Extensive CD4 and CD8 T Cell Cross-Reactivity between Alphaherpesviruses

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The epidemiology of infections with members of the Alphaherpesvirinae subfamily is geographically and temporally complex, showing variation between regions and over time. Close to 100% of the United States population is seropositive for VZV as a result of infection or vaccination. Since commencement of universal vaccination with attenuated VZV in 1995 (1), the relative proportion of persons with natural and vaccine-induced VZV immunity is shifting, with uncertain consequences for VZV transmission and recurrence. The age-specific incidence of recurrent varicella infection (zoster) is increasing in the United States (2). Pediatric varicella vaccination is not practiced in most countries, where primary varicella remains ubiquitous (1). Seronegative adults remain susceptible to primary varicella; curiously, VZV seropositivity among adults is considerably <100% in some areas near the equator (3). Conversely, HSV seroprevalence is higher in some equatorial regions (4) than in the United States. Among United States adults aged 14 to 45 y, 50% are infected with HSV-1, and 16% are infected with HSV-2. As with VZV, HSV infection and resulting seroconversion are thought to be permanent as a result of latent infection of neural ganglia. Modest decreases have occurred in the age-specific prevalence of HSV-1 over recent decades (5). Reflecting this, more individuals are commencing sexual activity while seronegative for HSV-1. Indeed, HSV-1 accounts for the majority of clinical first-episode genital herpes both in the United States and in Northern Europe (6).

The immune-boost hypothesis of Hope-Simpson (7) suggests that periodic re-exposure to wild-type VZV stimulates favorable immune responses that inhibit zoster. These antigenic encounters may be decreasing as an unintended consequence of pediatric vaccination (7, 8). However, the causal link between varicella vaccination and zoster is controversial (9). The relative order of acquisition of immunity to HSV-1 and VZV is likely heterogeneous within populations. Varicella vaccine, where used, is recommended at 12–15 mo of age. HSV-1 seroprevalence also increases rapidly during the first few years of life. Overall, infection and vaccination patterns with HSV-1, HSV-2, and VZV vary with location and age group and are changing dynamically within communities, creating a complex pattern within which diverse immune interactions may operate to modulate the clinical manifestations of these infections.

Given that HSV and VZV have 65 homologous genes (10), it is rational that immunity related to VZV infection or vaccination could exert heterologous effects on HSV-1 or HSV-2 infection and vice versa. Boosting of Ab levels to HSV by VZV infection, and the reciprocal, occur in primary and recurrent infection (11–13), but far less is known about T cell responses. Our group observed T cell reactivity to HSV in HSV-1/HSV-2–seronegative persons. This could be due to VZV cross-reactivity, although a limited number of HSV-2–reactive CD4 clones did not exhibit this property (14, 15). This report focuses on T cell cross-reactivity to structurally related, sequence-homologous peptides. More broadly, T cell cross-reactivity includes recognition of unrelated peptides, in the context of either the index or unrelated MHC molecules, and is now thought to underlie minor histocompatibility Ag graft rejection, HLA-linked drug hypersensitivity, and, possibly, heterologous immunity effects between unrelated organisms. The T cell repertoire seems to be less diverse than the non-self peptide set, requiring ubiquitous cross-reactivity to minimize gaps in non-self recognition.

Zoster is the target of the only licensed therapeutic vaccine. This attenuated varicella strain modestly boosts serum Ab and VZV-specific CD4 T cells (16). It is thought to work via T cells, because shingles risk correlates with HLA-region single-nucleotide polymorphisms (17) and with age-related declines in VZV-specific CD4 T cells (18). The apparent correlation of efficacy with Ab responses could reflect CD4 T cell help (19). A recent study found that only one in eight (12.5%) persons had a vaccine-related CD8 T cell boost (20). Indeed, very little is known overall about the CD8 response to VZV. CD8 T cell lysis of VZV-infected cells has never been demonstrated. Concerns about the live vaccine’s safety in immune-compromised persons, its limited, 50–70% efficacy (21, 22), and the identification of CD4 T cell responses to VZV glycoprotein E (open reading frame [ORF]68 = ORF68 product) have motivated development of an ORF68 subunit vaccine in a novel adjuvant platform. This vaccine stimulates high CD4/Ab responses and appears very efficacious (23). Cross-reactivity between glycoprotein E or other ORFs shared between VZV and HSV might inform extension of this subunit vaccine format to HSV immunotherapy.

We developed proteome-wide tools to study CD8 and CD4 T cell responses to every protein in each alphaherpesvirus that infects humans. Responder cells are prepared by enriching and expanding memory virus-specific polyclonal T cell lines that contain multiple epitope-specific populations. In this article, we extend previous virus-specific reports in which we discovered many viral T cell Ags and epitopes (16, 24–26) to delineate and measure T cell cross-reactivity across the Alphaherpesvirinae subfamily.

Participants were healthy adults. Some were studied previously (Supplemental Table I). HSV-1 immunity was studied in participants 21 and 23 (25, 27); zoster vaccine immunity was studied in participants 1, 2, 3, 4, and 26 (16); T cell responses to HSV-2 was studied in subjects 30–34 (15); and drug reactions were studied in subject 35 (28). Subjects 6–19 and 26–29 were genetically confirmed HSV-1–infected monozygotic twins (29, 30), with consecutive numbers denoting twin pairs. PBMCs were cryopreserved. VZV (16) and HSV-1 and HSV-2 IgGs were detected as reported (16, 31).

Cell lines were Mycoplasma negative. VZV isolated from zoster was expanded in human embryonic tonsil (HET). Trypsin-recovered fibroblasts were cryopreserved for a cell-associated stock of wild-type VZV. VZV strain OKA from Zostavax (Merck, Kenilworth, NJ) was cryopreserved as infected cells and used to make UV cell-associated whole-VZV Ag (16). To create a PCR template for truncation analyses, VZV DNA was prepared as described (25). HSV-1 17⁺ and HSV-2 186 Ags and mock Vero (for HSV) and HET Ags were similarly prepared (32). HSV-1, HSV-2, and VZV ORFeome-covering proteins were described previously (16, 24, 26). For CD4 epitope mapping, regions of VZV ORF24 and ORF68 were cloned into pDEST203 (25) (primers available on request). Proteins were expressed by in vitro transcription translation (IVTT) (16). For CD8 assays, VZV (16), HSV-1 (25), and HSV-2 (24) ORFs in pDONR207 or pDONR221 (Invitrogen, Carlsbad, CA) were moved to pDEST103 (25). In-frame fusion to GFP was sequence confirmed. New or previously obtained (15, 16, 33) peptides (Sigma, St. Louis, MO; GenScript, Piscataway, NJ) were dissolved in DMSO. Peptide/primers were from GenBank: HSV-1, JN555585.1; HSV-2, JN561323.1; and VZV, NC_001348.1. HLA-A*0201–restricted peptide recognized by CMV-specific TCR was NLVPMVATV (34).

Stimulation of PBMCs with whole VZV and expansion with IL-2 to create polyclonal VZV-reactive cell lines were described (16). For some subjects, we stimulated PBMCs for 18 h with whole UV-treated VZV (1:100). Live CD3⁺CD4⁺ lymphocytes were sorted into CD137^low and CD137^high populations and expanded once with PHA and then once with anti-CD3 mAb (25). HSV-1–reactive polyclonal CD4 T cell lines were created using CD137 (25). CD4 T cell clones from persons who are seronegative for HSV-1 and HSV-2 were described (14, 15).

HSV-1–reactive polyclonal CD8 T cell lines were created as described (25). Briefly, HSV-1–infected HeLa cells were added to myeloid dendritic cells, which were coincubated with autologous CD8⁺ PBMCs. After 20 h, CD3⁺CD8⁺CD137^high cells were sorted and polyclonally expanded. For HSV-2, a similar workflow used an HSV-2 UL41–deletion mutant. For polyclonal peptide-specific CD8 T cell lines or clones, PBMCs or the polyclonal CD8 T cell lines were stained with PE-labeled tetramers of HLA-A*0201 and either HSV-1 UL25 372-380 or HSV-1 UL40 184-192 (35). CD8⁺tetramer^high cells were expanded polyclonally or plated in limited dilution and expanded with PHA and then anti-CD3 (25).

V-CDR3 regions of TCRA and TCRB mRNA from T cell clones were sequenced using 5′ RACE (Clontech, Mountain View, CA) (36). TCR expression plasmid was created by placing TCRA/TCRB V-CDR3 (GenBank KT955849/KT955850) into pMP71flex (34). An extracellular murine TCRB-C epitope allows detection of transduced cells with anti-TCRB (H57-597; eBioscience, San Diego, CA). TCR cassettes were inserted into pRRL.PPT.MP.GFPpre (37). Lentiviral stocks were created by cotransfection of HEK293 and used to transduce anti-CD3/anti-CD28–stimulated (Dynal, Grand Island, NY) CD8 PBMCs, which were expanded using anti-CD3 (38). For bulk CDR3 sequencing, 10⁶ polyclonal HSV-2–reactive CD8 T cells or 3 × 10³ tetramer-sorted cells were submitted to Adaptive Biotechnologies (Seattle, WA) for TCRB clonoSEQ (39).

PBMCs were stimulated with UV-treated VZV Ag, media, or mock control and analyzed for CD4 T cell expression of IFN-γ and IL-2 (40). To test expanded CD4 T cell lines, equal numbers of responders and CFSE-labeled autologous PBMCs used as APCs were added to test stimuli for 18 h (16). Live CFSE⁻CD3⁺CD4⁺ cells were analyzed for IFN-γ and IL-2 (25). Analyses were performed using a FACSCanto II (Becton Dickinson, Franklin Lakes, NJ) and FlowJo software (TreeStar, Ashland, OR). Proteome-spanning proliferation assays admixed 5–10 × 10⁴ polyclonal expanded CD4 T cells with Ag and an equal number of gamma-irradiated autologous PBMCs in 96-well U-bottom plates (in duplicate for recombinant Ags, in triplicate for whole viral Ags) (35), measuring incorporation of [³H]thymidine (cpm) at 72 h (32). Results are expressed as net cpm = mean cpm for experimental Ag − mean cpm for negative control Ag or as stimulation index (SI) = mean cpm for experimental Ag/mean cpm for control Ag. Ags were UV-treated VZV, HSV-1, or HSV-2 strain 186 or mock preparations (1:100), PHA (1.6 μg/ml; Remel, Kansas City, KS), or IVTT proteins (1:1000–1:2000). To set the cutoff, cpm values from 27 IVTT-expressed Plasmodium falciparum proteins were used (16). Other assays used 1 μg/ml peptide in triplicate with 0.1% DMSO control. For studies of HSV-2–reactive CD4 T cell clones from HSV-1/HSV-2–seronegative persons (14, 15), ORF-level specificity was assigned using responder clones seeded at 2 × 10⁴ cells/well in duplicate with 1 × 10⁵ autologous, gamma-irradiated PBMCs/well as APCs, and each HSV-2 protein (24) at 1:2000 (24) using [³H]thymidine incorporation as the readout at 72 h. Alternatively, HSV-2 peptide specificity was determined using a peptide set covering a portion of the HSV-2 proteome (15). Subsequent assays to study VZV cross-reactivity used responder cells and APCs, as above, with whole HSV-2 and VZV Ags, selected IVTT-synthesized HSV-2 and VZV proteins, or 1 μg/ml peptide, with relevant negative controls.

For whole virus, killing assays used dermal fibroblasts (DFBs) infected by coculture (3:1) with VZV-infected HET. After several coculture passages (3:1) of uninfected DFB (UN-DFB) with infected DFB (IN-DFB), IN-DFB were used at >50% cytopathic effect, typically 3 d after passage. eGFP-VZV showed fluorescent viral infection well outside of visible plaques. Killing was measured by VITAL-FR (41) using UN-DFB and IN-DFB labeled with 5 μM Far Red and 10 μM CFSE (Invitrogen), respectively. DFBs (infected or uninfected in separate wells) were replated in 24-well plates in triplicate (5 × 10⁵/well), and HSV-1–reactive CD8 T cell effectors (1 × 10⁶/well) were added 4 h later. After 24 h, the trypsin-recovered contents of one uninfected and one infected DFB well were paired, mixed, and fixed, and dye-labeled cells were counted by flow cytometry. For each tube, the ratio R between the percentage of IN-DFB (green) and the percentage of UN-DFB (red) was calculated. Killing was calculated as (R [no T cells added] − R [T cells added])/R (no T cells added). For each E:T combination, we report the mean and SD of three calculated values derived from six pairs/12 wells. To test cytokine synthesis, HSV-1–reactive CD8 T cell effectors were coincubated with autologous or HLA-mismatched EBV–lymphocyte continuous line (LCL) or DFB. LCLs were infected with HSV-1 strain E-115 at a multiplicity of infection of 10 for 24 h, and DFBs were infected with vOKA, as above. APC and responder cells were plated (2.5 × 10⁵/well of each cell type) in 96-well U-bottom wells when EBV-LCL were used as APC, or in 48-well plates when DFB were used as APC. Cells were stained with Violet LIVE/DEAD and Abs to CD8, CD3, IL-2 and IFN-γ, fixed, and analyzed by flow cytometry (25).

To test processing/presentation of full-length VZV ORFs, Cos-7 cells were cotransfected with HLA cDNA and HSV-1, HSV-2, or VZV ORFs cloned into pDEST103 to create artificial APCs, coincubated with CD8 T cell lines, and assayed for IFN-γ secretion (25). In a modification used for Ag discovery, polyclonal HSV-2–reactive CD8 T cells were screened with Cos-7 cells cotransfected with HLA-B*1502 cDNA and each HSV-2 ORF, as described (25).

To test recognition of peptides, polyclonally expanded CD8 T cell lines (see above) were incubated with autologous LCL and 1 μg/ml peptide and processed for intracellular cytokine staining (ICS) to detect IFN-γ accumulation (25). To screen candidate peptide-reactive CD8 T cells, responders (one quarter of a clonal microculture, or 5 × 10⁴ polyclonal cells) were coincubated for 48 h with an equal number of HLA-appropriate EBV-LCLs and 1 μg/ml peptide, followed by supernatant IFN-γ ELISA. Tests of cloned TCRs used 1 × 10⁵ each lentivirally transduced CD8 T cells and EBV-LCL/well and 1 μg/ml peptide.

For [³H] assays with ORFeome panels, a 1% false positivity threshold was set as cpm > median plus 2.3*median absolute deviation (MAD). MAD = 1.4826*median(abs[qi − mq]), where qi is the log₁₀ for each control well, and mq is the median of control quantities. The threshold for positivity is mq + Z*MAD, where Z is the standard quantile, such that the probability of exceeding abs(Z) is the false positive rate: p(qi > mq + Z*MAD) = p(false positivity) (42, 43). The control quantities were derived from a panel of 27 P. falciparum proteins run alongside the viral ORFeomes within each assay. T cell clonality indices were estimated as described (44, 45). CD4 T cell cross-reactivity to HSV-1 and VZV ORFs were analyzed using InStat 3.10 (GraphPad, La Jolla, CA).

Investigations conformed to Declaration of Helsinki principles and were approved by the relevant institutional review boards. Informed written consent was received from participants prior to inclusion in the study.

The abundance of VZV-reactive CD4 T cells in blood is <0.2%, even after boosting with attenuated VZV, whereas HSV-specific CD4 T cells are more abundant, in the 0.2–3.0% range (16, 40). This may reflect more frequent HSV reactivation. We created VZV-reactive CD4 T cell lines before and after VZV vaccination (16). These cells showed brisk IFN-γ/IL-2 responses to HSV-1, in addition to VZV, whether obtained before or after VZV vaccination and regardless of the presence or absence of HSV infection (Fig. 1). The proportion of VZV-reactive CD4 T cells that showed cross-reactivity toward HSV-1 ranged from 18 to 100%, as judged by the ratio between reactivity to the two whole-virus preparations. To explore reciprocal cross-reactivity, we created HSV-1–reactive CD4 T cell lines from seven pairs of HSV-1–seropositive, HSV-2–seronegative monozygotic twins (participant characteristics are listed in Supplemental Table I) using HSV-1 restimulation and CD137 selection (25). These cell lines had positive HSV-1 SIs (>5.0 using [³H]thymidine-proliferation assays) for 13 of 14 donors (93%). Among these, 12 of 13 (92%) were also positive for VZV, with a good correlation for mean cpm values between viruses (R² = +0.48). We also compared SI and net cpm values for HSV-1 and VZV whole-viral Ags as very rough estimates of CD4 T cell cross-reactivity. The mean value of the SI for VZV was 42% of that for HSV-1, whereas the mean δ cpm value for VZV was 47% of that for HSV-1.

After showing cross-reactivity to whole-viral Ags, we tested polyclonal virus-reactive T cells for responses to proteome-covering recombinant protein sets. Polyclonal CD4 HSV-1– or VZV-reactive cell lines were obtained with or without using activation marker CD137 expression (25, 27). From a participant seropositive for HSV-1, HSV-2, and VZV (subject 5), VZV-reactive CD4 T cells were rare (0.1–0.2%) by direct ex vivo cytokine staining and CD137 analyses (Fig. 2A). However, bulk-expanded CD3⁺CD4⁺CD137^high cells were enriched ∼100-fold for VZV reactivity (Fig. 2B).

The alphaherpesvirus protein sets (16, 24, 25) include 77 HSV-1 and HSV-2 and 70 VZV ORFs. They drive CD4 T cell proliferative and cytokine responses at 1:1,000–1:36,000 dilutions with autologous PBMCs as APCs (16, 24–27). Polyclonal VZV-driven CD137^high CD4 T cells show proliferation to many VZV proteins (Fig. 2C). Among the eight VZV proteins stimulating the strongest proliferative responses in this subject, responses to five HSV proteins (UL40, homolog of VZV ORF18; US8, homolog of VZV ORF68; UL39, homolog of VZV ORF 19; UL29, homolog of VZV ORF 29; and UL23, homolog of VZV ORF 36) were also detected (Fig. 2C). Additional cross-reactive and VZV-only responses were noted (not labeled in Fig. 2C to preserve readability). In contrast, although responses to VZV ORFs 8, 50, and 67 were positive, the HSV-1 and HSV-2 homologs did not stimulate proliferation. No instances of reactivity to HSV-1 or HSV-2 proteins were noted for HSV proteins that were homologs of nonreactive VZV proteins. Overall, among the 30/70 (43%) VZV proteins that stimulated CD4 responses for this donor, HSV-1 cross-reactivity was noted for eight proteins (27%).

Reciprocal cross-reactivity by HSV-1–reactive CD4 T cells toward their VZV homolog was measured in HSV-1–reactive polyclonal cell lines enriched from monozygotic twins (n = 18) (subjects 6–19 and 26–29, Supplemental Table I) (Fig. 2D). In aggregate, among the 18 donors and 65 proteins with homologs in HSV-1 and VZV, HSV-driven cell lines recognized HSV-1 proteins on 322 occasions. Among these, cross-reactivity to both the HSV-1 and VZV protein was noted 33 times (Fig. 2D). Thus, 10.2% of the Ag-level CD4 responses to HSV-1 in this population were cross-reactive toward VZV. Among the 65 proteins with homologs in HSV-1 and VZV (upper portion of Fig. 2D), 13 (20%) showed shared recognition for the HSV-1 and VZV homologs for at least one participant. There was an apparent relationship between the breadth of potentially cross-reactive HSV-1 Ags recognized and the presence of VZV cross-reactivity. Among the 14 persons with cross-reactivity to at least one VZV protein, the mean (± SD) number of HSV-1 ORFs recognized was 14 ± 8.3, whereas among the 4 persons without a positive response to a discrete VZV protein, the values were 9.2 ± 3.1 (p = 0.025, two-tailed Mann–Whitney U test). However, among the 14 persons with CD4 cross-reactivity to at least one VZV protein, there did not appear to be a correlation between the number of potentially cross-reactive HSV-1 proteins recognized and the number of VZV proteins recognized (Spearman rank correlation, r = +0.06, p = 0.83).

These cross-reactive proteins, using HSV-1 nomenclature, included enzymes and factors involved in DNA replication (UL5, UL9, UL12, UL29, UL40), structural capsid (UL19) and tegument (UL21, UL46, UL47, UL49) proteins, and two proteins involved in nuclear egress (UL31 and UL34). The protein homolog set with the most prevalent cross-reactive recognition was HSV UL40/VZV ORF18, the small subunit of ribonucleotide reductase, for which we detected cross-reactivity among 8 of 18 subjects (44%). Cross-reactivity in HSV-1 ICP4, an essential repressor/transactivator encoded by gene RS1 in HSV and ORF62 in VZV, was detected in 7 of 18 subjects.

Five VZV ORFs (ORF1, ORF2, ORF13, ORF32, ORF57) have no known HSV homolog (10, 46), whereas nine HSV ORFs (γ34.5, UL45, US2, US4, US5, US6, US8.5, US11, US12) have no known VZV homolog. Among these nine (mid-lower portion of Fig. 2D), plentiful reactivity to some HSV-1 proteins was detected, particularly for the HSV vaccine candidate glycoprotein D (15/18 subjects, gene US6, lower portion of Fig. 2D) (47). There were no instances of cross-reactivity to any of the five VZV proteins that do not have HSV-1 homologs (bottom of Fig. 2D) in any donor.

We validated selected hits from the ORFeome scans to the peptide level. We sought to add sequence-related but nonidentical HSV/VZV cross-reactive CD4 epitopes to the known sequence-identical epitope (48), and to compare the functional avidities of the T cells for VZV and HSV peptide homologs. We used polyclonal CD4 CD137^high-origin responder cells (Fig. 2B) or UV-treated VZV/IL-2–driven polyclonal cell lines from subjects 2 and 25 (Supplemental Table I) from our previous vaccine study (16) as responders. Cross-reactive epitopes were defined by two strategies. The first used existing (25, 33) HSV-1 and HSV-2 overlapping peptide sets (OLPs) covering the HSV homolog of a reactive VZV ORF to obtain an initial hit, followed by titration assays with the VZV and HSV-1/HSV-2 homolog peptides. The second started with truncated VZV proteins (primer details on request) and progressed to synthetic peptides within reactive VZV fragments. Autologous PBMCs were required as APCs for IVTT-expressed polypeptide Ags, and EBV-LCLs were used as APCs for peptides.

An example of each strategy is detailed for polyclonal CD137^high-origin VZV-reactive CD4 T cells and VZV ORFs 29 and 68. Each was cross-reactive when studied as full-length polypeptides with their HSV-1 and HSV-2 homologs (Fig. 2C). ORF68 truncation showed several reactive fragments, with aa 264–420 being the strongest (Fig. 3A). ORF68 is a promising vaccine candidate, and data consistent with multiple ORF68 CD4 epitopes per person is not unexpected based on previous studies (23, 49). We limited the search for cross-reactive peptides to the aa 348–420 region because VZV and HSV proteins are less divergent therein than in the other active VZV ORF68 fragments. CD4⁺ T cell reactivity was present for 15-mers 388–402 and 396–410 (Fig. 3B). The HSV-1 and HSV-2 homologs of VZV 388-402, QPMRLYSTCLYHPNA, with sequences AEMRIYESCLYHPQL and ADMRIYEACLYHPQL, respectively, were active (Fig. 3C). The HSV homolog of VZV 396-410, which is identical between HSV-1 and HSV-2, was inactive at 1 μg/ml (data not shown). Dose-response (Fig. 3C) showed equal potency for both HSV homologs of VZV 388–402. As an example of the second strategy, we tested HSV-2 UL29 OLPs and detected strong responses to HSV-2 UL29 529-543. Comparison of the homologous alphaherpesvirus peptides (Fig. 3D) showed divergence for potency, with the HSV-2 peptide showing brisk responses down to 1 pg/ml. These HSV peptides differ from their VZV homolog only at aa 1 and 12.

Additional cross-reactive CD4 peptide epitopes (Table I, Supplemental Fig. 1) were obtained by the truncation strategy for VZV ORF24 and by the OLP strategy for VZV ORFs 29, 31, 19, 18, and 9. Only peptides active at ≤1 μg/ml concentration in two separate assays in duplicate or triplicate are reported. For the epitope in VZV ORF24/HSV UL34, detailed truncation analyses of the active 15-mer were performed, defining 11-mer VZV ORF24 84–94 as the shortest active peptide (Supplemental Fig. 2). Interestingly, every cross-reactive epitope that we identified is nonidentical between HSV and VZV, at least at the 13-mer to 15-mer level (Table I). Overall, 11 peptide sets driving cross-reactive HSV/VZV CD4 T cell responses were discovered. Six epitopes show identity between the HSV-1 and HSV-2 homologs of the reactive VZV peptide, whereas five display divergence between the biologically active HSV-1 and HSV-2 peptides. In the case of VZV ORFs 29 and 31, the same polyclonal responder cell population responded to two contiguous 15-mer peptides, overlapping by 11 aa, from each virus. Because CD4 T cells can recognize peptides as short as 8 aa, we conservatively estimate the discovery of at least nine cross-reactive CD4 T cell peptides if these overlapping pairs are counted as a single epitope each. VZV ORF18 contained a minimum of three spatially separated and, thus, distinct epitopes that were cross-reactive with their homologs in HSV UL40. With the exception of the VZV ORF29 epitope (Fig. 3D), the visually estimated EC₅₀ tended to be equivalent between peptides from different viruses and mostly were in the 0.1-μg/ml range.

We previously characterized healthy immune-seronegative (IS) adults who repeatedly test negative for Abs to HSV-1 and HSV-2 but have direct ex vivo CD4 T cell–proliferative responses to whole killed HSV-2 Ag (14, 15). Limiting-dilution cloning of bulk T cell cultures after one cycle of whole HSV-2 stimulation of PBMCs from these individuals readily yields HSV-2–specific CD4⁺ T cell clones. Most of these individuals have had negative, serial studies of genital tract swabs using a sensitive PCR assay for HSV-2 DNA, in contrast to the almost universal finding of at least some low-level HSV-2 shedding in seropositive persons (50), consistent with a true lack of infection. Many have been in sexual relationships with persons with HSV-2 infection, such that mucosal exposure leading to Ag exposure and T cell sensitization was hypothesized to lead to sensitization. In an earlier report, we evaluated four CD4 T cell clones from an IS individual reactive with HSV-1 UL21, UL29, UL46, and UL47 (14). None reacted with a commercial VZV Ag preparation, despite the presence of a genomic homolog of each HSV-2 ORF in VZV (10).

To examine the hypothesis that some T cell reactivity to HSV in HSV-seronegative persons could be due to Alphaherpesvirinae cross-reactivity, two strategies were used. In the first approach, 12 CD4 T cell clones reactive with whole UV-treated HSV-2 were screened against both HSV-2 (24) and VZV genome-covering ORF polypeptide sets. Two clones from subject 34 (Supplemental Table I) cross-reactive against HSV-2 UL2 and the VZV homolog ORF59 were observed (representative clone 6 from subject 34, Fig. 3E). The 10 additional HSV-2–reactive CD4 T cell clones from IS subjects 30–33 reacted with eight other HSV-2 proteins but do not react with their VZV homolog (data not shown). By this approach, one in nine (11%) HSV-2 proteins recognized by clones from HSV-seronegative persons could be ascribed to possible VZV cross-reactivity.

The second method used a panel of 31 CD4 T cell clones from eight IS participants and determined their reactivity to HSV-2 15-mer peptides by screening a partial proteome-covering peptide set at 1 μg/ml peptide, similar to (15) (data not shown). A total of 21 peptides was recognized among these clones, which were then tested for reactivity to whole VZV. Using a conservative criterion of a net 5000 cpm of [³H]thymidine incorporation, 8 of 31 clones (26%) recognizing 6 of 21 distinct HSV-2 peptides (29%) had proliferative responses to VZV (Fig. 3F). The HSV-2 specificities (Supplemental Table II) included HSV-2 UL27, encoding envelope glycoprotein gB2, a candidate viral entry protein and vaccine Ag. Several clones reacted with the 15-mer peptide HSV-2 protein ICP4 1024-1038, containing a known (48) cross-reactive VZV ORF62 CD4 T cell epitope in a region with 14 contiguous amino acids identical to the HSV-2 15-mer. A separate CD4 T cell clone was specific for a second HSV-2 ICP4/VZV ORF62 epitope. Two clones with VZV cross-reactivity recognized distinct epitopes in HSV-2 major capsid protein UL19. Not all clones reactive with HSV-2 UL19 1233-1247 cross-reacted with whole VZV (data not shown), implying that cross-reactivity may vary with T cell clonotype. The reactive HSV-2 peptides and their putative VZV cross-reactive homologs are each relatively similar in sequence to each other (Supplemental Table II).

The cross-reactive peptide in HSV-2 UL29 was particularly interesting. We noted convergence of epitopes discovered using VZV restimulation of PBMCs to obtain polyclonal responder cells and using HSV-2 to obtain CD4 T cell clones from HSV-seronegative persons. The VZV ORF29 527-541/HSV-2 UL29 529-543 peptide pair was immunogenic for VZV-stimulated polyclonal CD4 cells from an HSV-1/HSV-2/VZV triple-seropositive subject (Table I); as noted above, the HSV-2 peptide was extraordinarily potent in this context (Fig. 3D). CD4 reactivity to the same HSV-2 peptide was detected independently (Fig. 3F, Supplemental Table II) in subject 33, a 56-y-old who was HSV-1 and HSV-2 seronegative (Supplemental Table I).

We previously showed 100-fold enrichment of polyclonal HSV-1–specific CD8 T cells from PBMCs by dendritic cell–based cross-presentation and selection of CD137^high activated cells and confirmed specificity with polypeptide, peptide, and tetramer assays (25). We began CD8 T cell cross-reactivity studies using these polyclonal T cell lines and whole VZV-infected APCs in cytokine and cell-killing assays. Unlike the CD4 T cell data above, we have not reliably enriched polyclonal VZV-specific memory CD8 T cells from PBMCs by direct or cross-presentation; therefore, we have no reciprocal cross-reactivity data for VZV-reactive polyclonal CD8 T cells against HSV.

For a representative HSV-1/VZV-seropositive subject (subject 21, Supplemental Table I), 36.9% of polyclonal HSV-1–driven CD8 T cells expressed IFN-γ and/or IL-2 in response to autologous HSV-1–infected LCLs, with a background of 1.7% for uninfected APCs (Fig. 4A). A lower level (16.8%) of cytokine response was detected for HSV-1–infected HLA-mismatched allogeneic EBV-LCLs used as APCs. This was not simple alloreactivity, because the same cell line was only 0.5% stimulatory in the absence of HSV-1 infection (see 21Discussion). The CD8 T cell line exhibited 10.9% cytokine responses to VZV-infected autologous DFBs, no response to uninfected negative-control stimulators, and no response to VZV-infected DFBs from the same HLA-mismatched subject (subject 36, Supplemental Table I) used for EBV-LCLs (Fig. 4A, 4B). Thus, ∼30% of the HSV-1-reactivity in polyclonal CD8 cultures from this subject showed cross-reactivity to VZV. We conclude that CD8 T cells derived using HSV-1 have significant cross-reactivity against VZV and that VZV-infected DFBs are competent for Ag presentation to these effectors.

We measured the ability of cross-reactive CD8 T cells to kill VZV-infected cells using monospecific effectors purified using HLA-A*0201–containing tetramers formulated with known HSV CD8 T cell epitopes. One tetramer included HSV-1 UL40 181-189 (25), previously described (20) as HSV–VZV cross-reactive, and the other tetramer included HSV-1 UL25 372-380 (33). HSV infection downregulates HLA class I in DFBs (31) and eliminates CD8 T cell recognition, so we typically use EBV-LCLs as CD8 T cell targets to study HSV-specific, HLA-restricted lysis (51). VZV also downregulates DFB HLA class I, but because EBV-LCLs are not permissive for VZV replication, we had to try DFBs as APCs for VZV-killing experiments involving viral infection (52). We observed strong cytotoxicity toward VZV-infected DFBs for both VZV vOKA and a wild-type VZV (Fig. 4C). Killing was HLA restricted.

From the preceding data, we predicted that some VZV ORFs should be processed to antigenic peptides and presented to HSV-1–driven polyclonal CD8 T cell lines. To test this, we transiently cotransfected Cos-7 cells with subject-specific HLA class I cDNA and with individual viral ORFs. The viral genes were expressed as fusions with eGFP under the control of a strong constitutive promoter, whereas cell surface HLA expression after coexport with Chlorocebus aethiops β2-microglobulin was documented by flow cytometry (25, 26, 38). Polyclonal CD8 T cell responders from subject 21 showed HLA-A*0201–restricted cross-reactive IFN-γ secretion responses to the VZV ORF homologs of HSV UL25 and UL40 (Fig. 5, top). Application of the HSV-1 CD8 polyclonal restimulation and ORFeome screen platform (26, 53) to HSV-2 revealed an additional cross-reactive ORF. In studies focused on subject 35 and HLA-B*1502–restricted T cells, HSV-2–specific CD8 T cells recognized full-length HSV-2 UL48 and the HSV-1 and VZV ORF10 homologs after cotransfection with HLA-B*1502 cDNA (Fig. 5, bottom). We conclude that VZV ORF 10, 18, and 34 polypeptides are intracellularly processed to peptide epitopes recognized by HSV-1– or HSV-2–reactive CD8 T cells.

We previously defined 39 HSV-1 peptides recognized by polyclonal HSV-1–driven CD8 T cell lines (25). We tested each VZV homolog of these HSV-1 CD8 epitopes (n = 30) alongside the HSV-1 peptide, using the presence of a VZV homolog protein and six or more identical amino acids as selection criteria. Using IFN-γ ICS, we detected cross-reactive responses in 4 of 39 (10%) epitopes. Our data (not shown) for an HSV UL40/VZV ORF18 cross-reactive CD8 T cell epitope confirm the earlier report by Chiu et al. (20) that these ORFs contain a cross-reactive HLA-A*0201–restricted epitope. Novel cross-reactive CD8 epitopes were detected in HSV-1/VZV ORFs UL25/ORF34 (subjects 21 and 23, two distinct epitopes) and UL29/ORF29 (subject 23) (Table II). Although background IFN-γ was above zero for some responder lines, the levels observed after peptide addition were reproducibly higher. Each novel cross-reactive epitope had strong predicted (54) binding to the relevant HLA class I molecule for both the HSV and VZV peptide homologs (Table II).

Transfection assays (Fig. 5) indicated the presence of at least one cross-reactive HLA-B*1502–restricted epitope in HSV-1 UL48/VZV ORF10. Inspection of the sequences and HLA-binding prediction (55) suggested that HSV-2 UL48 160-168 and the homologous VZV ORF10 164-172, with sequences ELRAREESY and ELRAREEAY, respectively, were candidate cross-reactive peptides. Initial mapping with HSV-1–overlapping peptides defined an HSV UL48 CD8 epitope in this region (data not shown). Peptide truncation (Fig. 6A) mapped the nonamer peptide epitope to UL48 160-168, a region identical between HSV-1 and HSV-2. Addition of C terminal amino acids reduced activity, whereas an N-terminal alanine did not change potency. The antigenic region is sequence identical in HSV-1 and HSV-2. For the polyclonal responders, the VZV nonamer ELRAREEAY had a lower maximal response compared with the HSV peptide ELRAREESY, but both were definitely and repeatedly positive (Fig. 6B, data not shown).

The CD8 T cell response to individual viral peptides is usually polyclonal (56). The higher IFN-γ responses for HSV than for VZV peptides among polyclonal responders suggested that these populations contained cross-reactive and HSV monoreactive clonotypes. To study cross-reactivity at the clonal level, peptide-HLA tetramers (33) constructed for HSV-1 UL25 372-380/HLA-A*0201 were used to stain peptide-specific cells. CD8⁺tetramer⁺ T cells were selected from PBMCs or from polyclonal HSV-1–reactive CD8 T cell lines, cloned by limiting dilution, expanded, and then tested in a split-well format. For subject 21, all 36 clones derived from the pre-expanded polyclonal line were cross-reactive, whereas for subject 24, all reactive clones were HSV monoreactive. Clones derived directly from PBMCs showed discrete HSV-1–only reactivity or HSV-1/VZV cross-reactivity (Fig. 6E).

Separate populations of CD8 T cells, with or without cross-reactivity, were also observed for the B*1502-restricted HSV UL48 epitope. Tetramers of B*1502 with the HSV or VZV peptide were differentially fluorochrome labeled. Each stained a well-separated subset of polyclonal, HSV-2–driven CD8 T cells by single staining (data not shown). When used simultaneously, we observed HSV/VZV double tetramer-positive and HSV tetramer-only–positive groups (Fig. 6C). These were sorted for TCRB CDR3 sequencing. The cross-reactive cells had a single dominant (68%) TCRB CDR3 using TCRBV12 and TCRBJ01-01*01 encoding CASNRQGARNTEAFF (Table III). The HSV monoreactive cells appeared more polyclonal, with two TCRB CDR3 sequences accounting for 45 and 31% of reads, using distinct TCRBV genes but the same TCRBJ 01-06*01 gene. Two subdominant clonotypes had related CDR3 sequences (CASSQYRVDEQFF and CASSQLRRETQYF) and used similar gene segments. The major clonotypes in each group were rare in the other. The tetramer-staining and CDR3 sequence data indicate that alphaherpesvirus-specific monoreactive and cross-reactive CD8 T cells are distinct.

To confirm TCR cross-reactivity, TCRA and TCRB CDR3 regions were cloned from cross-reactive CD8 T cell clone TCC17 (Fig. 6E) into a lentivirus (34, 37). The TCRA and TCRB used TRAV13-1*02/TRAJ18*01 and TRBV28*01/TRBJ2-7*02, encoding CDR3 sequences CAATRGSTLGRLYF and CASSSAMTSGSSYEQYF. Transduced T cells specifically recognized both the HSV-1 and VZV peptides, but not a CMV peptide, with a reciprocal pattern for the control CMV-specific TCR (Fig. 6F).

In this report, we demonstrate recognition of VZV at the virus, ORF, and peptide levels by polyclonal HSV-1–driven CD4 responder T cells. Reciprocally, we detect responses to HSV-1 virus, Ag, and peptide by CD4 T cells reactivated using VZV. This is largely recapitulated for CD8 T cells, for which polyclonal HSV-1–reactive cells recognize VZV-infected cells, proteins, and peptides. Various methods for measuring the degree of CD4 and CD8 T cell cross-reactivity each yield numbers in the 10–50% range. We propose that this degree of cross-reactivity is likely to be clinically significant in some situations and is suitable for exploitation during vaccine design.

Homo sapiens harbor two simplex viruses, HSV-1 and HSV-2, that are antigenically related, including T cell cross-reactivity (51, 56, 57). Betaherpesvirinae and Gammaherpesvirinae that infect humans are more distantly related (58). Earlier analyses detected single instances of CD4 and CD8 T cell HSV/VZV cross-reactivity (20, 48), with CD8 cross-reactivity extending to EBV. We have not detected cross-reactivity to herpesvirus peptides outside of Alphaherpesvirinae (data not shown), but our data support HSV/VZV cross-reactivity as a generalizable phenomenon. Some of the cross-reactive epitopes appear to be immunodominant: for example HLA-*0201–restricted responses specific for HSV UL25 (Figs. 4, 5) and cross-reactive with VZV ORF34 can be quite large in direct ex vivo PBMC tetramer stains (33). For most of the epitopes in this study, further research is required to determine their place within immunodominance hierarchies.

Clinically or epidemiologically important reciprocal interactions during the natural history of primary and recurrent infections by HSV and VZV have not been described, but, arguably, have not yet been carefully sought. Perhaps the best opportunity to determine whether HSV-specific immunity might modify VZV infection is zoster. Almost all adults harbor latent VZV, but only ∼30% of adults experience clinical zoster. The reasons for this heterogeneity are incompletely understood. Analysis of specimens from the placebo arms of the zoster-prevention vaccines (21–23), by serotesting for HSV-1 and HSV-2, could determine whether HSV-infected persons have variation in zoster incidence or postherpetic neuralgia. Another puzzling clinical phenotype is the variable efficacy of the current vaccine, which protects 50–70% of persons from shingles. It is possible that the currently licensed live attenuated VZV vaccine could be more active in HSV-infected persons, because abundant HSV-specific memory CD4 and CD8 T cells are present, and the subset that cross-reacts with VZV might be boosted by the vaccine.

Negative immune interactions are possible. Memory responses have a lower threshold for activation than does priming, so prior infection could favor expansion of pre-existing cross-reactive memory clonotypes, referred to as original antigenic sin (59). Longitudinal cross-reactivity assays in persons in the process of primary seroconversion to one virus and with prior immunity to the other, as well as viral accurate proteome-wide comparisons of the relative contributions of monoreactive versus cross-reactive T cell memory from mono- and dually-infected persons, may detect this phenomenon. Cross-modulation due to T cell exhaustion from chronic infection is also possible. Essentially all HSV-2–infected persons have intermittent reactivations (50). VZV is thought to reactivate far less frequently. Despite reactivations, HSV-specific CD4 T cells show brisk proliferative and polyfunctional cytokine responses (40), and CD8 T cells proliferate well to Ag (25, 60). There is some evidence that proliferation, cytokine secretion, and cytotoxic potential may vary with HSV severity and fine epitope specificity (61, 62). VZV-specific CD4 T cells are polyfunctional and proliferate well during VZV quiescence (16), show characteristics of exhaustion when obtained during zoster, and revert to a nonexhausted phenotype after recovery (63, 64). HSV–VZV cross-reactive CD8 T cells studied with a previously identified tetramer showed a spectrum of exhaustion markers between persons and irrespective of HSV immune status (20).

Proteome-wide reagents allowed us to detect many examples of Alphaherpesvirinae T cell cross-reactivity. Our genome-covering screening assays are, by nature, high throughput, and it is possible that positive responses may vary near the cutoffs that are determined using irrelevant Ags. Sampling of microbe-specific cells isolated from blood using CD137-based sorting could also lead to variable results for rare specificities. Synthetic peptides allow us to validate and extend the observations made using our virtual libraries. We focus on Ags in candidate vaccines and those showing both CD4 and CD8 T cell cross-reactivity. VZV ORF68 is a virulence factor (65), neutralizing Ab target (66), and vaccine candidate showing >95% efficacy as an adjuvanted subunit (23). Bergen et al. (67) detected T cell responses to VZV ORF68 388-400 and, based on sequence, speculated that it might be HSV cross-reactive. We now provide data that this is true (Fig. 3C). Similar to Bergen et al. (67), we showed that individuals can recognize multiple ORF68 epitopes (Fig. 3A).

A subunit vaccine containing truncated HSV-2 ICP4 has partial efficacy against recurrent HSV-2 (68, 69). We observed that HSV-1–specific CD4 T cell lines frequently recognized HSV-1 ICP4 and VZV ORF62 (Fig. 2D; last gene in upper section among genes with homologs in both viruses). Ouwendijk et al. (48) detected HSV–VZV ORF62 918-927, sequence LLLSTRDLAF, as a minimal HSV–VZV cross-reactive epitope. We recovered CD4 T cell clones from HSV-uninfected persons that recognized HSV-2 ICP4 1024-1038, QGVLLLSTRDLAFAG, homologous to VZV ORF62 915-929, RGVLLLSTRDLAFAG. These clones recognize whole VZV, thus likely detecting the epitope described above. A CD4 T cell clone reactive with HSV-2 ICP4 505-519 also cross-reacts with whole VZV (Supplemental Table II). ICP4/ORF62 are also CD8 T cell targets (68). These data support the use of ICP4 in vaccines.

In this article, we extend HSV–VZV CD4 T cell cross-reactivity to nonidentical peptides. The shortest active peptides in the cross-reactive HSV UL34/VZV ORF24 epitope are different. Most of the other proven (Fig. 3, Table I, Supplemental Fig. 1) and probable (Supplemental Table II) cross-reactive peptide sets also contain several variant amino acids. Thus, whole-virus and ORF-level cross-reactivity (Figs. 1, 2) likely integrates identical and nonidentical epitopes. Heterogeneity for mono- and cross-reactivity T cell clonotype level, as shown for CD8 T cell (Fig. 6C, 6E), is also likely for CD4 responses.

Vaccine Ags recognized by both T cell subsets are rational. We report that VZV ORF29/HSV UL29 and VZV ORF18/HSV UL40 contain cross-reactive CD4 and CD8 T cell epitopes. VZV ORF18/HSV UL40 is an enzyme that is a prevalent CD4 T cell Ag for both viruses and contains additional documented epitopes (16, 25). Chiu et al. (20) determined that CD8 T cells purified with tetramers of HLA-A*0201 and a VZV ORF18 peptide could cross-react against HSV-1 and HSV-2 homologs. We used a similar tetramer, containing the HSV-1 variant epitope that we initially described (25), to extend these findings to recognition of full-length, naturally processed viral proteins (Fig. 5) and whole virus (Fig. 4A, 4B). To our knowledge, this is the first demonstration of CD8 T cell recognition of VZV-infected cells by CD8 T cell effectors. The recognition of HSV-1 by these polyclonal effectors had a component that appeared not to be HLA restricted (Fig. 4A, far right panel). We hypothesize that this is due to transmission of HSV-1 from infected fibroblasts to CD8 T cells during this prolonged assay, followed by T cell recognition of infected T cells. We extended CD8 effector functions to cytotoxicity, showing that HSV UL40/VZV ORF18-reactive and HSV UL25/VZV ORF34-reactive CD8 T cells kill VZV-infected fibroblasts in a virus- and HLA-restricted manner (Fig. 4C).

VZV ORF10/HSV UL48 is a third dually CD4/CD8 cross-reactive target. The CD8 data in this report (Fig. 6–C, Table II) are complemented by our prior reports that the VZV, HSV-1, and HSV-2 UL48 homologs contain multiple CD4 and CD8 T cell epitopes recognized by cells from blood, genital skin biopsies, and human trigeminal ganglia (16, 24, 26, 32, 70–72). Several of the minimal CD4 epitopes defined in our previous work are sequence conserved between these viruses.

There is increasing interest in adoptive T cell therapy for severe viral infections in immune-compromised hosts (39). CD4 and CD8 T cell therapy were explored with natural and engineered T cells, and clinical-grade CD8 T cell products were developed for VZV (73). We purified polyclonal HSV/VZV cross-reactive CD8 T cells restricted by HLA-A*0201, the most common HLA class I allele, present in 40–50% of persons in diverse ethnic groups (Fig. 5); these cells can be expanded rapidly as in our polyomavirus study (74). We show in this article that TCR-engineered cells using TCRs recovered from CD8 clones show HSV/VZV cross-reactivity. This approach holds promise as an off-the-shelf method to provide cross-reactive CD8 T cells to HLA-defined patients. Interestingly, tetramer sorting–based recovery of cross-reactive versus monoreactive CD8 clones for the purpose of TCR harvesting was somewhat dependent on the cell source used. Although both direct ex vivo PBMCs and cell lines yielded cross-reactive clones, the additional recovery of HSV monoreactive clones directly from PBMCs (Fig. 5E) suggested that this cell source might best capture TCR diversity.

TCR sequencing also implies structural information concerning peptide-HLA contacts. The cross-reactive HSV UL48/VZV ORF10 peptides are identical at aa 1–7 and 9. From TCRB CDR3 sequencing (Fig. 6C supplying cells sequenced in Table III), we conclude that TCRB CDR3 amino acids contribute to differential recognition of the HSV and VZV peptide variants. This is consistent with crystal structures of TCR-HLA-peptide (75, 76). We are testing the hypothesis that TCRA CDR3 sequences may be similar for mono- and cross-reactive cells. Interest in HLA-B*1502–restricted TCRs and epitopes is enhanced by the high risk for severe T cell–mediated skin reactions in HLA-B*1502⁺ persons receiving carbamazepine (28). Abacavir, a cause of severe, HLA-linked toxicity (77), binds the peptide-binding groove in an allele-specific manner (78) and alters the HLA-bound peptide repertoire (79). We hope to use full TCR sequences from HLA-B*1502–restricted T cells to elucidate HLA-B*1502–related hypersensitivity.

T cell cross-reactivity is likely quite ubiquitous, given the large size of the set of potential T cell epitopes and the reduced complexity of TCRs (80). T cell responses among apparently uninfected persons and mice can have memory phenotypes and be linked to specific responses to sequence-unrelated peptides present in environmental flora (81, 82). In contrast, the cross-reactivity in this study is likely due to sequence-related, homologous peptides present in phylogenetically related pathogens. However, although we observed cross-reactivity in both dually HSV–VZV–infected persons and VZV-monoinfected persons, it does not explain all of the reactivity to HSV that we observed in HSV-uninfected persons. Thus, priming by more distantly related Ags may account for some of our observations. We have not performed reciprocal studies to measure whether VZV-seronegative persons, who are quite uncommon in most populations, can harbor VZV-reactive T cells, possibly as a result of HSV cross-reactivity.

In summary, we demonstrated bidirectional, CD4 and CD8 T cell cross-reactivity between HSV and VZV, detected cross-reactivity for many protein homolog sets, defined several novel cross-reactive fine T cell epitopes, and provided quantitative estimates for the extent of cross-reactivity. The clinical significance of these observations should be testable. Several viral ORFs that elicit both cross-reactive CD4 and CD8 T cells have been discovered that can be prioritized as subunit vaccine candidates, and synthetic immunogens that link cross-reactive moieties can be envisioned. Licensed primary varicella and shingles vaccines are live attenuated VZV strains, but we have not had the chance to study whether these elicit or boost cross-reactive T cells. Cross-reactivity due to VZV infection can account for a proportion of T cell reactivity to HSV that was noted in HSV seronegative persons. Additionally, we provide the first evidence, to our knowledge, that specific CD8 T cells can recognize VZV-infected skin cells and reconstitute CD8 T cell cross-reactivity using expression of cloned TCRs to provide a prototype for parsimonious adoptive T cell therapy of multiple viral infections. Using current proteome-wide Ag and TCR tools to dissect T cell responses, and recognizing that most microbes occur in large, related families, we now appreciate that cross-reactivity can make a considerable contribution to overall pathogen-specific responses.

We thank D. Knipe for HSV-2 186, J. Blaho for HSV-2 UL41 deletion, P. Kinchington for eGFP-VZV, A. Cent for VZV isolate and serology, D. Mueller and L. Zhao for technical assistance, J. Cao for tetramers, A. Magaret for statistical assistance, C. Linnemann for pMP71flex, and D. Sommermeyer for pRRL.PPT.MP.GFPpre. The University of Washington Virology Clinic recruited participants and obtained specimens.

D.M.K., L.J., K.J.L., and A.W. are coinventors on institutional patents concerning HSV vaccines. D.M.K. received research funding from Sanofi Pasteur for HSV vaccine immunogenicity testing. A.W. received research funding from Vical, Agenus, and Genocea for HSV vaccine clinical trials.