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. Author manuscript; available in PMC: 2011 Mar 15.
Published in final edited form as: J Immunol. 2010 Feb 17;184(6):3250–3259. doi: 10.4049/jimmunol.0900722

Detailed Characterization of T Cell Responses to HSV-2 in Immune Seronegative Persons1

Christine M Posavad *,†,2, Michael Remington †, Dawn E Mueller *, Lin Zhao †, Amalia S Magaret *,†, Anna Wald *,†,‡,§, Lawrence Corey *,†,‡
PMCID: PMC2877513  NIHMSID: NIHMS188855  PMID: 20164419

Abstract

In 2003 we described a small cohort of subjects (n=6) who possessed no detectable serum antibodies to HSV-1 or HSV-2, no clinical or virological evidence of mucosal HSV infection yet possessed consistently detectable HSV-specific T cell responses measured primarily by lymphoproliferative (LP) and CTL assays to whole HSV-2 antigen. We termed these persons immune seronegative (IS). This report characterizes the T cell responses in 22 IS subjects largely recruited from studies of HSV-seronegative subjects in ongoing sexual relationships with HSV-2-seropositive (HSV-2+) partners using pools of overlapping peptides spanning 16 immuno-prevalent HSV-2 proteins. Overall, 77% of IS subjects had HSV-specific LP responses, 85% had IFN-γ ELISPOT responses to at least 1 HSV-2 peptide pool, and 55% had both LP and IFN-γ ELISPOT responses. In some cases, IFN-γ ELISPOT responses were in excess of 500 spot-forming cells per 106 PBMC and persisted for over 5 years. While HSV-2+ subjects (n=40) had frequent responses to glycoproteins, tegument and immediate-early (IE) proteins of HSV-2, T cell responses in IS subjects were directed primarily at UL39 and the IE proteins ICP4 and ICP0. These data suggest that the antigenic repertoire of T cells in IS subjects is skewed compared to HSV-2+ subjects and that IS subjects had more frequent T cells responses to IE proteins and infrequent T cell responses to virion components. Understanding the mechanism(s) by which such responses are elicited may provide important insights in developing novel strategies for preventing acquisition of sexually acquired HSV-2.

Keywords: human, T cells, viral, epitopes

INTRODUCTION

Genital herpes is a common sexually transmitted disease caused predominantly by herpes simplex virus type 2 (HSV-2). Virus transmission is primarily through sexual contact, typically during asymptomatic HSV shedding episodes from infected partners. The clinical spectrum of HSV disease ranges from asymptomatic to frequently recurring infections; severe manifestations of the disease can include persistent and extensive mucosal lesions in immunocompromised subjects suggesting a role for the adaptive immune system in preventing HSV infection and disease. With an estimated US seroprevalence rate of 17% among 14-49 year olds (1), the recognition of genital herpes as a worldwide pandemic, and the increased risk that HSV-2 plays on HIV-1 acquisition (2, 3), the development of an HSV vaccine is instrumental to disease control. To date, a prophylactic or therapeutic vaccine for HSV-2 remain elusive due in large part to a lack of understanding of the adaptive immune correlates of HSV infection and disease severity.

We have described a cohort of HSV-seronegative subjects with prior exposure to HSV with no clinical or virological evidence of HSV infection but who possess HSV-specific T cell responses (hereafter referred to as immune seronegative or IS3) (4) suggesting that some persons may be resistant to HSV infection and/or disease. These subjects are similar to those who appear to be resistant to HIV-1 or hepatitis C virus (HCV) even in face of multiple high-risk exposures (5-10). While various innate and adaptive immune mechanisms of resistance have been proposed to confer protection to these viruses in these resistant cohorts, no single genetic or immunologic parameter has yet been able to fully explain this phenomenon.

In order to gain insight into the adaptive immune correlates of HSV resistance, we characterized HSV-specific T cell responses in HSV-1 and HSV-2 seronegative subjects who were in sexual relationships with HSV-1 and/or HSV-2 infected partners. A total of 22 subjects, or 29% of all HSV-seronegative subjects tested, were identified as IS and a detailed analysis of their T cell responses using peptides from a subset of immuno-prevalent HSV-2 proteins and IFN-γ ELISPOT, intracellular cytokine staining (ICS) and flow cytometry are described.

MATERIALS AND METHODS

Subjects

We sought to evaluate the frequency in which we could detect HSV-specific T cell responses among persons who were seronegative to HSV-1 and HSV-2 (HSVneg). All subjects were enrolled into University of Washington Institutional Review Board approved protocols and provided written informed consent. All HSVneg subjects and subjects seropositive to HSV-1 (HSV-1+), HSV-2 (HSV-2+) or to both viruses (HSV-1+/2+) who served as controls in the T cell assays for the IS subjects were enrolled into 1 of 4 studies performed at the Virology Research Clinic (VRC), University of Washington, Seattle, WA. HLA class I and II typing was performed at the Puget Sound Blood Center.

Subjects were recruited from both high risk and “lower” risk cohorts for acquiring HSV-2 infection. High risk cohorts included sexually active adults who considered themselves at risk of genital herpes acquisition and enrolled in a genital HSV vaccine or were HSVneg sexual partners of persons with documented HSV-1 and/or HSV-2. “Lower risk” cohorts included persons specifically recruited as HSVneg to serve as negative control subjects for validating HSV-specific T cell assays. The first study enrolled 25 healthy, HSVneg subjects and involved testing the safety and immunogenicity of a candidate HSV-2 DNA vaccine (11).

An initial PBMC sample was evaluated for HSV-specific T cell responses as described below. If a positive response to HSV was detected, the subject was recontacted, reconsented and additional PBMC were collected at 3-6 months intervals for up to 5 years.

Serology

HSV Western blot to detect antibodies to HSV-1 and HSV-2 was performed as previously described (12, 13). Antibodies to CMV indicating prior CMV infection were assessed using a commercial enzyme immunoassay kit for detection of total antibodies to CMV (Abbott CMV Total AB EIA, Abbott Laboratories, Abbott Park, Illinois) (14). The assay was performed and interpreted according to manufacturer recommendations.

Cells and Viruses

PBMC were isolated by Ficoll-Hypaque and cryopreserved within 8 hours of venipuncture as previously described (15). HSV-1 strain E115 and HSV-2 strain 333 were used where indicated.

HSV-specific LP responses and T cell clones

Cryopreserved PBMC were thawed and 1×105 cells were incubated in triplicate in 200 μl RPMI-H in 96-well round-bottomed plates with 1:500 dilution of UV-inactivated (UV) HSV-1, UV-HSV-2, mock antigen (Ag) or PHA-P (0.4 μg/ml) as previously described (16). After 5 days, 1μCi [3H]thymidine (New England Nuclear) was added to each well for 18 hrs and its incorporation into DNA was measured. Δcpm were calculated by subtracting cpm from mock Ag wells from cpm from UV-HSV wells. An LP response was considered positive if the Δcpm were >5,000 cpm. HSV-2 specific T cell clones were generated and expanded from PBMC using UV-HSV-2 for CD4 T cell clones or HSV-2 infected dendritic cells for CD8 T cell clones as previously described (4). The CD4 or CD8 phenotype of HSV-specific T cell clones was confirmed by flow cytometry.

Synthetic peptides and peptide pools

Open reading frames (ORFs) for peptide synthesis (n=16) were selected from among the known ORFs of HSV-2 based on studies of the prevalence of CD8+ T cell responses in subjects with genital herpes (17, 18) (Table I). ORFs were mostly virion proteins (capsid, tegument, glycoprotein) or immediate-early (IE) proteins. Amino acid sequences were derived from the HSV-2 strain HG52 genome (GenBank accession no. NC-001798). Peptides were 15 amino acids long and overlapping by 11 amino acids and synthesized in crude form by either CBI/Mimotopes (San Diego, CA) (gD-2, ICP0, ICP4, ICP22, ICP27, UL39) or New England Peptide LLC (Gardner, MA) (UL19, UL25, UL35, UL46, UL47, UL49, UL11, UL27, UL29, US5) and lyophilized. Each peptide was dissolved in 10 mg/ml in sterile endotoxin-free DMSO (Sigma) and stored at 4°C. The mass of each peptide was approximately 4-5 mg. Control peptide pools included the CEF pool (CBI/Mimotopes) comprising immunodominant CD8+ T cell epitopes within CMV, EBV and influenza (15, 19) and the CMV pp65 peptide mix containing 138 15 mers overlapping by 11 amino acids (Becton Dickinson (BD) Biosciences, San Jose, CA).

TABLE I.

HSV-2 peptide pools used in IFN-γ ELISPOT assay

No. HSV-2 ORF ORF classa Common name # peptidesb # pools # peptides/pool
1 US6 gp gD-2 96 1 96
2 RL2 IE ICP0 198 2 98,100
3 US1 IE ICP22 101 2 50,51
4 UL54 IE ICP27 126 2 63,63
5 RS1 IE ICP4 330 4 82,82,83,83
6 UL39 other ICP6 283 3 94,94,95
7 UL19 capsid VP5/ICP5 341 4 85,85,85,86
8 UL25 capsid 144 2 72,72
9 UL35 capsid VP26 26 1 26
10 UL46 tegument VP11/12 178 2 89,89
11 UL47 tegument VP13/14 172 2 86,86
12 UL49 tegument VP22 74 1 74
13 UL11 tegument 22 1 22
14 UL27 gp gB 224 3 74,87,63
15 UL29 other ICP8 297 3 99,99,99
16 US5 gp gJ 21 1 21
Totals 2,633 34 range: 21-100
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a

Open reading frames (ORF) for study encoded glycoproteins (gp), immediate early (IE), capsid, tegument or other proteins.

b

15 mers overlapping by 11 amino acids.

Peptide pools (library and array) were prepared for each ORF. Briefly, library pools were prepared by grouping peptides linearly across an ORF and were used to screen PBMC. A total of 2,633 peptides were synthesized and combined into pools composed of peptides ranging from 21-100 peptides/pool (median 85 peptides/pool) (Table I). Array pools, used to deconvolute library pools that were positive in the PBMC screen, were prepared by arranging the peptides within a single library pool in a row-and-column format and pooling the peptides in each column or row.

IFN-γ ELISPOT

The IFN-γ ELISPOT assay was performed as previously described (20) with modifications. PBMC were thawed in R10 [RPMI 1640 supplemented with 10% FBS, 2 mM l-glutamine, 25 mM HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) buffer, 50 μg/ml streptomycin, 50 U/ml penicillin containing 50 U/ml benzonase (Novagen, Madison, WI)], washed and rested overnight in R10 at 37°C, 5% CO2 before assay. Using the IFN-γ ELISPOT kit (Mabtech, Cincinnati, OH) according to the manufacturer's instructions, PBMC were plated at 2×105 cells/well. Peptides were added to the wells at a final concentration of 1 μg/ml for overnight stimulation. Wells containing R10 alone or R10 and DMSO served as negative controls, and those containing 1 μg/ml PHA-P (Murex, distributed by Remel, Lenexa, KS) served as positive controls. Negative controls were tested in 4 replicate wells, whereas HSV-2, CMV and CEF peptide pools and PHA were tested in duplicate wells. Spots were counted and analyzed using the automated Bioreader® (Biosys GmbH, Germany). Responses were considered positive if (1) the spot forming cells (SFC)/well were 4 times greater than the mean SFC in the 4 DMSO wells and (2) the SFC/well was > 11 or 55 SFC/106 PBMC. Results are expressed as SFC per 106 PBMC.

ICS and flow cytometry

HSV-2 peptide-specific T cell responses from IS subjects were phenotyped ex vivo using a 6-color ICS panel in a 96-well plate format modified from (21). Briefly, PBMC were thawed were rested overnight in R10 media followed by stimulation with DMSO (negative control), SEB (positive control), HSV-2 peptide pools (1 μg/ml), individual HSV-2 15-mers (1 μg/ml), individual HSV-2 9-mers (1 μg/ml), CEF or CMV peptide pools. During the 6-hr incubation at 37°C, Brefeldin A (10 μg/ml, Sigma, St. Louis, MO) and the co-stimulatory antibodies CD28 and CD49d (each at 1 μg/ml, BD Biosciences) were included. Antibodies CD4-FITC, CD8 PerCP-Cy5.5, IFN-γ APC, and IL-2 PE were purchased from BD Biosciences, CD3 ECD was purchased from Beckman-Coulter (Marseille, France) and the LIVE/DEAD Fixable Violet Dead Cell Stain was purchased from Invitrogen/Molecular Probes (Eugene, OR). Samples were collected from 96-well plates using High Throughput Sample (HTS, BD) device for analysis by the LSRII and all FACS analyses were performed using FlowJo® software (Treestar, Inc; OR).

RESULTS

Clinical and demographic characteristics of IS subjects

We performed HSV-specific LP and/or IFN-γ ELISPOT assays in 77 subjects seronegative for HSV-1 and HSV-2 (HSVneg), 22 (29%) of whom demonstrated a T cell response to HSV in either the HSV-specific LP (17 of 22, 85%), IFN-γ ELISPOT (17 of 20, 85%) or both T cell assays (12 of 22, 55%) (Table II). In contrast, 55 HSVneg subjects (71%) demonstrated no T cell responses to HSV in either T cell assay (data not shown; Figure 1B). Of the 22 IS subjects, 9 were identified from those screened in an HSV DNA vaccine study (11), 1 from a study designed to validate an ICS assay, and 12 identified as HSV-seronegative sexual partners of HSV-2 infected persons (Table II and III). The median age of the IS subjects was 36 years, 77% were male and they had a median of 8 lifetime sexual partners (range 1-30) (Table III). Information regarding the HSV serostatus of current sexual partners was available on 16 of the 22 IS subjects and of these, 13 had current sexual partners who were HSV-2 seropositive (Tables II and III).

TABLE II.

HSV-specific LP and IFN-γ ELISPOT in IS subjectsa

Subject Studyb HSV serology of current sexual partner HSV-specific LP responses #pos/#testedc IFN-γ ELISPOT responses #pos/#testedd HLA typing
A B DRB DQB
1 Trans. 1-/2+ 1/1 nde 01, 28 08, 35 nd nd
2 DNA 1+/2- 3/3 2/3 03, 31 44, 1524 11, 15 03, 06
3 Trans. 1+/2+ 1/3 0/2 01, 02 18, 37 11, 15 03, 06
4 DNA unknown 1/2 5/5 01, 11 08, 35 nd nd
5 DNA unknown 3/3 3/5 03, 30 4405, 39 11, 13 03
6 DNA unknown 1/3 4/4 01 08 nd nd
7 DNA 1+/2- 0/3 2/4 24, 31 07, 14 15 06
8 DNA unknown 2/2 nd 02 07, 44 04 3
9 DNA unknown 0/3 4/4 03, 74 35 nd nd
10 Trans. 1+/2+ 1/2 1/1 24, 30 13, 51 07, 15 02, 06
11 Trans. 1+/2+ 0/3 1/1 01, 68 35, 37 10, 11 05, 07
12 Trans. 1-/2+ 2/2 1/2 01, 03 37, 60 10, 13 05, 06
13 DNA 1-/2+ 3/3 1/3 01, 11 35, 51 04 03
14 Trans. 1-/2+ 0/1 1/1 02, 11 44, 56 01, 12 03, 05
15 Trans. 1+/2+ 1/2 0/3 32, 68 14, 51 13 02, 06
16 IS 1+/2+ 1/2 3/3 01, 24 44, 57 01, 04 03, 05
17 IS 1+/2+ 1/2 2/5 11, 29 07, 18 01, 15 05, 06
18 IS 1+/2- 1/2 1/1 11, 25 18, 38 11, 15 03, 06
19 IS 1-/2+ 3/3 3/5 01, 30 13, 37 01 05
20 IS 1-/2+ 3/3 0/2 02, 31 40, 55 04, 11 033
21 IS 1-/2+ 1/3 1/1 02, 24 15, 39 08, 13 04, 06
22 ICS val. unknown 0/1 1/1 nd nd nd nd
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a

HSV-specific LP and ELISPOT results are displayed for all 22 IS subjects. Shading means no response detected.

b

IS subjects were identified from those screened in a DNA vaccine study (DNA), a HSV-2 transmission study (Trans.), a study to identify IS subjects (IS), or a study to validate an ICS assay (ICS val.).

c

HSV-LP response considered “+” if dCPM >5,000 CPM.

d

ELISPOT was considered positive if subject had a positive response to at least one HSV-2 peptide pool.

e

nd, insufficient PBMC for testing.

Figure 1.

Figure 1

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(A) No evidence of HSV-1 or HSV-2 seroconversion in IS subjects. Sera drawn on study entry, day 0 (d0), or during the course of study from IS3, IS12 and IS19 were analyzed for the presence of antibodies to HSV-1 (1) and HSV-2 (2) by WB. Controls included sera from HSV-1+ (HSV-1), HSV-2+ (HSV-2), HSV-1+/2+ (HSV-1/2) and HSVneg (neg) subjects. (B) Response rates of IS and non-IS subjects to HSV-2 and control peptide pools. PBMC from 20 IS and 46 non-IS subjects were screened for IFN-γ ELISPOT responses using peptide pools from CMV, CEF, or 34 pools of overlapping peptides representing 16 different HSV-2 ORFs (Table I). The % of subjects with positive responses to the different antigens is displayed; for CMV responses, only CMV-seropositive subjects were included (n=6 for IS subjects and n=6 for non-IS subjects). (C) Breadth of HSV-2 peptide pool response in IS and HSV-2+ subjects. The percentage of IS subjects (n=20) or HSV-2+ subjects (n=40) who responded to 0-12 different HSV-2 peptide pools is displayed. (D) Diversity of HSV-2 peptide-specific responses in IS subjects. The response rates of IS subjects (n=20) to individual HSV-2 proteins is displayed. (E) Diversity of HSV-2 peptide-specific responses in HSV-2+ subjects. The response rates of HSV-2+ subjects (n=40) to individual HSV-2 proteins is displayed. *Peptide pools recognized more frequently in HSV-2+ subjects compared to IS subjects.

TABLE III.

Demographic Profile of HSV-seronegative subjectsa

HSV-seronegative
IS non-IS
Total subjects (n) 22 55
% of all HSV-seronegative subjects 29% 71%
Identified/enrolled in:
DNA vaccine study 9 (36%) 16 (64%)
Transmission Study 6 (37%) 10 (63%)
IS study 6 (35%) 11 (65%)
ICS Validation Study 1 (5%) 18 (95%)
median age (years) 36 (range 22-62) 34 (range 20-72)
gender:
female 5 (23%) 26 (47%)
male 17 (77%) 29 (53%)
race 21 white, 1 black 55 white
Previous/current partner with HSV: n=16 n=21
HSV-1 only 3 (19%) 3 (14%)
HSV-2 only 7 (44%) 12 (57%)
HSV-1 and HSV-2 6 (37%) 6 (29%)
No. of lifetime sexual partners 8 (range 1-30) 5 (range 1-30)
CMV serology 8/22 (36%) 7/35 (20%)
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a

HSV-seronegative subjects were classified as IS if they possessed HSV-specific LP and/or IFN-γ ELISPOT responses

HSV WB was performed on serum obtained from multiple blood draws from each IS subject (median 4 HSV WB/subject, range 2-8) over the course of study and all serum samples were negative for antibodies to HSV-1 and HSV-2. As illustrated in 3 representative HSV WB, sera from IS3, IS12 and IS19 lacked antibodies to HSV-1 and HSV-2 for up to 3 years after enrollment (Figure 1A).

T cell responses to HSV-2 in IS subjects

LP responses to HSV-2 antigen were detected in 17 of the 22 IS subjects (77%) from at least 1 blood draw: 5 (29%) of these subjects had positive LP responses to HSV-2 in 3 consecutive blood draws (Table II). Repeat LP assays using HSV-1 and HSV-2 antigens were performed in 12 of the IS subjects and 6 exhibited responses to both viruses while 6 had responses to HSV-2 only (data not shown).

Sufficient PBMC were available from 20 IS subjects to measure IFN-γ ELISPOT responses to 34 HSV-2 peptide pools representing 16 HSV-2 proteins (Table I). Of the 20 IS subjects, 17 (85%) had IFN-γ ELISPOT responses to at least 1 HSV-2 peptide pool including 12 IS subjects with HSV-2 LP responses (Table II). Five IS subjects had IFN-γ ELISPOT responses but no LP responses (IS7, IS9, IS11, IS14 and IS22), while 3 subjects had LP responses but no IFN-γ ELISPOT responses (IS3, IS15 and IS20). Two subjects (IS1 and IS8) had LP responses but sufficient PBMC to test by IFN-γ ELISPOT were not available. Thus, HSV-specific LP responses were detected in 77% of IS subjects tested, HSV-2 peptide-specific IFN-γ ELISPOT responses were detected in 85% of IS subjects tested, and 55% of IS subjects tested had positive responses measured by both assays (Table II). By definition, none of the 55 non-IS subjects possessed detectable LP responses to HSV antigens and PBMC from 46 non-IS subjects were tested by IFN-γ ELISPOT using all 34 peptide pools and none were positive (Figure 1B). IS and non-IS subjects had similar response rates to the CEF control peptide pool which were 70% and 67%, respectively. Positive IFN-γ ELISPOT responses to CMV were measured in 6 of 6 (100%) CMV-seropositive IS subjects and 5 of 6 (83%) CMV-seropositive non-IS subjects (Figure 1B).

IFN-γ ELISPOT responses to HSV-2 peptide pools in IS subjects: magnitude, diversity, and persistence

The magnitude of positive responses to HSV-2 peptide pools in IS subjects ranged from 55 spot-forming cells (SFC)/106 PBMC to 1,750 SFC/106 PBMC; the median SFC/106 PBMC was 130 SFC/106. This is similar to what we have observed in 40 HSV-2+ subjects including 14 HSV-2+ partners of the IS subjects where the median SFC/106 PBMC was 180 (range 55 to 1,821 SFC/106 PBMC). In terms of breadth of response, IS subjects recognized between 0 and 6 individual HSV-2 peptide pools (median response of 2 pools) (Figure 1C). This figure underestimates the number of individual epitopes recognized as many of the IS subjects responded to multiple epitopes within individual HSV-2 peptide pools (see below). HSV-2+ subjects tended to have a more diverse T cell response to HSV-2 and recognized between 0 and 12 individual HSV-2 peptide pools (median response of 3 HSV-2 peptide pools) (Figure 1C).

The most frequently recognized HSV-2 protein in IS subjects was UL39 (ICP6) which was recognized in 11 of 20 IS subjects tested (55%) (Figure 1D). Responses were also frequent to ICP4 (35%), ICP0 (25%), UL19 (15%) and UL29 (15%) (Figure 1D). We detected no responses to UL25, UL35, UL46 or UL11 (Figure 1D). These data are in contrast to what we observed in HSV-2+ subjects who had frequent responses to gD-2 (43%), UL46 (25%), UL49 (28%) and gB-2 (UL27) (20%) in addition to those proteins frequently recognized in IS subjects [UL39 (50%), ICP4 (38%) and ICP0 (40%)] (Figure 1E). These data suggest that the antigenic repertoire of T cells in IS subjects is skewed compared to HSV-2+ subjects and that IS subjects had more frequent T cell responses to HSV-2 IE proteins and infrequent T cell responses to virion components (tegument and glycoproteins).

The diversity and magnitude of HSV-2 peptide-specific responses was highly variable between individual IS subjects as displayed in Figure 2. Robust responses (>1,000 SFC/106 PBMC) were measured in 4 IS subjects (Figure 2 IS7, IS9, IS10 and IS11), while weak responses (<100 SFC/106 PBMC) were measured in 3 IS subjects [Figure 2 IS19, IS21 and IS22 (data not shown)]. Diverse responses were measured in 3 IS subjects: IS2 recognized 4 peptide pools, IS5 recognized 5 peptide pools and IS10 recognized 6 peptide pools (Figure 2). Responses to a single HSV-2 peptide pool were measured in 8 IS subjects [Figure 2 IS6, IS11, IS16, IS17, IS18, IS19, IS21 and IS22 (data not shown)] although additional HSV-2 peptide pools were positive in subsequent blood draws from 4 of these subjects (IS6, IS16, IS17 and IS19). Of the 20 IS subjects tested, 5 recognized more than 1 sub-pool for a single HSV-2 protein; IS9 and IS10 recognized at least 2 sub-pools of UL39, IS4 recognized 2 sub-pools of ICP4, and IS7 and IS10 recognized 2 sub-pools of ICP0 (Figure 2). No IFN-γ ELISPOT responses were measured in 3 IS subjects, IS3, IS15 and IS20, and no PBMC were available for assay in 2 IS subjects (IS1, IS8).

Figure 2. Diversity and magnitude of IFN-γ ELISPOT responses to HSV-2 peptide pools in individual IS subjects.

Figure 2

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PBMC from IS subjects were screened for IFN-γ ELISPOT responses to the 34 HSV-2 peptide pools and data from 16 IS subjects with positive responses are displayed. The individual IS subjects are displayed at the bottom right of each graph. Dashed lines represent the threshold for positivity (4x median of DMSO control wells and >55 SFC/106 PBMC).

IFN-γ ELISPOT analyses were performed on subsequent PBMC samples collected from 15 of the IS subjects (2 to 5 blood draws over a period of between 1 month and 5 years) and 4 HSV-2+ subjects (2 blood draws over a period of between 6 and 14 months). Five of the 15 IS subjects (33%) had positive IFN-γ ELISPOT responses in PBMC from all subsequent blood draws while 7 subjects had at least one additional PBMC collected with detectable responses (Table IV) . In 8 of the 12 IS subjects, responses to the same peptide pool were detected in more than 1 blood draw. Interestingly, the responses to the same peptide pools were of similar magnitude over time (with the exception of the ICP0-A pool in subject IS19 that decreased 12-fold over a 3-year period), and persisted from 27 days (UL39-B in IS16) to over 5 years (ICP4-D in IS5) (Table IV). Overall, 22 of the 32 positive responses to the HSV-2 peptide pools detected in the 12 IS subjects were detected in a single blood draw (Table IV) suggesting that many of the HSV-2 peptide-specific responses in IS subjects were transient and/or fell to frequency levels below the limit of detection of our IFN-γ ELISPOT assay. In support of the latter, we have isolated multiple T cell clones directed at multiple different HSV-2 peptide pools from blood draws from the 3 IS subjects where ex vivo IFN-γ ELISPOT assays were negative (IS3, IS15 and IS20). CD4 T cell clones directed at gB-2, UL39 and ICP0 were isolated from IS20, CD4 T cell clones directed at gB-2, gD-2 and UL19 were isolated from IS3 and CD4 T cell clones directed at ICP4 were isolated from IS15; CD8 T cell clones directed at ICP4 were isolated from IS3. The expansion of HSV-2 specific T cell clones from PBMC that tested negative for HSV-2 peptide-specific T cell responses by ex vivo IFN-γ ELISPOT suggests that HSV-2 peptide-specific T cells were present but below the limit of detection of the IFN-γ ELISPOT assay.

TABLE IV.

Longitudinal IFN-γ ELISPOT Responses in IS and HSV-2+ Subjectsa

Subject Study
Day		 # of SFC/106 PBMC to each of the following HSV-2 Peptide Pools:
gD-2 ICP0A ICP0B ICP22A ICP27B ICP4A ICP4C ICP4D UL39A UL39B UL39C UL19B UL19-C UL19D UL25B UL46B UL47A UL49 UL27A UL29B UL29C
IS2 0 215b 105 70 90
1,638 100
1,945
IS4 0 115
14 140
34 60 185
51 145
350 90 155
IS5 0
22 100 105 110 155
343
1,865 95
1,927 95
IS6 0 285
51 240
155 555
450 760 450
IS7 0 1,280 1,760
161
366 60
707
IS9 0 1,340 115 845 15
140 80
163 110
1,767 150
IS11 0 1,200
57 520
343 1,040
IS12 0 130 890 135
28
IS13 0
302 205
400
IS16 0 60
27 105
IS17 0 110
364
651 105
764
IS19 0 60
28 1,315 240 230 95
56
91
406
1,148 105 60
HSV-2+ A 0 119 74 329 154 96 179
358 129 286 291 169
HSV-2+ B 0 200 163
314 58
HSV-2+ C 0 275
440 61
HSV-2+ D 0 83 130
185 130
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a

PBMC were obtained from multiple blood draws from IS and HSV-2+ subjects and screened by IFN-γ ELISPOT using HSV-2 peptide pools. Only those HSV-2 peptide pools recognized by these subjects are displayed; no other HSV-2 peptide pools were positive in these 16 subjects. Blank cells represent a negative response to the peptide pool.

b

Shading denotes peptide pools positive from more than 1 blood draw in the same subject.

A similar pattern of T cell reactivity was observed in 4 HSV-2+ subjects; 3 of the 4 HSV-2+ subjects had positive responses to the same HSV-2 peptide pool in both blood draws (HSV-2+ subjects A, B, and C) that were of similar magnitude and of the 12 total positive responses in these 4 HSV-2+ subjects, 6 were detected in only 1 blood draw (Table IV).

Peptide deconvolution and phenotype of HSV-2 peptide-specific T cells from IS subjects

Sufficient PBMC were available from 11 IS subjects to determine the individual peptides recognized by the HSV-specific T cells. Table V lists the T cell peptides deduced by IFN-γ ELISPOT to contain epitopes recognized by HSV-specific T cells from the IS subjects. There were 4 peptides that were recognized in more than 1 IS subject; ICP4996-1010 was recognized in IS4 and IS5, ICP0636-650 and ICP0648-662 were both recognized in IS7, IS9 and IS10, and ICP4761-775 was recognized in IS9 and IS13 (Table V). Interestingly, IS9 and IS13 both expressed HLA B35 (Table III), suggesting that this is the restricting allele for ICP4761-775.

Table V.

HSV-2 peptides recognized by IS subjects

Subject
 ORF
 Peptide
 Sequence
 CD4/CD8
IS2 gD-2 gD-2245-259 LPRFIPENQRTVALY CD4
 
 gD-2
 gD-2272-286
 YTSTLLPPELSDTTN
 CD4
IS4 ICP4 ICP41001-1015 GLSCLLAALGNRLCG CD8
ICP4 ICP41009-1023a LGNRLCGPATAAWAG CD8
ICP4 ICP41109-1123 ARDLRRTVLASGRVF CD8
ICP4 ICP41161-1175 FGPDTLVPMSPREYR CD8
 
 ICP4
 ICP41169-1183
 MSPREYRRAVLPALD
 CD8
IS5 UL27 (gB-2) gB-2141-155 IAVVFKENIAPYKFK ndb
 
 ICP4
 ICP41009-1023
 LGNRLCGPATAAWAG
 CD8
IS6 UL49 UL4945-59 PMRARPRGEVRFLHY CD8
 
 UL29
 UL29993-1007
 GNDRVFQAGNWASLL
 nd
IS7 ICP0 ICP0156-170 IPLRNTCPLCNTPVA nd
ICP0 ICP0636-650 SASGAGERRETSLGP nd
 
 ICP0
 ICP0648-662
 LGPRAAAPRGPRKCA
 nd
IS9 ICP0 ICP0636-650 SASGAGERRETSLGP nd
ICP0 ICP0648-662 LGPRAAAPRGPRKCA nd
UL39 UL39309-323 PTVGTDPGYPVPLEL nd
UL39 UL391093-1107 TLPASTLVRLLVHAY nd
 
 ICP4
 ICP4777-791
 FQNQSLRPLLADTVA
 nd
IS10 ICP0 ICP0636-650 SASGAGERRETSLGP nd
ICP0 ICP0648-662 LGPRAAAPRGPRKCA nd
UL29 UL2989-103 HYSPSVYVFHGGRHL nd
 
 UL29
 UL2993-107
 SVYVFHGGRHLAPST
 nd
IS11
 UL19
 UL19785-800
 HNTQARAADAADDRPH
 CD8
IS12 ICP22 ICP2217-31 NHAPPSPPPGSRKRR nd
 
 UL39
 UL39109-123
 TAVVALGGTSGPSAT
 nd
IS13
 ICP4
 ICP4777-791
 FQNQSLRPLLADTVA
 nd
IS19 UL39 UL3917-31 SERQEPREPEVAPPG nd
UL39 UL3925-39 PEVAPPGGDHVFCRK nd
UL39 UL39309-317 PTVGTDPGY CD8
UL39 UL39313-327 TDPGYPVPLELTPEN nd
ICP0 ICP05-23 PGTSSRADPGPERPPRQTP nd
ICP0 ICP017-30 RPPRQTPGTQPAAPH nd
ICP0 ICP0123-142 VCAVCTDEIAPPLRCQSFP nd
ICP0 ICP0135-149 LRCQSFPCLHPFCIP nd
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a

Shaded cells indicate epitopes recognized in more than 1 IS subject.

b

nd: not determined due to lack of PBMC or response below level of detection of ICS assay.

In some cases, peptides were confirmed and the phenotypes of HSV-2 peptide-specific T cells were determined by ICS and flow cytometry by incubating PBMC with the peptides deduced in the IFN-γ ELISPOT assay. Figure 3 displays the histograms from 5 subjects where the HSV-2 peptides were confirmed by ICS and flow cytometry. IS11 had CD8 T cells that recognized UL19785-800 (Figure 3B), IS6 had CD8 T cells that recognized UL4945-59 (Figure 3D) and IS4 had CD8 T cells that recognized ICP41001-1015 (Figure 3F). From IS19 (HLA A1,30; B13,37), the 9-mer peptide UL39309-317 contained within UL39309-323 and predicted by the Immune Epitope Database (www.immuneepitope.org) as binding to HLA A30 was confirmed by ICS and flow cytometry as being a CD8 epitope which was recognized by 0.78% of all gated CD8 T cells (Figure 3H). The CD4 T cell epitope gD-2245-259 was confirmed by ICS and flow cytometry from IS2 (Figure 3J). The phenotypes of T cells responding to additional HSV-2 peptides are listed in Table V. Not all peptides could be confirmed by ICS and flow cytometry likely due to the lower sensitivity of the ICS assay compared to the IFN-γ ELISPOT.

Figure 3. Phenotype of HSV-2 peptide-specific T cell responses measured by ICS and flow cytometry.

Figure 3

Open in a new tab

PBMC from 5 IS subjects were incubated with HSV-2 peptides deduced from IFN-γ ELISPOT as T cell epitopes in order to confirm the peptides and determine the phenotype of the responding T cells. PBMC from IS11 were incubated with DMSO only (A) or peptide UL19785-800 (B), PBMC from IS6 were incubated with DMSO only (C) or peptide UL4945-59 (D), PBMC from IS4 were incubated with DMSO only (E) or peptide ICP41001-1015 (F), PBMC from IS19 were incubated with DMSO only (G) or peptide UL39309-317 (H), and PBMC from IS2 were incubated with DMSO only (I) or peptide gD-2245-259 (J). The percentage of CD8+/IFN-γ+ (A-H) or CD4+/IFN-γ+ (I and J) T cells is displayed within each plot.

DISCUSSION

Our study markedly extends the knowledge and solidifies the concept that there is a population of persistently HSV-seronegative adults who demonstrate consistently detectable T cell responses to HSV-2. Although we did not undertake a population-based approach, we did identify 22 subjects, or 29% of all subjects screened, who were seronegative for HSV-1 and HSV-2 by repeated WB analyses but possessed HSV-specific LP and IFN-γ ELISPOT responses. Over 1/3 of HSV-seronegative subjects enrolled into 2 different HSV-2 discordant couples studies were identified as IS suggesting that the IS phenotype is common among HSV-seronegative partners of HSV-2 infected persons.

We identified CD4 and CD8 T cell epitopes to multiple HSV-2 proteins that were present at similar frequencies in PBMC obtained from sequential blood draws spanning several years. Some IS subjects recognized a broad range of HSV-2 epitopes and some peptide-specific responses were present at high frequencies. Of the 16 HSV-2 ORFs used to screen for IFN-γ ELISPOT responses in the IS subjects, UL39 (ICP6) was the most commonly recognized HSV-2 protein and T cells directed at ICP6 were present in 55% of IS subjects followed by ICP4 (35%), ICP0 (25%), UL19 (15%) and UL29 (15%). No responses were measured to UL25, UL35, UL46 and UL11 and single positive responses in individual IS subjects were seen to gD-2, ICP22, ICP27, UL47, UL49, UL27 (gB-2) and US5. These data are in contrast to what we observed in 40 HSV-2+ subjects (including 14 HSV-2+ partners of the IS subjects); while responses were frequent to UL39 (ICP6), ICP4 and ICP0, responses were also frequent to HSV-2 glycoproteins (gB-2 and gD-2) and tegument proteins (UL46 and UL49). This pattern of T cell reactivity in HSV-2+ subjects is consistent a recent study from our group, the most comprehensive study of immunodominant CD8 epitopes to HSV-2 reported to date, which demonstrated that the highest frequencies of CD8 responses in HSV-2+ subjects (n=21) were directed at UL39, UL25, UL27, ICP0, UL46 and UL47 in decreasing order using a CD8 IFN-γ ELISPOT assay and peptide pools representing 48 HSV-2 ORFs (17). In a study characterizing T cell responses by IFN-γ ELISPOT responses to IE proteins in HSV-2+ subjects, CD8 responses were found to UL49, ICP0 and ICP4 but not to ICP27, ICP22 or gD-2, while CD4 responses were mostly directed at UL49, gD-2, ICP4 and ICP0 (18). While T cell epitopes were frequent to UL39 in the current IS study, a relative lack of T cell responses to gB-2, gD-2, UL46 and UL49 in IS subjects suggests that the antigenic repertoire of T cells in IS subjects is skewed compared to HSV-2+ subjects. Differential recognition of CD8 T cell epitopes has been described in HIV-exposed persistently seronegative subjects compared to HIV-infected subjects (22) although how these T cells provide enhanced resistance to these subjects is not clear. The skewing of the T cell response to HSV-2 in IS subjects compared to HSV-2+ subjects may be related to the differences in exposure to HSV antigens in the 2 different cohorts. The preponderance of T cell responses directed at IE proteins in IS subjects suggests that IS subjects have been exposed to replicating virus since IE proteins are the first proteins made during the virus infectious life cycle and are not present in infectious virions. T cells directed at IE proteins would be engaged early in the infectious life cycle and may be able to kill the virally-infected cell before the production of infectious progeny and thus advantageous to the host. If some of the IS subjects are infected with HSV-2 in the absence of seroconversion, the presence of T cells directed at IE proteins at the neural-epidermal junction would provide the quickest defense against the virus spreading to the periphery and may explain why we did not detect any HSV DNA at mucosal sites in IS subjects (4). Although HSV-2+ subjects possess T cells directed at IE proteins, it is possible that these T cells do not localize to the sites of HSV-2 reactivation and thus cannot contain the spread of infectious virus to the periphery. Studies to assess whether T cells directed at IE proteins are present at local sites of HSV-2 exposure in IS and HSV-2+ subjects are underway and may shed light on the role of these T cells in protection from HSV-2 .

Although HSV-specific LP responses, indicative of CD4 T cell responses, were detected in 77% of the IS subjects, we identified and confirmed only 2 CD4 T cell epitopes in a single IS subject (Figure 3J; Table V). In our original description of IS subjects, we identified several HSV-specific CD4 T cell clones in an IS subject that were directed at multiple epitopes including ones contained in UL21, UL29, UL46 and UL47 (4). Additionally, CD4 T cell responses to whole HSV-2 antigen were detectable by ICS in several IS subjects (4). These results suggest that unlike CD4 T cell responses to whole HSV-antigen, CD4 T cell responses to individual HSV-2 peptides in IS subjects were beneath of level of detection of the ICS assay and that T cell cloning will be required to expand the cells in order to characterize them. In support of this, analysis of CD4 T cell clones generated from PBMC from the 3 IS subjects with positive LP/negative IFN-γ ELISPOT responses to HSV-2 suggests that CD4 T cell responses directed at multiple T cell proteins (gB-2, gD-2, UL19, ICP0, ICP4 and UL39) were present in these subjects. These data also suggest that the frequencies and magnitudes of HSV-specific CD4 T cells are lower than observed in HSV-2 infected subjects where 84% of subjects possessed HSV-2 peptide-specific CD4 T cell responses as measured by ICS and flow cytometry (Laing and Corey, unpublished observations). In contrast, most HSV-2 T cell epitopes that we detected by ICS and flow cytometry in the IS subjects were recognized by CD8 T cells and in many cases, the frequencies of these individual peptide responses were of high magnitude (up to 0.78% of all gated CD8 T cells, Figure 3H).

While differences in antigenic recognition of T cells in resistant versus infected HIV populations have been observed as mentioned above, differences in other aspects of T cell immunity to HIV in exposed seronegative (ESN) subjects versus HIV-infected subjects have been reported. These include but are not limited to (1) a skewing of naïve and central memory cells (23), (2) secretion of mainly IL-2 from gag-specific T cells in ESN compared to mainly IFN-γ from gag-specific T cells in HIV-infected subjects (23), (3) an increase in late effectors and natural killer cells in ESN compared to HIV-infected subjects (23), (4) greater proliferative activity of CD4 T cells to p24 in ESN compared to HIV-infected subjects (24), (5) elevated levels of CD4 T cells and RANTES expression in the genital mucosa of HIV-resistant Kenyan commercial sex workers compared to HIV-infected CSW (25), to name a few. The impact of these differences on preventing HIV infection in the ESN however is not known and different mechanisms have been linked to different ESN cohorts. The IS cohort we have described represents a unique and a novel population in the HSV disease setting to assess potential immune mechanisms involved in HSV resistance; human clinical studies have previously focused on various innate and adaptive immune mechanisms related to the variability in the clinical course of HSV disease expression in HSV-infected subjects (16, 26-30) as opposed to mechanisms related to the resistance of HSV infection in HSV-seronegative subjects at risk of infection.

A possible explanation for the presence of HSV-specific T cell responses in the IS subjects is that these responses were primed by related or unrelated cross-reactive T cells such as those that have been observed between different strains of influenza virus or between unrelated viruses such as influenza virus and hepatitis C virus (HCV), EBV or HIV (reviewed in (31)). We feel that this mechanism is an unlikely explanation for most of the IS subjects we identified since the majority possessed T cells directed at multiple epitopes within HSV-2. This mechanism seems more probable in subjects with single HSV-2 epitope responses as we observed in 4 of the IS subjects and experiments to determine potential cross-reactive epitopes may shed light on a potential immune mechanism of resistance in these subjects.

In summary, IS subjects are common among HSV-seronegative sexual partners of HSV-infected individuals and most IS subjects possess frequent and persistent T cell responses to multiple HSV-2 antigens. This suggests that HSV-2 exposure at mucosal sites can result in the exclusive priming of HSV-2 specific T cell responses. Studying the quantitative and qualitative aspects of T cell immunity in these subjects may invoke new concepts for the correlates of protection against genital HSV-2 infection and the rational design of protective HSV-2 vaccines.

ACKNOWLEDGEMENTS

We thank Steven De Rosa and Helen Horton for expert advice on IFN-γ ELISPOT and ICS and flow cytometry. For excellent data management, we thank Stacy Selke. We thank Selin Caka for subject scheduling and James Reith and Elizabeth Morrigan for assistance with Institutional Review Board protocols. We thank Kerry Laing for supplying peptides to UL19, UL25, UL35, UL46, UL47, UL49, UL11, UL27, UL29 and US5. We are grateful to Anne Cent and Rosemary Obrigewitch for performing HSV and CMV serological assays.

Footnotes

1

This work is supported by National Institutes of Health Grants AI-049394, AI-030731 and AI-042528. Research support was provided by Pfizer.

3

Abbreviations used in this paper: IS, immune seronegative, HCV, hepatitis C virus, LP, lymphoproliferation, ICS, intracellular cytokine staining, HSVneg, seronegative to HSV-1 and HSV-2, ORF, open reading frame, IE, immediate-early, SFC, spot-forming cell, WB, Western blot, ESN, exposed seronegative.

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