asymptomatic human cd4 cytotoxic t-cell epitopes identified from

JOURNAL OF VIROLOGY, Dec. 2008, p. 11792–11802 Vol. 82, No. 230022-538X/08/$08.00�0 doi:10.1128/JVI.00692-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Asymptomatic Human CD4� Cytotoxic T-Cell Epitopes Identifiedfrom Herpes Simplex Virus Glycoprotein B�

Aziz Alami Chentoufi,1 Nicholas R. Binder,1 Noureddine Berka,2 Guillaume Durand,3 Alex Nguyen,1Ilham Bettahi,1 Bernard Maillere,3 and Lbachir BenMohamed1,4*

Laboratory of Cellular and Molecular Immunology, The Gavin S. Herbert Eye Institute, University of California Irvine, School ofMedicine, Irvine, California 92697-43751; Tissue Typing Laboratory, Calgary, Alberta T2L2K8, Canada2; CEA, iBiTecS,

Service d’Ingenierie Moleculaire des Proteines (SIMOPRO), Gif Sur Yvette F-91191, France3; and Center forImmunology, University of California Irvine, Irvine, California 92697-14504

Received 28 March 2008/Accepted 2 September 2008

The identification of “asymptomatic” (i.e., protective) epitopes recognized by T cells from herpes simplexvirus (HSV)-seropositive healthy individuals is a prerequisite for an effective vaccine. Using the PepScanepitope mapping strategy, a library of 179 potential peptide epitopes (15-mers overlapping by 10 amino acids)was identified from HSV type 1 (HSV-1) glycoprotein B (gB), an antigen that induces protective immunity inboth animal models and humans. Eighteen groups (G1 to G18) of 10 adjacent peptides each were first screenedfor T-cell antigenicity in 38 HSV-1-seropositive but HSV-2-seronegative individuals. Individual peptides withinthe two immunodominant groups (i.e., G4 and G14) were further screened with T cells from HLA-DR-genotyped and clinically defined symptomatic (n � 10) and asymptomatic (n � 10) HSV-1-seropositive healthyindividuals. Peptides gB161–175 and gB166–180 within G4 and gB661–675 within G14 recalled the strongestHLA-DR-dependent CD4� T-cell proliferation and gamma interferon production. gB166–180, gB661–675, andgB666–680 elicited ex vivo CD4� cytotoxic T cells (CTLs) that lysed autologous HSV-1- and vaccinia virus(expressing gB)-infected lymphoblastoid cell lines. Interestingly, gB166–180 and gB666–680 peptide epitopes werestrongly recognized by CD4� T cells from 10 of 10 asymptomatic patients but not by CD4� T cells from 10 of10 symptomatic patients (P < 0.0001; analysis of variance posttest). Inversely, CD4� T cells from symptomaticpatients preferentially recognized gB661–675 (P < 0.0001). Thus, we identified three previously unrecognizedCD4� CTL peptide epitopes in HSV-1 gB. Among these, gB166–180 and gB666–680 appear to be “asymptomatic”peptide epitopes and therefore should be considered in the design of future herpes vaccines.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) areubiquitous viruses that infect a majority of people worldwide(3, 22, 68). Shedding of reactivated HSV is estimated to occurat rates of 3 to 28% in adults who harbor latent HSV in theirsensory neurons (32, 66–68). However, the vast majority ofthese individuals do not experience recurrent herpetic diseaseand are designated “asymptomatic patients” (22, 40, 68). Incontrast, in some individuals (symptomatic patients), reactiva-tion of latent virus leads to induction of “pathogenic” HSV-specific CD4� and CD8� T cells (22, 68) and recurrent dis-ease, ranging from rare episodes occurring every 5 to 10 yearsto outbreaks occurring monthly or even more frequentlyamong a small proportion of subjects (22, 26, 60). Interestingly,for genital herpes, symptomatic and asymptomatic patientshave similar virus shedding rates (68). It is likely that the sameis true for ocular and oro-facial herpes, since shedding rates intears and saliva of asymptomatic individuals have been re-ported to be as high as 33.5% (22, 29, 40, 42). Latently infectedpatients are at risk of developing severe immunopathology,such as blinding herpetic stromal keratitis (HSK) (primarilydue to HSV-1), painful genital ulcerations (primarily due to

HSV-2), and in rare cases, fatal HSV encephalitis (reviewed inreference 72).

Considering the wealth of data addressing the role of T cellsin animal models, it is surprising how few reports explore theimmunologic basis of symptomatic and asymptomatic HSVinfections in humans. The HSV-1-specific CD4� T-cell re-sponses in the cornea are much more likely to cause pathologythan those in the genital tract or anus/buttocks region. Indeed,the involvement of CD4� T cells that produce Th1 cytokines(interleukin-2 [IL-2] and gamma interferon [IFN-�]) in HSKhas been established with the mouse model of ocular herpes,and corneal herpetic diseases can be abrogated by depletion ofCD4� T cells or neutralization of Th1 cytokines (24, 25, 50, 51,62, 63). HSV-specific CD4� T cells are key participants in thedevelopment of recurrent herpetic sores in symptomatic indi-viduals (14, 21, 39, 46, 47, 65, 75, 79). Paradoxically, HSV-specific CD4� T cells may also be important in controlling theseverity of genital herpes infection (1, 47, 49). CD4� T-cellinfiltrates appear to correlate with HSV clearance from muco-cutaneous genital lesions (38). Individuals with severe immu-nodeficiency involving a lack of CD4� T cells can have severerecurrent herpes with a longer duration of symptomatic lesions(17, 43, 57), thus supporting the thought that CD4� T cells areone of the main mediators of protective immunity againstrecurrent herpes (34, 47, 75, 76). Early in the development ofrecurrent lesions, HSV-1-specific IFN-�-producing CD4� Tcells that display a cytotoxic activity predominate in the mono-nuclear infiltrate surrounding HSV-1-infected epidermal cells,

* Corresponding author. Mailing address: Laboratory of Cellularand Molecular Immunology, University of California Irvine MedicalCenter, 101 The City Drive, Bldg. 55, Room 202, Orange, CA 92868.Phone: (714) 456-7371. Fax: (714) 456-5073. E-mail: [email protected].

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which strongly express HLA-DR (47). For many years, theassociation of HLA-DR haplotypes with susceptibility to bothlabial and genital herpes has been appreciated (27, 41, 44, 55).Among the HLA-DR alleles, DRB1*04, DRB1*07, DRB1*11,and DRB1*13 are most frequent in the populations studied(26, 35, 41, 55). DRB1*13 is frequently seen in symptomaticHSV infection, while DRB1*04 is most frequent in individualswith asymptomatic HSV infection (41). Obvious and immedi-ate consequences of variations in HLA-DR allele distributionare changes in functional epitopes presented to the host’sCD4� T cells (26, 27, 35, 41, 44, 47, 49, 55).

We hypothesize that the clinical spectrum of herpes, rangingfrom asymptomatic infection to frequently distressing out-breaks, may be reflected in T-cell recognition of different setsof epitopes of one or several HSV protein antigens (Ags).Thus, recognition by CD4� T cells of a set of viral epitopesdesignated “symptomatic” might be associated with severe im-munopathological diseases, while recognition of “asymptom-atic” epitopes might, in turn, lead to immunoprotection. How-ever, identification of HSV target Ags and the sets of epitopesrecognized by “asymptomatic” versus “symptomatic” CD4� Tcells (i.e., “protective” versus “pathogenic” T cells) is far frombeing completed. Due to the lack of an animal model with ahigh incidence of recurrent eye disease, it is currently notfeasible to demonstrate that “symptomatic” T-cell epitopes arepathogenic. However, if people with a history of severe recur-rent disease (i.e., symptomatic people) tend to develop T cellsthat recognize a subset of epitopes (i.e., symptomatic epitopes)that differ from those recognized by T cells from asymptomaticpeople (i.e., asymptomatic epitopes), it would be logical toexclude such symptomatic epitopes from vaccines on thegrounds that they may enhance rather than diminish recurrentdisease.

Results from a number of studies indicate that HSV glyco-protein B (gB), one of the major HSV Ags that produce pro-tective immunity in both animal models and humans (7, 10, 12,14, 19, 45, 61), is recognized by CD4� T cells from bothsymptomatic and asymptomatic HSV-seropositive humans (14,39, 45, 75–77). In the late 1980s and early 1990s, Zarling andcoworkers generated gB-specific CD4� T-cell clones fromHSV-2-seropositive severely symptomatic patients who had re-current HSV-2 genital infections approximately every 8 weeksand from HSV-1-seropositive symptomatic patients who suf-fered frequent recurrent oral lesions (75–77). gB-specificCD4� T cells have also been cloned from HSV-seropositivehealthy asymptomatic individuals (76, 77). Five of these CD4�

cytotoxic T-lymphocyte (CTL) clones lysed autologous HSV-1-or both HSV-1- and -2-infected lymphoblastoid cell lines(LCLs), and their cytotoxicity was restricted to HLA-DR mol-ecules (73, 76). In another study, HSV-specific CD4� CTLclones were recovered from recurrent herpes lesions of fivepatients (33, 38). In the late 1990s and early 2000s, Cunning-ham and coworkers reported that gB is a major target forIFN-�-producing CD4� CTLs recovered from skin lesions (47,49). Although the above studies suggest that gB is a target ofboth “symptomatic” and “asymptomatic” CD4� T cells, nospecific “symptomatic” or “asymptomatic” gB epitopes haveever been defined.

In this study, we hypothesize that different sets of HSV-1 gBepitopes might be recognized by CD4� T cells from symptom-

atic versus asymptomatic individuals. We used the PepScanpeptide library strategy and identified the following four pre-viously unrecognized CD4� T-cell peptide epitopes fromHSV-1 gB: gB161–175, gB166–180, gB661–675, and gB666–680.Among these, gB166–180, gB661–675, and gB666–680 were targetedby CD4� CTLs that lysed autologous HSV-1- and vacciniavirus (expressing gB [VVgB])-infected LCLs. Interestingly,gB166–180 and gB666–680 appeared to be recognized preferen-tially by CD4� T cells from HSV-1-seropositive healthy“asymptomatic” individuals, while gB661–675 appeared to berecognized preferentially by CD4� T cells from severely“symptomatic” individuals. We propose that an effective im-munotherapeutic herpes vaccine should exclude the potential“symptomatic” gB661–675 epitope.

MATERIALS AND METHODS

Study population. From August 2003 to October 2007, we screened 283HSV-1- and/or HSV-2-seropositive individuals. Among these, a cohort of 79immunocompetent individuals who were seropositive or seronegative for HSV-1and/or HSV-2, with an age range of 18 to 63 years (median, 31 years), wereenrolled in the present study. Figure 1 shows the characteristics of this studypopulation with respect to sex, age, HSV serology, HSV disease, and HLA-DRfrequency distribution. Forty-eight individuals were white, 31 were nonwhite(African, Asian, Hispanic, and others), 43 were females, and 36 were males. Allpatients were negative for human immunodeficiency virus and hepatitis B virusand had no history of immunodeficiency. Thirty-eight patients were HSV-1seropositive and HSV-2 seronegative, among which 28 patients were healthy andasymptomatic (no history of recurrent HSV disease). The other 10 patients weredefined as HSV-1-seropositive symptomatic patients who suffered from frequentand severe recurrent oral and/or oro-facial lesions, with 2 patients having hadwell-characterized HSK. Control individuals (n � 24) were seronegative for bothHSV-1 and HSV-2 and had no history of ocular HSK, genital lesions, or oro-facial herpes disease. All subjects were enrolled at the University of CaliforniaIrvine under an institutional review board-approved protocol. All subjects pro-vided written informed consent.

HSV-1 and HSV-2 seropositivity screening. The sera collected from 79 pa-tients were tested for HSV-1 and HSV-2 status by use of HerpeSelect immuno-globulin G1 (IgG1) and IgG2 HSV enzyme-linked immunosorbent assay(ELISA) kits (Focus Diagnostics, Cypress, CA) as previously described (18). Thesensitivities and specificities of these ELISAs were 91.2% (HSV-1) to 96.1%(HSV-2) and 92.3% (HSV-1) to 97% (HSV-2), respectively. Although theseassays generally give clear-cut results, in some instances the stereotyping was alsovalidated by Western blotting as previously described (53).

FIG. 1. Characteristics of the study population. A cohort of 79immunocompetent individuals were enrolled in the present study andwere characterized with respect to sex, race, age, HSV serology, andHSV disease (A). (B) The individuals were HLA-DR typed, and HLA-DRB1 allele frequency was calculated by the following formula: allelefrequency � n/N, where n is the number of samples positive for the Ag,and N is the total number of samples.

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PepScan peptide library screening and synthesis. gB from strain 17 (GenBankaccession number P10211) was used in this study. One hundred seventy-ninesynthetic peptides encompassing the entire sequence of HSV-1 gB (904 aminoacids [aa]) were produced by using the multipin DKP system (Mimotopes).Figure 2A and B show examples of the 10 overlapping peptides forming group 4(G4) and group 14 (G14). Peptides were synthesized as 15-mers overlapping by10 aa with adjacent peptides. Synthesis was performed using classical 9-fluorenyl-methoxy carbonyl/tert-butyl chemistry. After side chain deprotection with a mix-ture of trifluoroacetic acid, ethanedithiol, and anisole (38:1:1), the peptides werecleaved in ammonium bicarbonate buffer (0.1 M, pH 8.4) with 40% (vol/vol)acetonitrile via a cyclization mechanism, leaving a cyclo (lysylpropyl) moiety atthe C terminus. Finally, the peptides were freeze-dried and stored as a drypowder at �80°C. Working stock solutions of peptides were made at 1 mg/ml inphosphate-buffered saline–10% dimethyl sulfoxide and were stored at �20°C.

HLA-DR genotyping. Peripheral blood mononuclear cells (PBMCs) wereHLA typed genomically after isolation of their DNA by use of a Qiagen DNA kit.The amount of DNA obtained was quantified with a spectrophotometer. Olerupand Zetterquist (52) previously described the localization, sequences, lengths,and specificities of the sequence-specific primers used for typing the DRB1,DRB3, DRB4, and DRB5 alleles. HLA-DRB1 genotyping was performed usingDynal Reli SSO kits (Invitrogen). In brief, a target sequence of 272 bp in thesecond exon of the DRB1, DRB3, DRB4, and DRB5 genes was amplified byPCR. Genotyping of HLA-DRB1 alleles was carried out with a direct DNAprobe test that, after PCR, uses a nucleic acid hybridization method for thedifferentiation of over 210 distinct alleles. This was performed using 54 specificprobes immobilized on paper strips and was able to distinguish the polymorphicsequence motifs in the second exon of the DRB1, DRB3, DRB4, and DRB5genes. HLA-DRB1 allele frequency was calculated by the following formula:allele frequency � n/N, where n is the number of samples positive for the Ag andN is the total number of samples.

Peptide binding assays specific for HLA-DR molecules. HLA-DR moleculeswere immunopurified from homologous Epstein-Barr virus cell lines by affinitychromatography using the monomorphic monoclonal antibodies (MAbs) L243and B7/21 (64). Binding to HLA-DR molecules was assessed by a competitiveELISA using biotinylated gB peptides as previously reported (64). Unlabeledforms of nonherpesvirus peptides were used as references to assess the validityof each experiment. The sequences of these reference peptides and their 50%inhibitory concentrations (IC50s) are as follows: HA 306-318 (PKYVKQNTLKLAT) for DRB1*0101 (1 nM), DRB1*0401 (5 nM), and DRB1*1101 (11 nM),YKL (AAYAAAKAAALAA) for DRB1*0701 (10 nM), A3 152-166 (EAEQLRAYLDGTGVE) for DRB1*1501 (35 nM), MT 2-16 (AKTIAYDEEARRGLE) for DRB1*0301 (129 nM), and B1 21-36 (TERVRLVTRHIYNREE) forDRB1*1301 (52 nM).

PBMC preparation and enzyme-linked immunospot (ELISPOT) assay.PBMCs were isolated as we described previously (13). Approximately 175 ml ofdonor blood was drawn into a yellow-top Vacutainer tube (Becton Dickinson).The serum was isolated and centrifuged for 10 min at 800 � g. The PBMCs wereisolated by gradient centrifugation using leukocyte separation medium (Cellgro).

The cells were then washed in phosphate-buffered saline and resuspended incomplete culture medium. Aliquots of freshly isolated PBMCs were also cryo-preserved in liquid nitrogen for future testing.

T-cell stimulation was measured by IFN-� production in peptide-stimulatedPBMCs, using a BD-ELISpot IFN-� kit (BD-Pharmingen) as we previouslydescribed (13). Briefly, 3 � 105 PBMCs were stimulated in ELISPOT plates(Millipore) for 2 days with 10 �g/ml of pooled peptides (10 peptides per pool),10 �g/ml of an individual gB peptide, heat-inactivated HSV-1 (strain McKrae)(multiplicity of infection [MOI] of 5), or 1 �g/ml phytohemagglutinin (PHA) asa positive control. The plates were precoated with anti-human IFN-� capture Abin a humidified incubator at 37°C with 5% CO2. The spot-forming cells (SFCs)were developed as described by the manufacturer (BD-ELISpot IFN-� kit; BD-Pharmingen) and were counted using a stereoscopic microscope.

T-cell activation and proliferation. PBMCs were stained with carboxyfluores-cein succinimidyl ester (CFSE) (2 mM) and then incubated with individual gBpeptides (10 �g/ml) for 5 days. The cells were then washed and stained for CD4molecule expression. For HLA restriction, CFSE-labeled cells were stimulated inthe presence or absence of 10 �g/ml of anti-DR (L243; BD-Pharmingen, CA) oranti-DQ (SPV-L3; AbD Serotec, Oxford, United Kingdom) blocking Ab. Cyclingcells were then analyzed by flow cytometry, and their absolute number wascalculated using the following formula: (number of events in double-positivecells � number of events in gated lymphocytes)/total number of events acquired.To assess CD4� T-cell activation, PBMCs were stimulated with individual gBpeptides (10 �g/ml) for 24 h and then analyzed by flow cytometry for theexpression of the T-cell early activated marker CD69 after double staining withfluorescent anti-human CD69 and anti-human CD4 Abs.

Generation of gB peptide-specific CD4� T-cell lines. PBMC-derived CD4� Tcells were stimulated with mitomycin C-treated (50 �g/ml) autologous PBMCs(106/ml) incubated for 3 h with individual peptides (10 �g/ml). IL-2 (5 ng/ml;R&D Systems) was added to the culture every other day starting on day 3. Onday 7, cells were restimulated with mitomycin C-treated autologous individualpeptide-pulsed PBMCs (106/ml), and IL-7 (20 ng/ml) was added every other day.On day 14, cultures were expanded and restimulated as described above foranother week and then used as effector cells for CD107 assay.

Target cells. LCLs were generated as we recently described (13). Briefly, LCLswere derived by stimulating PBMCs with PHA (1 �g/ml) in RPMI 1640 plus 10%fetal calf serum (FCS) for 3 days. Half of the medium was then replaced withfresh medium containing IL-2 (10 ng/ml) and IL-7 (20 ng/ml). On day 6, theLCLs were infected overnight with VVgB, an empty vaccinia virus control(VVC), or HSV-1, each at an MOI of 10. The next day, infected LCLs werewashed three times and treated with mitomycin C (50 �g/ml) for 30 min. TheLCLs were then washed three more times before a 4-h incubation with effectorCD4� T cells at the indicated effector/target ratio. Cytotoxic activity was de-tected in a CD107a/b degranulation assay as we recently described (13). In orderto demonstrate whether the strong T-cell responses detected against syntheticpeptides gB161–175, gB166–180, gB661–675, and gB666–680 can be detected fromnaturally processed gB, PHA-stimulated blasts were infected with HSV-1, VVgB,

FIG. 2. The PepScan library was designed from the entire HSV-1 gB sequence (strain 17) (GenBank accession number P10211). (A) Sequencesof the 10 overlapping 15-mer gB peptides in G4. (B) Sequences of the 10 overlapping 15-mer gB peptides in G14.

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or VVC and used as target cells. The response to VVC was subtracted from theresponse to VVgB (not shown).

T-cell cytotoxicity assay. In order to detect cytolytic CD4� T cells recognizinggB peptides in freshly isolated PBMCs, we used the CD107a/b cytotoxicity assayas we recently described (13). On the day of the assay, nonstimulated or gBpeptide-stimulated PBMCs were incubated at 37°C for 5 to 6 h with BD-GolgiStop, costimulatory Abs anti-CD28 and anti-CD49d (1 �g/ml), and 10 �l ofCD107a-fluorescein isothiocyanate (CD107a-FITC) and CD107b-FITC. At theend of the incubation period, the cells were harvested into separate tubes,washed once with fluorescence-activated cell sorting (FACS) buffer, and thenstained with Cy-phycoerythrin-conjugated anti-human CD4 for 30 min. The cellswere then washed again and analyzed using a FACScan flow cytometer (BectonDickinson, San Diego, CA).

Statistical analysis. Data are expressed as means � standard errors. Statisticalsignificance was estimated by analysis of variance followed by Dunnett’s test toidentify differences between groups. Differences were considered significantwhen P values were �0.05. All P values were two-tailed unless stated otherwise.

RESULTS

Prevalence of gB epitope-specific IFN-�-producing CD4� Tcells in healthy HSV-seropositive patients. We first assessedthe ability of HSV-specific CD4� T cells from 38 HSV-1-seropositive, HSV-2-seronegative individuals to respond to aPepScan library of 179 gB peptides divided into 18 groups of 10peptides each (G1 to G18). In an IFN-� ELISPOT assay, weused fresh PBMC-derived CD4� T cells isolated directly exvivo to minimize artifacts that might arise from in vitro re-stimulation of CD4� T cells. A preliminary analysis of IFN-�-producing CD4� T cells revealed that 20 or more spots/106

CD4� T cells gave a 97% probability of defining a positiveresponse. Of the 540 separate ELISPOT wells used to screen20 HSV-seronegative individuals, only 16 wells (3%) exceeded20 SFCs/106 CD4� T cells. Of the 566 separate ELISPOTassays used to screen the 38 seropositive individuals, 96 (17%)had readings above 20 SFCs/106 CD4� T cells. A level of 20SFCs/106 CD4� T cells was therefore used as the cutoff pointfor subsequent analyses.

The number of IFN-�-producing CD4� T cells specific for

each group of gB peptides was determined for each individualand ranked as follows: strong responses, 100 SFCs/106

PBMCs); medium responses, 50 and �100 SFCs/106 PBMCs;low responses, 20 and �50 SFCs/106 PBMCs; and no re-sponse, �20 SFCs/106 PBMCs. Positive IFN-�-producingCD4� T-cell responses were compared with responses in con-trol wells without Ag. Figure 3 shows the frequency distribu-tion of CD4� T-cell responses induced by 18 groups of gBpeptides and heat-inactivated HSV-1 (McKrae) or PHA as apositive control. Based on these criteria, significant IFN-�-producing CD4� T-cell responses were detected for all butfive groups of gB peptides (G2, G10, G11, G16, and G18)(Fig. 3).

The highest frequency of IFN-�-producing CD4� T-cell re-sponses was detected against G4 (47.5%), while 42% of indi-viduals responded to G14 and G9. In comparison, only 5 to38% of individuals showed positive T-cell responses to theremaining 16 groups of peptides (G1, G2, G3, G5, G6, G7, G8,G10, G11, G12, G13, G15 G16, G17, and G18) (5%). Thehighest magnitude of response, against G4 and G14, was alsorecorded for 11% of HSV-1-seropositive individuals (Fig. 3).The lowest magnitude of response was detected against G18.All of the donors responded similarly to the PHA and heat-inactivated HSV-1 positive controls. The G6 group of peptidesappeared to be recognized by a large percentage of individualsand to stimulate the highest magnitude of T-cell responses.However, G6 induced a significantly smaller proportion ofresponses in the 100 SFC range than did G4 (5% versus11%). In addition, a smaller percentage of individuals re-sponded to G6 than to G4 and G14 (38% versus 47.5% and42%). Besides G6, the G9 group of peptides appeared to benext, after G4 and G14, to be recognized by a large percentageof individuals. However, G9 induced a smaller proportion ofresponses in the 100 SFC range than G4 and G14 did (0%versus 11%). In addition, although a majority of individuals

FIG. 3. Prevalence of gB peptide-specific T-cell responses in HSV-1-seropositive responders. Eighteen groups (pools of 10 adjacent peptidesper group) of peptides from the HSV-1 gB PepScan library were analyzed by ELISPOT assay for the capacity to elicit IFN-� production bystimulated CD4� T cells from 38 HSV-1-seropositive, HSV-2-seronegative individuals. The results represent the frequency distribution of gBpeptide-specific responders in the presence of 10 �g/ml of each group of gB peptides or with heat-inactivated HSV-1 (strain McKrae) (MOI �5) or PHA (1 �g/ml) as a positive control. The categories of immune response strength are as follows: no/low response, 0 to 20 SFCs; mediumresponse, 20 to 50 SFCs; high response, 50 to 100 SFCs; and strong response, 100 SFCs.



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responded to G9, their T-cell responses were at a low magni-tude (i.e., 37% of individuals responded in the range of 20 to50% SFCs) compared to the responses to G4 and G14 (31%and 15%). The borderline negative responses obtained withG6 and G9 were confirmed by the inability of individual pep-tides within G6 and G9 to stimulate significant CD4� T-cellresponses, based on both the magnitude and percentage ofresponders (not shown). Thus, both G6 and G9 do not containan immunodominant epitope and were therefore excludedfrom further studies. Altogether, these results indicate that G4and G14 might contain one or several immunodominant gBepitopes.

gB161–175, gB166–180, gB661–675, and gB666–680 are immuno-dominant peptide epitopes. To identify the immunodominantCD4� T-cell epitopes within G4 and G14, we analyzed theability of the 10 individual peptides forming each group toinduce HSV-specific CD4� T-cell responses in four HSV-se-ropositive individuals with different HLA-DR haplotypes.Both activation (expression of the CD69 early activationmarker) and proliferation (CFSE incorporation) were fol-lowed by FACS of gated CD4� T cells, as described in Mate-rials and Methods. Among the 10 peptides forming G4, pep-tides gB161–175 and gB166–180 induced the strongest CD4�

T-cell activation (Fig. 4A) and proliferation (Fig. 4B), withgB166–180 being the immunodominant peptide epitope withinthis group. In G14, peptides gB661–675 and gB666–680 inducedthe strongest CD4� T-cell activation (Fig. 4C) and prolifera-tion (Fig. 4D). Overall, T-cell responses induced by individualpeptides within G4 were higher than those induced by peptidesin G14. As a positive control, CD4� T-cell responses to PHAwere similar in all tested donors. Altogether, the results showthat peptides gB161–175 and gB166–180 among G4 peptides andpeptides gB661–675 and gB666–680 among G14 peptides are im-munodominant peptide epitopes. In addition, the results indi-cate that regardless of HLA-DR haplotype, gB161–175, gB166–180,gB661–675, and gB666–680 induced significant magnitudes ofCD4� T-cell activation (expression of the CD69 early activa-tion marker) and proliferation (CFSE incorporation), pointingto the promiscuity of these epitopes.

In order to depict the magnitude and frequency of CD4�

T-cell responses induced by each HSV-1 gB peptide, we em-ployed multiple immunological assays. Thus, to rank as animmunodominant peptide epitope, the peptide must inducestrong responses (i.e., T-cell activation, proliferation, CD69upregulation, and IFN-� production) in at least four differentdonors. Looking at these immunological criteria, gB686–700 did

FIG. 4. T-cell stimulation with individual gB peptides from the dominant groups (G4 and G14). (A and C) Induction of CD69 (early activationmarker) expression in gB peptide-specific CD4� T cells. (B and D) Absolute numbers of dividing CD4� T cells after 5 days of stimulation with10 �g/ml of individual peptides from groups G4 and G14.



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induce T-cell stimulation but failed to induce cell division (Fig.4B). Similarly, although gB176–190 induced a moderate prolif-eration of T cells, it also induced one of the lowest and leastsignificant T-cell stimulations (Fig. 4C). Therefore, both gB176–190

and gB686–700 peptides were ranked as negative peptides, andgB166–180 andgB666–680 were ranked as immunodominant pep-tide epitopes.

In vitro binding to soluble HLA-DR molecules confirmsgB161–175, gB166–180, gB661–675, and gB666–680 as strong peptideepitopes. The four immunodominant gB responder peptides(i.e., gB161–175, gB166–180, gB661–675, and gB666–680), as well asfour nonresponder control peptides (i.e., gB871–895, gB886–900,and gB890–904), were synthesized and tested in vitro for bindingto seven soluble HLA-DR molecules (Table 1). This panel ofavailable HLA-DR molecules consists of the most commonHLA class II haplotypes, regardless of ethnicity (78). The rel-

ative binding capacity (IC50 [nanomolar]) for each peptide wascalculated as the concentration of competitor peptide requiredto inhibit 50% of the binding of an allele-specific biotinylatedpeptide (indicator peptide), as described in Materials andMethods. Based on an upper threshold of 250 nM, whichcharacterizes high-affinity peptide binders (11, 78), gB161–175,

gB166–180, gB661–675, and gB666–680 bound to three or moredifferent HLA-DR molecules, while the remaining peptideepitopes did not bind to any of the HLA-DR molecules tested.This suggests that the gB161–175, gB166–180, gB661–675, andgB666–680 peptide epitopes contain at least one universal T-cellepitope or several overlapping epitopes presented by multipleHLA-DR molecules.

T-cell responses to peptides gB161–175, gB166–180, and gB661–675

are HLA-DR dependent. In order to ascertain that T-cell re-sponses to the four immunodominant gB peptides are

FIG. 5. CD4� T-cell stimulation with individual gB peptides from the dominant groups (G4 and G14) in the presence or absence of anti-DRblocking MAb. (A) Absolute numbers of dividing CD4� T cells stimulated for 5 days with 10 �g/ml of individual peptides gB161–175, gB166–180,gB661–675, and gB666–680 in the absence (solid bars) or presence (open bars) of anti-DR blocking Abs. (B) CD4� T cells from three HLA-DRB1-genotyped donors were incubated with 10 �g/ml of gB peptide in the absence (solid bars) or presence (open bars) of anti-DQ blocking MAb. Thegraph shows the means plus standard deviations of the absolute numbers of dividing CD4� T cells in the presence of the indicated gB peptidessubtracted from the value without peptide. The results are representative of three experiments (n � 3 patients).

TABLE 1. In vitro binding capacities of HSV-1 gB-derived epitope peptides for soluble HLA-DR moleculesa

PeptideHLA-DR binding capacity (IC50 nM�)b

DR1 (B1*0101) DR3 (B1*0301) DR4 (B1*0401) DR7 (B1*0701) DR11 (B1*1101) DR13 (B1*1302) DR15 (B1*1501)

gB161–175 98 0.3 0.4 4 85 1,925 0.4gB166–180 9,759 18 224 289 1,690 233 139gB661–675 5,333 5 45 9 254 1,925 2gB666–680 436 3 15 612 131 1,925 7gB871–895 9,759 774 1,826 1,021 1,690 1,925 283gB886–900 9,759 774 1,826 1,021 1,265 1,925 283gB890–904 9,759 774 1,826 1,021 1,690 1,925 283

a HSV-1 gB-derived peptides were subjected to ELISAs specific for HLA-DR molecules as described in Materials and Methods. Reference nonherpesvirus peptideswere used to validate each assay. Data are expressed as relative activities (ratio of the IC50 of the peptides to the IC50 of the reference peptide) and are the meansof two experiments. Peptide epitopes with high-affinity binding to HLA-DR molecules have IC50s below 250 and are shown in bold. “” indicates peptide epitopes thatfailed to bind to tested HLA-DR molecules.

b Population coverage was 8%, 6%, 21%, 15%, 12%, 14%, and 6% for DR1, DR3, DR4, DR7, DR11, DR13, and DR15, respectively.



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HLA-DR dependent, HLA blocking experiments were per-formed where antigen-presenting cell presentation of individ-ual peptides to CD4� T cells was assessed in the presence andabsence of MAbs specific to the monomorphic region ofHLA-DR or HLA-DQ molecules. Figure 5 shows that CD4�

T-cell proliferation induced by gB161–175, gB166–180, and gB661–675

was significantly (P � 0.005) abrogated in the presence of ananti-HLA-DR blocking MAb (4A) but not in the presence ofan anti-HLA-DQ MAb (4B). CD4� T-cell proliferation in-duced by gB666–680 was not affected by either anti-HLA-DRMAb or anti-HLA-DQ MAb. These results were confirmed forHSV-seropositive individuals with six different HLA-DRB1haplotypes. Altogether, these results show that T-cell re-sponses to peptides gB161–175, gB166–180, and gB661–675 areHLA-DR dependent.

gB166–180-, gB661–675-, and gB666–680-specific CD4� T cellsdisplay lytic activity. In order to detect whether CD4� T cellsspecific to the gB161–175, gB166–180, gB661–675, and gB666–680

peptides display cytotoxic activity, we used the CD107a/b de-granulation assay on freshly isolated T cells. Figure 6 showsthat peptides gB166–180, gB661–675, and gB666–680 were able toinduce significant degranulation/mobilization of CD107 on thesurfaces of specific CD4� T cells. gB166–180 induced the highestcytotoxic activity, suggesting that this peptide contains a dom-inant CTL epitope. gB161–175 was unable to elicit CD4� cyto-toxic T cells, suggesting a helper function of this epitope.

gB166–180, gB661–675, and gB666–680 peptide epitopes are nat-urally processed and presented from native gB. Because CD4�

CTLs were generated by in vitro stimulation with syntheticpeptides, it was of interest to ascertain whether they recog-nized processed HSV gB-derived native epitopes in addition tothe artificial synthetic peptide epitopes. Therefore, we ana-lyzed the ability of CD4� T-cell lines generated by gB161–175,gB166–180, gB661–675, and gB666–680 peptides to lyse HLA-DR-positive autologous HSV-1- and VVgB-infected LCLs. Figure7 shows that CD4� T-cell lines specific to gB166–180, gB661–675,

and gB666–680 peptides undergo significant CD107a/b degran-ulation when incubated with HLA-DR-positive autologous tar-get cells infected with VVgB or HSV-1 (strain McKrae) (P �0.005). As a control, no cytolytic activity was detected whengB166–180-, gB661–675-, and gB666–680-specific CD4� T-cell lineswere incubated with target cells infected with empty vacciniavirus (VVC) not expressing gB (not shown). gB161–175 wasunable to elicit CTL activity even after several rounds of T-cell-line expansion. Altogether, these results indicate that gB166–180,gB661–675, and gB666–680 are targets of CD4� CTLs that areable to recognize naturally processed gB epitopes.

T cells from asymptomatic and symptomatic patients rec-ognize different sets of gB peptide epitopes. Finally, we as-sessed whether CD4� T cells from symptomatic and asymp-tomatic patients recognized different sets of gB epitopes.gB166–180 and gB666–680 T-cell peptide epitopes were stronglyrecognized by CD4� T cells from 10 of 10 asymptomatic pa-tients but not by CD4� T cells from 10 of 10 symptomaticpatients (P � 0.0001; analysis of variance posttest) (Fig. 8).Conversely, CD4� T cells from symptomatic patients prefer-entially recognized gB661–675 (P � 0.0001) (Fig. 8). These find-ings greatly strengthen our hypothesis that T cells from symp-tomatic and asymptomatic patients recognize different sets ofherpesvirus epitopes. Interestingly, the “asymptomatic” gB166–180

and gB666–680 peptide epitopes appeared to be highly con-served within and between HSV-1 and HSV-2 strains (notshown) and showed high binding affinities for a range ofHLA-DR haplotypes (Table 1). Altogether, these results sug-gest that the “asymptomatic” gB166–180 and gB666–680 peptide

FIG. 6. CD107a and -b expression ex vivo by activated gB peptide-specific CD4� T cells. PBMCs from four different HLA-DR donorswere stimulated with the dominant gB peptides in the presence ofanti-CD28/49d, FITC-conjugated anti-CD107a and -b, and Golgi-Stopfor 6 h. All peptides were used at a final concentration of 10 �g/ml. Thegraph shows the means � standard deviations of the percentages ofCD107a/b� and CD4� T cells in the presence of gB peptides or PHAfor donors 1, 3, 7, and 8.

FIG. 7. CD4� T-cell lines generated by immunodominant CD4�

T-cell peptide epitopes recognized infected target cells expressing na-tive gB. gB161–175, gB166–180, gB661–675, and gB666–680 peptide epitope-specific CD4� T-cell lines were generated from HLA-DR-positiveindividuals, and their cytotoxic activity (CD107a/b degranulation) wastested in the presence or absence of target cells infected with HSV-1 orwith VVgB. Target cells that were either uninfected or infected withVVC were used as negative controls. Dotted bars represent T-cellresponses to VVgB-infected target cells after subtraction of the per-cent recognition of VVC-infected control cells. Black bars representT-cell responses to HSV-1-infected target cells after subtracting thepercent recognition of uninfected control cells.



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epitopes are strong candidates to be included in the design offuture herpes vaccines, while the “symptomatic” gB661–675 pep-tide epitope should be excluded.

DISCUSSION

This study identified four human CD4� T-cell peptideepitopes from HSV-1 gB. Among these peptide epitopes,gB166–180 and gB666–680 appeared to be most highly recognizedby CD4� T cells from asymptomatic patients and were there-fore designated “asymptomatic epitopes,” while gB661–675 ap-peared to be most highly recognized by CD4� T cells fromsymptomatic patients and was therefore designated a “symp-tomatic epitope”.

CD4� T-cell infiltrates appear to correlate with HSV clear-ance from mucocutaneous lesions (38). The target HSVprotein Ags and derived epitopes remain to be identified. Anumber of studies indicate that gB is a major CD4� T-celltarget Ag in HSV-seropositive humans (14, 39, 45, 46, 75–77).Our studies suggest that gB epitopes are targets of both “symp-tomatic” and “asymptomatic” CD4� T cells, thus supportingprevious studies by Zarling et al., Koelle et al., and Cunning-ham et al. (33, 37, 38, 47–49, 73–77) showing that gB is targetedby T cells from both symptomatic and asymptomatic individu-als. Recently, it was reported that CD4� T-cell responses toHSV-2 gG differ in symptomatic and asymptomatic HSV-2-infected individuals (9, 16, 58). Similarly, HSV-1-infectedsymptomatic but not asymptomatic individuals respond toICP8 and VP16 (59). Of the three identified CD4� cytotoxicgB peptide epitopes described in the current report, gB166–180

and gB666–680 were recognized mostly by T cells from asymp-tomatic patients, while gB661–675 was recognized mostly by Tcells from symptomatic patients. These findings strengthen ourhypothesis that T cells from symptomatic and asymptomaticpatients recognize different sets of herpesvirus peptideepitopes. To our knowledge, this is the first report to show adifference in the sets of epitopes recognized in a specific HSV

protein by CD4� cytotoxic T cells from HSV-1-infected asymp-tomatic compared to symptomatic individuals. Although thefirst goal of the present report was to investigate whetherdifferent and discrete sets of HSV-1 gB epitopes are recognizeby CD4� T cells from symptomatic versus asymptomatic indi-viduals, an HLA association of CD4� T-cell responses in symp-tomatic versus asymptomatic individuals is possible. Indeed,the set of viral epitopes recognized by CD4� T cells fromsymptomatic individuals (i.e., “symptomatic epitopes”) mightbe associated with particular HLA haplotypes, while the dif-ferent set of viral epitopes recognized by T cells from asymp-tomatic individuals (i.e., “asymptomatic epitopes”) might beassociated with different HLA haplotypes. Investigating a pos-sible association between HLA haplotypes and herpes diseasewould be useful to address in future studies, using a largercohort of symptomatic and asymptomatic individuals.

It must also be remembered that humans are not immuno-logically naive and often have a large number of memoryCD4� T-cell populations that can cross-react with, and maydisproportionately contribute to the responses to, other infec-tious pathogens. These cross-reactive T cells can become acti-vated and modulate the immune response and outcome ofsubsequent heterologous infections, a phenomenon termedheterologous immunity (30, 69, 70). Therefore, we cannot ex-clude that some of the “symptomatic” and “asymptomatic”HSV-specific CD4� T cells identified in this study are cross-reactive with self or other pathogen-derived epitopes. Suchscenarios have been reported recently for murine heterologousinfection and may be true for humans, as shown for CD4�

T-cell responses to cytomegalovirus and other pathogens (15,54, 69, 70). Shedding of reactivated HSV is estimated to occurat rates of 3 to 33.5% in adults who harbor latent HSV-1 intheir sensory neurons (32, 66–68). However, the vast majorityof these individuals do not experience recurrent herpetic dis-ease and are designated “asymptomatic patients” (22, 40, 68).In contrast, in some individuals (symptomatic patients) reacti-vation of latent virus leads to either an induction of ineffective

FIG. 8. IFN-�-producing CD4� T-cell responses to gB peptide epitopes in HSV-seropositive symptomatic and asymptomatic patients. CD4�

T cells isolated from HLA-DR-genotyped sex-matched symptomatic (n � 10) and asymptomatic (n � 10) patients were stimulated for 3 days with10 �g/ml of the indicated gB peptide. IFN-�-producing CD4� T-cell responses specific to each epitope were determined as described in the legendto Fig. 2. Each circle represents one symptomatic (solid circle) or one asymptomatic (open circle) patient. “PHA,” T cells stimulated with themitogen PHA (positive controls). The table shows the HLA-DR alleles of the symptomatic and asymptomatic individuals tested in this study.



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or “symptomatic” HSV-specific T-cell immunity or a greaterimmunopathological response (20, 22, 68). Recurrent diseaseranges from rare episodes occurring once every 5 to 10 years tooutbreaks occurring monthly or even more frequently among asmall proportion of subjects (22). Interestingly, for genital her-pes, symptomatic and asymptomatic patients have similar virusshedding rates (68). It is likely to be the same for ocularherpes, since shedding rates in tears of asymptomatic individ-uals have been reported to be as high as 33.5% (22, 29, 40, 42).

Identification of the T-cell-mediated immune mechanism(s)by which asymptomatic patients control herpes disease andsymptomatic patients do not, or at least the viral epitopesinvolved, is critical for the rational advance of herpes vaccinedevelopment. Among the multitude and complex mechanismsthat might be in play are the following. (i) There may bedifferences in precursor frequencies, proliferative capacities,and functional properties of “symptomatic” versus “asymptom-atic” epitope-specific T cells. Indeed, the T-cell repertoires ofindividuals with the same major histocompatibility complexrestriction elements can vary significantly because of “heterol-ogous immunity” and “private specificity” (69, 70). (ii) Thedifferential level of infiltration/homing into sites of infection,i.e., corneas, genital areas, and/or sensory ganglia of T cells,specific to “symptomatic” versus “asymptomatic” epitopes mayaffect viral production and disease (23, 28, 38). (iii) “Asymp-tomatic epitopes” might trigger proliferation of “protective” Tcells within the sites of infection, while “symptomatic” epitopesmight trigger “pathogenic” T cells. (iv) The “symptomatic”epitopes may direct T-cell responses away from those that arebest suited to clear the viral infection, with minimal pathogenicreaction. (v). An immunopathogenic T-cell response mightoccur through stimulating low-affinity oligoclonal responsesthat inhibit broad-based high-affinity T-cell responses to otherwell-presented epitopes, thus deviating protective responses todamaging responses. (vi) There may be differences in effectorT cells lingering after recent shedding and/or disease versusmemory T cells maintained in the absence of antigenic expo-sure. (vii) Finally, T-cell cross-reactivity with epitopes fromother viruses, within or outside the herpesvirus family, can alsoplay a role in protective heterologous immunity versus damag-ing heterologous immunopathology (31, 71). Regardless of themechanism(s), if symptomatic individuals tend to generate Tcells that recognize a discrete set of “symptomatic” epitopesthat differs from the set of “asymptomatic” epitopes, it wouldbe logical to exclude such “symptomatic” epitopes from futureherpes vaccines on the grounds that they may enhance ratherthan diminish recurrent herpes diseases.

Rather than using only predictive computational algorithmsto map CD4� epitopes on HSV-1 gB, we instead employed asystematic strategy, including PepScan library scanning of thewhole 904-aa gB sequence followed by multiple functionalimmunological assays, such as in vitro binding of gB peptidesto a panel of HLA-DR molecules, ex vivo IFN-� ELISPOTassay, CD107 expression, and CD4� T-cell activation and pro-liferation analysis. Using these multiple screens, we identifiedfour HSV-1 gB peptides, gB161–175, gB166–180, gB661–675, andgB666–680, that induced IFN-�-producing CD4� T cells. Amongthese peptides, gB166–180, gB661–675, and gB666–680 recalledCD4� T cells that displayed cytotoxic activity. However, theresults reported here do not imply that these are the only

human CD4� T-cell epitopes present on gB. Indeed, the 10-aaregions where these 15-mer peptides overlap might by them-selves contain “junctional epitopes.” Shortening or lengthen-ing a peptide by one or a few amino acids might sometimesresult in missing junctional epitopes that might be present inthe overlapping regions. Thus, to look for these “junctionalepitopes,” we expect to synthesize and test a large number ofpeptide sequences, and the results from these experiments willbe the subject of a future report.

A majority of the world’s human population is infected withHSV-1 and/or HSV-2 and would most likely benefit from ther-apeutic vaccination designed to boost HSV-specific cellularimmunity (36). Although a primary goal of this study was toidentify human “asymptomatic” CD4� CTL peptide epitopeson HSV-1 gB, the ultimate goal is to build the knowledge ofHSV T-cell epitopes required for the development of a totallysynthetic, self-adjuvanting lipopeptide human vaccine. We nowplan to construct lipopeptide candidate vaccines incorporatingthe CD4� T-cell epitopes (but excluding the “symptomatic”epitopes) identified in this study together with human CD8�

T-cell epitopes we recently described for gD (13) and otherstructural and regulatory herpesvirus proteins (unpublisheddata). Such a combination would produce CD4�-CD8� chi-meric lipopeptide candidate vaccines similar to those we re-cently described for mice against ocular and genital herpesvi-rus infection (79; X. Zhang, A. A. Chentoufi, M. Wu, Z. Zhu,D. Carpenter, A. B. Nesburn, S. L. Wechsler, and L. Ben-Mohamed, submitted for publication). More importantly, eachlipopeptide vaccine construct will contain promiscuous CD4�

epitopes identified in this study, thus covering a majority of thepopulation rather than just those having specific HLA-DRalleles.

Although the high degree of HLA polymorphism is oftenpointed out as a major hindrance to the use of high-affinityepitope-based vaccines, this constraint can be dealt withthrough the inclusion of multiple supertype-restricted epitopes,recognized in the context of diverse related HLA alleles, and bydesigning vaccines with higher epitope densities (5, 8, 56, 80).Thus, an effective vaccine could include multiple “asymptom-atic” T-cell epitopes present in diverse herpesvirus proteinAgs, chosen to represent at least the HLA-DR supertypes,known to provide recognition in up to 95% of the globalpopulation, regardless of race and ethnicity (2, 4, 6, 56). Hence,bearing in mind the particular properties that would be re-quired in a possible human vaccine, we have conceived studiesto identify HLA class II “promiscuous” T-cell epitopes in HSVprotein Ags targeted by CD4� T cells from “asymptomatic”individuals. These epitopes, along with similarly identifiedHLA class I supertype-restricted “asymptomatic” CD8� CTLepitopes, would provide the rationale needed to develop mul-tiepitope CD4-CD8 vaccines that are broadly recognized in themajority of outbred racial and ethnic populations.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants EY14900,EY15225, and EY16663 from the NIH, by The Discovery Eye Foun-dation, by The Henry L. Guenther Foundation, and by a Research toPrevent Blindness Challenge grant. L. BenMohamed is an RPB Spe-cial Award Investigator.



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We thank Dennis Zaller (Merck & Co., Inc.) for providing theHLA-DR transgenic mice and David Koelle (University of Washing-ton) for providing the vaccinia viruses used in this study.

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