immu- HIV infection, where multiply exposed, uninfected persons have nogenicity of a candidate HSV-2 vaccine. We also enrolled 8 immuno- been described by many investigators. Resistance to HIV has been competent subjects who were HSV-1 seropositive only, 18 subjects who were HSV-2 seropositive, and 4 additional HSV-seronegative subjects not in many cases attributed to the presence of HIV-specific T cell enrolled in the vaccine study. All subjects were enrolled in a University of responses (4Ð13). More recently, T cell responses to hepatitis C Washington institutional review board-approved protocol. All subjects pro- virus (14, 15) and EBV (16) have been measured in the absence of vided written informed consent. detectable serum Ab. Whether these seronegative persons are in- Cells and viruses fected with the organism or have developed transient infection that results in viral elimination or containment of the infection is PBMC were isolated by Ficoll-Hypaque and cryopreserved. Lymphoblas- toid cell lines (LCL)3 were generated as previously described (17). HSV-1 unclear. strain E115 and HSV-2 strain 333 were used at an multiplicity of infection of 10 where indicated. Bulk CTL and LP assays

6 Departments of *Laboratory Medicine, †Medicine, and ‡Epidemiology, University of Cryopreserved PBMC (2 ϫ 10 /ml) were thawed and stimulated for 10 Washington, and ¤Program in Infectious Diseases, Fred Hutchinson Cancer Research days with ﬁxed autologous HSV-1 or -2 infected PHA blasts (1 ϫ 106/ml). Center, Seattle, WA 98109; and ¶Corixa Corp., Seattle, Washington 98104 NK cell-depleted bulk cultures or cultures depleted of NK cells and CD4ϩ ϩ 51 Received for publication October 10, 2002. Accepted for publication February or CD8 T cells were tested for lytic activity against Cr-labeled autol- 4, 2003. ogous or allogeneic LCL that were mock-infected or infected overnight with HSV-1 or -2 in a 4-h 51Cr release assay using 2 ϫ 103 targets/well in The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance triplicate in 96-well, round-bottom plates. NK cells were depleted using with 18 U.S.C. Section 1734 solely to indicate this fact. magnetic beads (Dynal Biotech, Great Neck, NY) coated with Abs to CD16 and CD56 just before use in CTL assays. 1 This work was supported by Grants AI01776 (to C.P.), AI49394 (to C.P.), AI42528 (to C.P., L.C., and A.W.), and AI30731 (to L.C., A.W., R.L.A., and D.M.K.) from the National Institute of Allergy and Infectious Diseases. 3 Abbreviations used in this paper: LCL, lymphoblastoid cell line; DC, dendritic cell; 2 Address correspondence and reprint requests to Dr. Christine M. Posavad, 1100 ICC, intracellular cytokine staining; IS, immune seronegative; pCTL, CTL precursor; Fairview Avenue North, Room D3-100, Seattle, WA 98109. E-mail address: pProlif, HSV-speciﬁc lymphoproliferative precursors; LP, lymphoproliferation; VZV, [email protected] varicella zoster virus.

Cryopreserved PBMC were thawed, and 1 ϫ 105 cells were incubated peptide) in an IFN-␥ ELISPOT assay (24). Individual peptides from pos- in triplicate with HSV-2 Ag (UV-inactivated HSV-2), mock Ag (1/500), or itive pools were tested in the same fashion to identify the epitopes. PHA (0.8 ␮g/ml), and HSV-2-specific LP was determined as previously described (18). Systemic Abs to HSV HSV-specific CTL and proliferative LDA Standard Western blots and ECL Western blots for the detection of HSV-1 and HSV-2 were performed on sera as previously described (25, 26). The frequency of HSV-1- and HSV-2-specific CD8ϩ CTL precursors (pCTL) was determined by limiting dilution analysis (LDA) with nega- ϩ Daily home sampling of mucosal sites and detection of HSV by tively selected CD8 T cells from cryopreserved PBMC using methods culture and PCR and calculations previously described (18, 19). The frequency of HSV-1- ϩ and HSV-2-specific CD4 lymphoproliferative precursors (pProlif) in Subjects were instructed on the daily sampling of genital and oral mucosal PBMC was determined as previously described (18, 20). sites as described in detail previously (27, 28). Dacron swabs placed in ␥ viral transport medium were delivered to the laboratory three times per Intracellular IFN- staining week. Viral isolation was performed as previously described (29, 30). PCR Intracellular IFN-␥ staining was performed as previously described (21) detection of HSV at mucosal sites was performed by quantitative real-time using cryopreserved PBMC (1 ϫ 106) incubated with 1 ␮g each of co- fluorescence-based PCR as previously described (31), using previously de- stimulatory Abs against CD28 and CD49d (BD Biosciences, San Jose, scribed primers and probes (32). Both the volume of sample used to extract CA). In every experiment a negative control (anti-CD28-CD49d) was in- DNA (400 ␮l) and the volume of purified DNA (20 ␮l) used in each 50-␮l cluded to control for spontaneous production of IFN-␥ as well as a positive PCR reaction were doubled compared with those used for the detection of control (staphylococcus enterotoxin B; final concentration, 1 ␮g/ml; Sig- HSV DNA in HSV-seropositive subjects (31) to detect extremely low copy ma-Aldrich, St. Louis, MO) to ensure that the cells were responsive. CD8 number of HSV. Samples were considered positive by PCR if they 1) Ͼ responses to HSV-2 were stimulated using GM-CSF/IL-4 dendritic cells contained 10 copies/reaction of HSV DNA, 2) could be repeated, and 3) Downloaded from (DC; 1 ϫ 105) that were mock-infected (negative control) or infected over- could be typed. night with HSV-2 (positive control). CD4 responses to HSV-2 were stimulated using a 1/100 dilution of mock or HSV-2 Ag. After 2-h incubation Results ␮ at 37¡C, the secretion inhibitor brefeldin A (10 g/ml; Sigma-Aldrich) was Cellular immunity to HSV in HSV-seronegative subjects enrolled added, followed by an additional 4-h incubation. The cells were then kept at 4¡C overnight. Cells were washed with PBS containing EDTA, followed in a candidate HSV-2 vaccine trial: identification of immune by incubation at room temperature for 10 min with FACSLyse (BD seronegative (IS) subjects Biosciences). Cells were washed and permeabilized using FACS permeabili- http://www.jimmunol.org/ zation solution (BD Biosciences) for 10 min at room temperature, washed, and Subjects who were seronegative to HSV-1 and -2 by Western blot stained for 30 min in the dark with CD4Ϫ or CD8-peridinin chlorophyll A were recruited into a phase I trial testing the safety and immuno- protein, CD69-PE, and IFN-␥-FITC (all from BD Biosciences). CD69-PE was genicity of a candidate HSV-2 vaccine at University of Washing- used to detect activated T cells. The cells were washed and resuspended in 1% ton. The initial screening visit evaluated past history of oral or ϫ 4 ϫ 5 paraformaldehyde in PBS. Between 5 10 and 1 10 events were ana- genital lesions, and blood was drawn for HSV Western blot anal- lyzed using a FACScan flow cytometer (BD Biosciences) and side scatter gating. The percentage of CD4ϩ or CD8ϩ T cells responding to HSV-2 was ysis (25). Only subjects without a history of oral or genital lesions defined for each donor by subtracting the percentage of CD69ϩ/IFN-␥ϩ cells who were HSV seronegative were eligible for enrollment. At the using mock Ag or mock-infected DC from the percentage of cells using enrollment visit blood was taken for determination of HSV-2-spe-

HSV-2 Ag or from HSV-2-infected DC. Responses were considered positive cific CTL and LP responses before immunization in 24 HSV-se- by guest on September 23, 2021 if the net percentage of CD69ϩ/IFN-␥ϩ cells was Ͼ0.05% of the total CD4 T cells or Ն10% of the total CD8 T cells. Background CD4 responses (anti- ronegative subjects (patients 1Ð24) as well as in four subjects in- CD28-CD49d alone or with mock Ag) were always Ͻ0.04%, and background fected with HSV-1 only (patients 30Ð33) and five subjects infected CD8 responses (anti CD28-CD49d alone or mock-infected DC) ranged from with HSV-2 (patients 40Ð44) who were used as controls (Fig. 1). 0 to 0.60% (median, 0.06%). Of the 24 HSV-seronegative subjects, four (patients 1, 3, 4, and Generation of PBMC-derived HSV-specific T cell clones 22) had positive HSV-2-specific CTL responses (Fig. 1a) and pos- ϩ itive HSV-2 specific LP responses (Fig. 1b). In addition, two of the HSV-specific CD4 T cell clones were established from PBMC as previ- 24 subjects (patients 16 and 19) had HSV-2-specific CTL re- ously described (20). HLA restriction of T cell clones was determined by inhibition of LP with mAbs to HLA class II DR, DQ, and DP as previously sponses in the absence of an HSV-specific LP response (Fig. 1). described (20). Clones were tested in an LP assay for reactivity to HSV-1, Weak positive LP responses to HSV-2 were measured in the ab- HSV-2, varicella zoster virus (VZV), EBV, CMV, and influenza virus. sence of an HSV-2-specific CTL response in three of the subjects CMV Ag was prepared as previously described (22) and was used at a final (patients 6, 8, and 14). Positive CTL and LP responses were de- dilution of 1/500, VZV Ag (Advanced Biotechnologies, Columbia MD) was used at a final dilution of 1/600, EBV-infected cell extract (Advanced tected in all nine HSV-infected subjects (Fig. 1, a and b). Biotechnologies) was used at a final concentration of 3 ␮g/ml, and Influ- enza Virus Vaccine USP (zonal purified, subvirion; Pasteur Merieux, Paris, Persistence of HSV-specific cellular immune responses in IS France) was used as a source of influenza Ag at a final concentration of subjects 1 ␮g/ml. Serial blood samples were obtained from the nine HSV-seroneg- HSV-2 expression library ative subjects with positive T cell responses to HSV-2 at screening. HSV-2 (333) genomic DNA fragments were ligated into the pET17b ex- HSV-2-specific bulk CTL and LP responses were measured in pression vector. Pools (20 clones/pool) of transformed Escherichia coli three to six separate blood samples over a period of 3Ð17 mo (Fig. (JM109 strain; Life Technologies, Gaithersburg, MD) were prepared and 2). The four subjects with positive CTL and LP responses at screened as previously described (23). E. coli clones prepared from posi- screening (patients 1, 3, 4, and 22) had consistently positive CTL tive pools were subsequently screened. Library inserts from positive clones were sequenced. Ags were identified by comparison of insert sequences and LP responses at all time points tested (Fig. 2). In contrast, the with the HSV-2 (HG52) genomic sequence (GenBank accession no. two subjects with positive CTL responses in the absence of an LP Z86099). response at screening (patients 16 and 19) had no evidence of Identification of T cell epitopes HSV-specific LP responses, but displayed CTL responses that lasted 6Ð7 mo and waned over time (Fig. 2). This observation Overlapping 15-mer peptides (five-amino acid overlap; Mimetopes, Clay- suggests that a Th response to HSV is required to maintain HSV- ton Victoria, Australia) spanning UL21, UL29, UL46, and UL47 were synthesized and dissolved in DMSO. Peptide pools (10 peptides/pool) were specific CTL responses. In addition, subjects with weak LP re- prepared and screened by coculture of T cells (2 ϫ 104/well), HLA- sponses in the absence of a CTL response at prevaccine time points matched DC (1 ϫ 104/well), and synthetic peptides (10 ng/ml of each (patients 6, 8, and 14) had negative LP and CTL responses to HSV 4382 T CELLS SPECIFIC FOR HERPES SIMPLEX VIRUS IN SERONEGATIVE SUBJECTS Downloaded from http://www.jimmunol.org/

FIGURE 1. HSV-2-specific cellular immune responses in blood samples at study entry from HSV-seronegative subjects recruited for a candidate HSV-2 vaccine study. a, HSV-2-specific CTL activity. PBMC were stimulated for 10 days with HSV-2-infected PHA blasts. NK-depleted bulk cultures were tested by guest on September 23, 2021 for HSV-2-specific CTL activity against mock-infected or HSV-2-infected LCL, and the net HSV-specific lysis was measured. E:T cell ratios were 100:1, 50:1, and 25:1 for all subjects except patients 3, 20, and 21, for whom these ratios were 50:1, 25:1, and 12.5:1. Patients 1Ð24 were HSV seronegative; patients 30Ð33 were infected with HSV-1 only (HSV-1ϩ/2Ϫ); patients 40Ð44 were infected with HSV-2 (HSV-2ϩ). A CTL response was considered positive if the net specific lysis (lysis of HSV-2 LCL minus lysis of mock LCL) was Ͼ10% at two or more E:T cell ratios (dashed line). b, HSV-2-specific LP responses. PBMC were stimulated for 6 days with UV-inactivated HSV-2 or mock Ag, and net HSV-specific proliferation was determined by measuring [3H]thymidine uptake. An LP response was considered positive if the net proliferation (counts per minute of HSV-2 Ag minus counts per minute of mock Responses considered positive. c, No evidence of HSV seroconversion in IS subjects. Sera drawn on different dates ,ء .(Ag) was Ͼ5000 cpm (dashed line from patient 3 were analyzed for the presence of IgG (G) or IgA (A) Abs directed at HSV-1 or -2 by ECL Western blot. ϩ, Controls are pooled sera from HSV-1- or HSV-2-seropositive subjects. Ϫ, Controls are pooled sera from HSV-seronegative subjects. Abs directed at specific HSV proteins are shown. in subsequent blood samples. HSV-seronegative subjects with ev- ultrasensitive assay; a representative ECL Western blot from IS idence of HSV-specific T cell responses (CTL and/or LP) in blood subject 3 is shown in Fig. 1c. All six IS subjects had normal levels at more than one time point are hereafter called IS, while those of serum IgG, IgA, IgM, IgE, and anti-tetanus Abs (data not HSV-seronegative subjects with no evidence of HSV-specificT shown), demonstrating that the IS subjects were not Ab deficient. cell responses or where one time point was positive for T cell Thus, by even the most sensitive assay available for detection of responses, but negative at subsequent time points, are hereafter Abs to HSV, no anti-HSV Abs were measured in serum from IS called HSV-seronegative. subjects, including those time points when HSV-specific T cell Sera from all HSV-seronegative subjects participating in the responses were detected in PBMC. vaccine trial were tested at multiple time points by Western blot ϩ for the detection of HSV-specific Abs, and none of the 24 subjects CD8 T cell responses to HSV in IS subjects displayed Abs to HSV-1 or HSV-2. Because of the consistent de- We quantitated the frequencies of CD8ϩ T cells specific for tection of HSV-specific T cell responses in the six IS subjects, we HSV-1 and -2 in the IS subjects by LDA (Fig. 3a) and intracellular repeated the Western blots on sera obtained from all time points in cytokine staining (ICC; see below). A high frequency of HSV-2- these subjects. No evidence of HSV-1 or -2 Abs was detected at specific CD8ϩ pCTL was measured in patient 3 (250/106). Low any time point tested (data not shown). We also examined sera frequencies of HSV-1- and HSV-2-specific CD8ϩ pCTL were from the IS subjects using an ECL Western blot, an assay that is measured in the other three IS subjects: patients 1 (18 and 22/106), 500-fold more sensitive than the standard Western blot and can 4 (17 and 16/106), and 22 (20 and 39/106). LDA in HSV-1-sero- detect HSV Abs with serum dilutions in excess of 106 (26). Both positive subjects with culture-proven HSV-1 ranged from 41Ð218/ IgG- and IgA-specific HSV Abs were evaluated. The six IS sub- 106 for HSV-1 and from 9Ð247/106 for HSV-2. Responses in jects exhibited no evidence of HSV-specific bands even by this HSV-2-seropositive subjects with culture-proven HSV-2 ranged The Journal of Immunology 4383 Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 2. Longitudinal analysis of HSV-specific cellular immune responses in HSV-seronegative subjects with positive cellular immune responses to HSV-2 at screening. HSV-2-specific bulk CTL responses (graphs in left column) and LP responses to HSV-2 (graphs in right column) were measured longitudinally in HSV-seronegative vaccine enrollees who had positive HSV-2-specific CTL and/or LP responses at screening. E:T cell ratios were 100:1, 50:1, and 25:1. a and b, patient 1; c and d, patient 3; e and f, patient 4; g and h, patient 6; i and j, patient 8; k and l, patient 14; m and n, patient 16; o and p, patient 19; q and r, patient 22. from 16Ð182/106 for HSV-1 and from 47Ð318/106 for HSV-2 (patient 49), and an IS subject (patient 3). HSV-2-specific CD8 (Fig. 3a). Among HSV-seronegative vaccine trial participants with responses were detected in all 12 HSV-2-infected subjects, ranging no previous demonstration of HSV-specific T cell responses, the from 0.10Ð1.93% of the total CD8ϩ T cells (median, 0.64%), 6 median frequency of HSV-specific pCTL was 1/10 in response to whereas the six HSV-seronegative subjects had CD8 responses to Ͻ 6 6 HSV-1 (range, 1Ð12/10 CD8s from PBMC) and 7/10 in re- HSV-2 ranging from 0.00Ð0.05% (median, 0.02%; Fig. 3d). These sponse to HSV-2 (range, Ͻ1Ð12/106 CD8s from PBMC). Thus, all ϩ data are consistent with the CTL responses in these subjects; all 12 four IS subjects had higher frequencies of HSV-specific CD8 ϩ HSV-2 subjects and none of the six HSV-seronegative subjects pCTL than those measured in HSV-seronegative subjects with no had HSV-2-specific CTL responses to HSV (Fig. 1a). All five IS detectable CTL responses in bulk stimulated cultures. ϩ subjects exhibited CD8 IFN-␥ responses to HSV-2 ranging from We determined the frequency of IFN-␥-producing CD8 T cells in response to HSV-2 by ICC in 5 of the IS subjects, 12 subjects 0.30Ð0.79% (median, 0.41%; Fig. 3d), consistent with the detec- with recurrent HSV-2 infection, and 6 HSV-seronegative subjects. tion of HSV-specific CTL responses in these subjects (Fig. 1a). ϩ Fig. 3b displays representative FACS plots of CD8 responses to These data confirm the existence of HSV-specific CD8 T cells in HSV-2 in an HSV-seronegative subject with no detectable in vitro IS subjects, including T cells with CTL activity and those that T cell responses to HSV (patient 26), an HSV-2 infected subject produce IFN-␥. 4384 T CELLS SPECIFIC FOR HERPES SIMPLEX VIRUS IN SERONEGATIVE SUBJECTS Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021

FIGURE 3. CD4ϩ and CD8ϩ T cell responses to HSV in IS subjects. a, HSV-specific CD8ϩ pCTL (left) and HSV-specific pProlif (right) were measured in five HSV-seronegative subjects with no HSV-specific CTL or LP responses (HSVϪ), in five subjects seropositive for HSV-1 only (1ϩ), in five subjects seropositive for HSV-2 (2ϩ), and in four IS subjects (patients 1, 3, 4, and 22). HSV-1-specific(f) and HSV-2-specific(Ⅺ) pCTL and pProlif were measured in each subject. b, Representative FACS plots of PBMC-derived CD8ϩ T cell responses to mock-infected or HSV-2-infected DC. The percentages of CD8ϩ/CD69ϩ/IFN-␥ϩ cells are shown for each panel. Patient 26 is HSV seronegative, patient 49 is HSV-2 infected, and patient 3 is an IS subject. c, Representative FACS plots of PBMC-derived CD4ϩ T cell responses to mock or HSV-2 Ag. The percentages of CD4ϩ/CD69ϩ/IFN-␥ϩ cells are shown for each panel. Subjects are as described in b. HSV-specific CD8ϩ (d) and CD4ϩ (e) T cell responses were measured by flow cytometry in HSV-2-infected (HSV-2ϩ), HSV-seronegative subjects (HSV-1Ϫ/2Ϫ), and IS subjects. Responses were considered positive if the net CD8ϩ/CD69ϩ/IFN-␥ϩ response was Ն0.10% of the total CD8ϩ T cells and the net CD4ϩ/CD69ϩ/IFN-␥ response was Ͼ0.05% of the total CD4ϩ T cells.

CD4ϩ T cell responses to HSV in IS subjects higher than those measured in HSV-seronegative subjects (Fig. We next quantitated HSV-speciﬁc CD4ϩ T cell responses by LDA 3a). The four IS subjects had pProlif frequencies ranging from (pProlif) and ICC. As with the CD8 LDAs, the IS subjects exhib- 19Ð286 cells/106 PBMC, which, with the exception of patient 22, ited responses similar to those of HSV-seropositive subjects and were within the ranges of those measured in HSV-1 (43Ð389/106

Table I. Characterization of HSV-speciﬁc CD4ϩ T cell clones from patient 1a

HSV-2-Specific Proliferation (cpm)b Cytokines (pg/ml)c HSV Ag Clone HSV-2 Anti-DR Anti-DQ Anti-DP IFN-␥ IL-2 IL-4 IL-5 Specificityd Ϫ 1.6 26,215 25,840 4 31,646 210 0 7 34 UL21281Ð300 1.20 24,010 27,837 35,040 59 7,900 70 237 178 UL29251Ð265 1.22 22,438 9,488 22,552 22,896 240 0 15 0 UL46621Ð629 1.24 12,829 467 12,938 14,299 295 0 21 280 UL47137Ð155 a HSV-specific CD4ϩ T cell clones were isolated from PBMC from patient 1. b HLA restriction of each clone was determined by inhibition of HSV-specific lymphoproliferation with mAb to HLA class II DR (anti-DR), DQ (anti-DQ), and DP (anti-DP). c Clones were stimulated with UV-HSV-2, and supernatants were tested for cytokines by ELISA; cytokines in supernatants from mock-stimulated clones were below the level of detection of cytokine ELISAs. d Expression cloning was used to determine the Ag specificity of CD4ϩ T cell clones. The specific epitope is contained with the peptide sequences that are subscripted. The Journal of Immunology 4385

FIGURE 4. Characterization of HSV-specific CD4ϩ T cell clones from patient 1 for CTL activity, in vitro cross-reactivity, and TCR affinity. a, CD4ϩ T cell clones from patient 1 were tested in a standard chromium release assay using mock-infected and HSV-2-infected autologous LCL as target cells; the E:T cell ratio was 10:1. Responses are shown as net HSV-2-specific lysis (percent specific lysis against HSV-2 LCL minus percent specific lysis against mock-infected LCL). b, CD4ϩ T cell clones from patient 1 were tested in a lymphoproliferation assay against HSV-1, HSV-2, VZV, CMV, EBV, and influenza virus Ag (Flu). Responses are the net proliferation (counts per minute of viral Ag minus counts per minute of mock Ag). c, Clone 22 from patient

1 was tested in a lymphoproliferation assay with the indicated concentrations of UL46621Ð639 peptide. Responses are net proliferation (counts per minute of viral peptide minus counts per minute of mock Ag). Downloaded from PBMC) and HSV-2 (28Ð484/106 PBMC) seropositive subjects below the level of detection in one of the three blood samples (data (Fig. 3a). not shown). ␥ ϩ We determined the frequency of IFN- -producing CD4 T cells ϩ in response to HSV-2 by ICC in five of the IS subjects, 13 HSV- Characterization of HSV-specific CD4 T cell clones from an IS 2-seropositive subjects, and six HSV-seronegative subjects. Fig. 3c subject displays representative FACS plots of CD4 responses to HSV-2 in HSV-specific CD4ϩ clones were isolated and expanded from an HSV-seronegative subject (patient 26), an HSV-2-infected sub- PBMC from patient 1, an IS subject in whom we had sufficient http://www.jimmunol.org/ ject (patient 49), and an IS subject (patient 3). HSV-2-specific CD4 PBMC for cloning and subsequent characterization. Of the 48 responses were detected in all 13 HSV-2-infected subjects, ranging clones from patient 1 that were screened for HSV-2-specificLP from 0.12 to 0.54% of the total CD4ϩ T cells (median, 0.35%), responses, 44 had net HSV-specific proliferation of Ͼ5000 cpm. whereas none of the six HSV-seronegative subjects had CD4 re- Ten randomly selected clones were restimulated and expanded sponses to HSV-2 above background (0.05%; Fig. 3e). These data with PHA and IL-2, and data from four representative clones are are consistent with the LP responses in these subjects; all 13 displayed in Table I and Fig. 4. Using mAbs to inhibit HLA class HSV-2ϩ subjects and none of the six HSV-seronegative subjects II molecules, we determined that clones 1.22 and 1.24 were

had HSV-2-specific LP responses to HSV (Fig. 1b). In contrast, HLA-DR restricted, clone 1.6 was DQ restricted, and clone 1.20 by guest on September 23, 2021 two of the five IS subjects exhibited IFN-␥ responses to HSV-2; was DP restricted (Table I). All four clones produced detectable patients 1 and 3 had 0.30 and 0.14% of CD4ϩ T cells specific for levels of IFN-␥ and IL-4 and/or IL-5 in response to HSV-2, in- HSV-2, respectively (Fig. 3e), consistent with the detection of dicative of a Th0 pattern of cytokine secretion. Expression cloning HSV-specific LP responses in these subjects (Fig. 1b). Although was used to determine the antigenic specificity of the CD4ϩ T cell patient 22 had persistent HSV-specific LP responses (Fig. 2), IFN- clones; all four clones recognized viral tegument proteins, includ- ␥-producing CD4ϩ cells were below the level of detection in this ing UL21, UL29, UL46, and UL47. The region of the protein IS subject (Fig. 3e). Patients 16 and 19, who each had transient containing the antigenic epitope recognized by the CD4ϩ T cell CTL responses in the absence of HSV-specific LP responses, also clones has been narrowed to 18Ð20 aa (Table I). The clones were had no detectable HSV-specific IFN-␥-producing CD4ϩ cells (Fig. also tested for HSV-specific CTL activity, and three of the four 3e). When we measured IFN-␥-producing CD4 cells longitudi- clones (1.6, 1.22, and 1.24) specifically lysed HSV-2-infected au- nally, CD4 responses to HSV-2 were detected in three of four tologous LCL, while clone 1.20 was not cytotoxic (Fig. 4a). blood draws taken over 13 mo from patient 3, although the re- Clones from patient 1 were tested for reactivity to HSV-2-responses were low (0.07Ð0.14%) (data not shown). CD4 responses lated human herpesviruses (HSV-1, VZV, CMV, and EBV) as well to HSV-2 in patient 1 persisted for 4 years, although they were as influenza virus, a virus unrelated to HSV. Clones 1.6, 1.22, and

Table II. PCR studies for mucosal HSV shedding among IS subjectsa

HSV PCR

HSV Cultures 1st sampling 2nd sampling Total Cultures/ Total PCRs/ Patient No. CTL/LP Oral Vul Pen PAPatient Oral Gen Oral Vul Pen PA Patient

1 ϩ/ϩ 0/79 0/79 158 3 ϩ/ϩ 0/25 0/25 0/25 75 0/51 0/51 0/26 0/25 0/25 178 16 ϩ/Ϫ 0/100 0/100 200 0/101 0/99 0/100 0/100 400 19 ϩ/Ϫ 0/99 0/102 0/102 303 0/100 0/100 0/102 0/101 0/102 505 22 ϩ/ϩ 0/112 0/111 0/111 334 0/114 0/114 0/114 342 Total/site 236 102 236 338 912 331 329 242 101 239 341 1,583

a During the ﬁrst sampling study, samples were collected for PCR from the oral and genital (Gen) sites. For the genital site, one swab was used to sample the penis (Pen) and perianal area (PA) from male subjects (patients 1, 3, 16, and 22), and one swab was used to sample the cervicovaginal, vulvar (Vul), and perianal area from the female subject (patient 19). In the second sampling study, samples were obtained for both viral cultures and PCR. 4386 T CELLS SPECIFIC FOR HERPES SIMPLEX VIRUS IN SERONEGATIVE SUBJECTS

1.24 were HSV type common because they proliferated in re- no evidence of HSV seroconversion in the IS subjects. In addition, sponse to HSV-1 and HSV-2, while clone 20 was HSV-2 type despite analysis of Ͼ1500 samples, we did not detect any infec- specific (Fig. 4b). The clones were not cross-reactive with VZV, tious HSV or HSV DNA at mucosal sites in the IS subjects we CMV, EBV, or influenza virus (Fig. 4b), supporting the hypothesis studied. Taken together, these novel findings indicate the existence that these clones were generated by exposure to or infection with of HSV-seronegative subjects with T cell immunity to HSV in the HSV. We next tested the TCR affinity of each clone by diluting absence of clinical or subclinical HSV infection and suggest that each peptide and measuring lymphoproliferation. The proliferative the characterization of T cell responses in these subjects may lend response of each clone was achieved at low peptide concentrations insight into the mechanisms involved in resistance to HSV infec- ϭϽ (EC50 1 ng/ml), providing further evidence that clones from this tion or inhibition of HSV disease expression. IS subject were not generated by cross-reactive epitopes. A represen- One possible explanation for the detection of HSV-specificT tative peptide dose-response experiment using clone 22 and the cell responses to HSV in IS subjects is that these responses were

UL46621Ð639 peptide is displayed (Fig. 4c). primed by cross-reacting Ags from related or unrelated organisms. CD8ϩ T cell cross-reactivity in humans has been recently docu- No evidence of HSV infection in IS subjects mented between nonhomologous viruses, including influenza A We next evaluated whether the IS subjects were infected with HSV virus with hepatitis C virus (33). While no study documents T cell in the absence of seroconversion. It was of course not possible to cross-reactivity between human ␣-herpesviruses (VZV and HSV), evaluate HSV infection in sensory neural root ganglia in IS sub- human CD4ϩ T cell cross-reactivity has been detected between jects. We could, however, evaluate mucosal reactivation of HSV human ␤-herpesviruses, namely CMV, HHV-6, and HHV-7, al- by daily sampling of genital and oral secretions. We asked the six though this is rare (34). In at least one IS subject (patient 1), T cell Downloaded from IS subjects to enroll in a daily mucosal shedding trial in which oral cross-reactivity seems unlikely, since four CD4ϩ T cell clones and genital secretions were sampled for HSV by both culture and isolated from this subject recognized four different epitopes from PCR, the most sensitive method of detecting mucosal HSV shed- four different HSV-2 proteins. Further, these CD4ϩ T cell clones ding. Four IS subjects (patients 3, 16, 19, and 22) collected sam- did not proliferate in response to VZV (the most closely related ples for viral culture and PCR detection, and one IS subject (pa- virus to HSV) or to CMV or EBV (other related human herpesvi- tient 1) collected samples for PCR only. Three subjects (patients 3, ruses). Moreover, the clones responded to low concentrations of http://www.jimmunol.org/ ϭϽ 16 and 19) performed two sampling studies for PCR (Table II). peptide (EC50 1 ng/ml), providing additional evidence that the Patient 4 declined participation in this study. Patient 16 agreed to clones were generated by exposure to or infection with HSV; the the collection of oral samples for one PCR sampling study only. concentration of peptide required to stimulate cross-reacting re- Subjects collected samples for PCR for 26Ð114 days and for viral sponses tends to be Ͼ1 ␮g/ml (35). Thus, our data strongly argue culture for 25Ð112 days (Table II). For individual subjects, 75Ð334 that the HSV-specific T cell responses identified in this IS subject viral cultures and 158Ð505 PCRs were collected for a total of 912 are not due to cross-reacting T cells, but are the result of exposure viral cultures (236 oral and 676 genital) and 1583 PCRs (573 oral to or infection with HSV. and 1010 genital) for all IS subjects (Table II). We anticipated that The sexual histories of the IS subjects related to potential HSV if virus was present, it would be at low levels, and thus we in- exposure suggest that at least three of the six IS have knowingly by guest on September 23, 2021 creased the sensitivity of the real-time PCR by doubling the sam- been exposed to HSV-1 or -2. However, considering 1) the high ple volume of purified DNA used in the assay. All 912 viral cul- percentage of subjects who are seropositive for HSV-1 or HSV-2 tures and all 1583 PCRs were negative for HSV. (36), 2) the number of previous sexual partners (range, 9Ð24), 3) the high percentage of HSV-2-infected subjects who do not rec- Demographics and sexual history of IS subjects: assessment of ognize the signs and symptoms of genital herpes (37), and 4) the exposure to HSV potential of HSV-infected subjects not to inform partners of their None of the IS subjects reported a history of oral or genital herpes. HSV status (38), it is highly likely that the other three IS subjects All six of the IS subjects were Caucasian. Patients 1, 3, 16, and 22 were exposed to at least one partner infected with genital herpes. were men, and patients 4 and 19 were women. The ages of the IS Analysis of T cell immunity to HSV in subjects with known and subjects ranged from 23Ð38 years. The six IS subjects reported documented exposure to HSV will provide stronger evidence of a number of lifetime sexual partners ranging from 9Ð24 (median, 10 link between exposure to HSV mucosally and acquired T cell im- partners). Three of the six IS subjects reported that they had part- munity to HSV in the absence of seroconversion. Currently, we are ners infected with HSV, two stated that previous partners were investigating couples discordant for HSV-2 where the susceptible infected with HSV-1 (patients 1 and 19), and one reported a cur- partner is seronegative for HSV-1 and -2. At present, we have rent partner with genital HSV-2 (patient 22). The human subjects identified four of 10 HSV-seronegative partners who possess HSV- review committee did not allow access to these partners. specific T cell responses in the absence of infection or seroconversion (C. M. Posavad and L. Corey, unpublished observations), corroborat- Discussion ing the hypothesis that exposure to HSV can induce HSV-specific We detected CD4ϩ and CD8ϩ T cells directed at HSV in 6 of 24, cellular immunity in the absence of seroconversion. or 25%, of HSV-seronegative subjects enrolled in a candidate To date we have no evidence of HSV infection in the IS subjects HSV-2 vaccine study. T cell responses were persistent in four of we have identified. In a recent study of 53 HSV-2-seropositive the IS subjects and lasted up to 4 years in one subject. Both cy- subjects who reported no history of genital herpes, 52 of 53 sub- totoxic and IFN-␥-producing CD4ϩ and CD8ϩ T cells specific for jects demonstrated HSV reactivation by PCR on the mucosal shed- HSV were detected in the IS subjects, although in general the ding sampling protocol we used in this study (37). If IS subjects frequencies of HSV-specific T cells tended to be lower in IS sub- are infected, we would have expected to detect HSV considering jects compared with HSV-seropositive subjects. CD4ϩ T cell the total number of days sampled (1583 days), especially with the clones were isolated and were shown to be specific for multiple doubling in sample volume we used. It is possible that 1) virus was HSV tegument proteins. No cross-reactivity to other herpesviruses, shed at too low a copy number (Ͻ10 copies/reaction) to be de- including the other human ␣-herpesvirus, VZV, was shown. Even tected by real-time PCR, 2) IS subjects reactivate HSV, but at even using the most sensitive assays for HSV-specific Abs, we obtained lower frequencies than we sampled, or 3) HSV does not reactivate The Journal of Immunology 4387 from latency in these subjects; for example, subjects are infected transmission of herpes simplex virus type 2 from men to women. JAMA 285: with a defective or less virulent or replication-defective mutant of 3100. 2. Corey, L., A. G. Langenberg, R. Ashley, R. E. Sekulovich, A. E. Izu, HSV (39). J. M. J. Douglas, H. H. Handsfield, T. Warren, L. Marr, S. Tyring, et al. 1999. Although the current study has described the immune response Recombinant glycoprotein vaccine for the prevention of genital HSV-2 infection: to HSV in IS subjects as a potential mechanism for resistance of two randomized controlled trials: Chiron HSV Vaccine Study Group. JAMA 282: 331. HSV infection or lack of disease, viral and genetic factors may also 3. Brown, Z. A., S. Selke, J. Zeh, J. Kopelman, A. Maslow, R. L. Ashley, H. Watts, contribute to the IS status. One genetic mechanism may be immu- S. Berry, M. Herd, and L. Corey. 1997. The acquisition of herpes simplex virus nologic determinants of disease severity that are associated with during pregnancy. N. Engl. J. Med. 337:509. 4. Clerici, M., J. V. Giorgi, C. C. Chou, V. K. Gudeman, J. A. Zack, P. Gupta, HLA expression. A prospective study of 146 subjects has identi- H. N. Ho, P. G. Nishanian, J. A. Berzofsky, and G. M. Shearer. 1992. Cell- fied several HLA alleles that are associated with HSV-2 infection mediated immune response to human immunodeficiency virus (HIV) type 1 in or with frequent symptomatic genital recurrences (40). Most sig- seronegative homosexual men with recent sexual exposure to HIV-1. J. Infect. Dis. 165:1012. nificantly, the presence of HLA-B8 and the absence of HLA-B27 5. Langlade-Demoyen, P., N. Hgo-Giang-Huong, F. Ferchal, and E. Oksenhendler. and -Cw2 were associated with symptomatic disease in HSV-2- 1994. Human immunodeficiency virus (HIV) nef-specific cytotoxic T lympho- infected subjects, and HLA-Cw4 was significantly associated with cytes in noninfected heterosexual contact of HIV-infected patients. J. Clin. Invest. 93:1293. HSV-2 infection (40). These HLA associations suggest that im- 6. Pinto, L. A., J. Sullivan, J. A. Berzofsky, M. Clerici, H. A. Kessler, A. L. Landay, munologic factors linked to the MHC influence the risk of HSV-2 and G. M. Shearer. 1995. ENV-specific cytotoxic T lymphocyte responses in HIV infection and disease expression. Four of the six IS subjects (pa- seronegative health care workers occupationally exposed to HIV-contaminated body fluids. J. Clin. Invest. 96:867. tients 1, 3, 4, and 16) did not express HLA-Cw4, consistent with 7. Rowland-Jones, S., J. Sutton, K. Ariyoshi, T. Dong, F. Gotch, S. McAdam, a decreased susceptibility to HSV-2 infection, although clearly D. Whitby, S. Sabally, A. Gallimore, T. Corrah, et al. 1995. HIV-specific cyto- Downloaded from many more IS subjects than we have identified will be required to toxic T-cells in HIV-exposed but uninfected Gambian women. Nat. Med. 1:59. 8. Rowland-Jones, S. L., T. Dong, L. Dorrell, G. Ogg, P. Hansasuta, P. Krausa, detect an association between HLA and IS status. J. Kimani, S. Sabally, K. Ariyoshi, J. Oyugi, et al. 1999. Broadly cross-reactive A second potential genetic factor may be mutations in genes HIV-specific cytotoxic T lymphocytes in highly-exposed persistently seronega- encoding HSV receptors, a situation analogous to the HIV system, tive donors. Immunol. Lett. 66:9. 9. Bernard, N. F., C. M. Yannakis, J. S. Lee, and C. M. Tsoukas. 1999. Human where resistance to HIV infection in some multiply exposed HIV immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte activity in HIV- seronegative persons has been associated with the homozygous exposed seronegative persons. J. Infect. Dis. 179:538. http://www.jimmunol.org/ inheritance of a defective HIV-1 coreceptor, CCR5 (41, 42). We 10. Goh, W. C., J. Markee, R. E. Akridge, M. Meldorf, L. Musey, T. Karchmer, M. Krone, A. Collier, L. Corey, M. Emerman, et al. 1999. Protection against have recently sequenced three known HSV receptors, herpesvirus human immunodeficiency virus type 1infection in persons with repeated expo- entry mediator, HVEM (HveA) (43), nectin-1 (HveC, PRR1) (44Ð sure: evidence for T cell immunity in the absence of inherited CCR5 coreceptor 46), and nectin-2 (HveB, PRR2) (47, 48), in patients 1, 3, and 4 to defects. J. Infect. Dis. 179:548. 11. Bienzle, D., K. S. MacDonald, F. M. Smaill, C. Kovacs, M. Baqi, B. Courssaris, determine whether coding polymorphisms in these genes could M. A. Luscher, S. L. Walmsley, and K. L. Rosenthal. 2000. Factors contributing cause altered susceptibility to HSV infection in IS subjects (49). to the lack of human immunodeficiency virus type 1 (HIV-1) transmission in Coding polymorphisms were detected in the HVEM and nectin-1 HIV-1 discordant partners. J. Infect. Dis. 182:123. 12. Kaul, R., F. A. Plummer, J. Kimani, T. Dong, P. Kiama, T. Rostron, E. Nhagi, genes, but these mutations were not exclusive to IS subjects, and K. S. MacDonald, J. J. Bwayo, A. J. McMichael, et al. 2000. HIV-1 specific the receptors encoded by these variant genes were indistinguish- mucosal CD8ϩ lymphocyte responses in the cervix of HIV-1-resistant prostitutes by guest on September 23, 2021 able from the wild-type forms tested in in vitro HSV entry activity in Nairobi. J. Immunol. 164:1602. 13. Schmechel, S. C., N. Russell, F. Hladik, J. Lang, A. Wilson, R. Ha, A. Desbien, assays (49). Additional subjects will be needed to determine and M. J. McElrath. 2001. Immune defence against HIV-1 infection in HIV-1- whether an association exists between HSV receptor polymor- exposed seronegative persons. Immunol. Lett. 79:21. phisms and susceptibility to HSV infection, especially in HSV- 14. Scognamiglio, P., D. Accapezzato, M. A. Casciaro, A. Cacciani, M. Artini, G. Bruno, M. L. Chircu, J. Sidney, S. Southwood, S. 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and L. Corey. 2000. Reactivation of genital herpes simplex virus type 2 infection mediators, HVEM, nectin-1 and nectin-2 in immune seronegative individuals. by guest on September 23, 2021 in asymptomatic seropositive persons. N. Engl. J. Med. 342:844. J. Infect. Dis. 185:36.