Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

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Christine Johnston, Jia Zhu, Lichen Jing, Kerry J. Laing, Christopher M. McClurkan, Alexis Klock, Kurt Diem, Lei Jin, Jeffrey Stanaway, Elizabeth Tronstein, William W. Kwok, Meei-li Huang, Stacy Selke, Youyi Fong, Amalia Magaret, David M. Koelle, Anna Wald and Lawrence Corey J. Virol. 2014, 88(9):4921. DOI: 10.1128/JVI.03285-13. Published Ahead of Print 19 February 2014.

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection Christine Johnston,a,g Jia Zhu,b,g Lichen Jing,a Kerry J. Laing,a Christopher M. McClurkan,a Alexis Klock,b Kurt Diem,b Lei Jin,b Jeffrey Stanaway,c Elizabeth Tronstein,a William W. Kwok,f Meei-li Huang,b Stacy Selke,b Youyi Fong,g Amalia Magaret,b,e,g David M. Koelle,a,b,d,g Anna Wald,a,b,c,g Lawrence Coreya,b,g Departments of Medicine,a Laboratory Medicine,b Epidemiology,c Global Health,d and Biostatistics,e University of Washington, Benaroya Research Institute at Virginia Mason,f and Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center,g Seattle, Washington, USA

ABSTRACT

IMPORTANCE

This detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8ⴙ T cells compared to control tissue, and HSV-specific CD4ⴙ T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.

H

erpes simplex virus 2 (HSV-2) infects over 500 million people worldwide (1) and is the leading cause of genital ulcer disease (2) and increases the risk of HIV acquisition (3). Herpetic genital ulcers in the immunocompetent person are localized with respect to time and anatomic site (4), consistent with reactivation of virus from the ganglion innervating this region. However, most HSV genital shedding is subclinical, with rapid onset and clearance (5, 6). Such subclinical reactivation may be more widespread in the genital area (7), consistent with frequent leakage of virus into the genital skin and mucosa with localized host containment (8, 9). Mathematical models suggest that observed shedding patterns are the result of overlapping shedding episodes from different anatomic sites (10) and that the amount of virus detected is both directly proportional to the number of infected cells and predictive of a long in vivo half-life of virus-infected cells (8). Additionally, models predict that greater concentrations of host T cells are associated with viral clearance whereas insufficient CD8⫹ T cell levels result in lesions (11); hence, T cell infiltration may be an in vivo marker of reactivation. Furthermore, tissue-resident memory T cells have been shown to persist at sites of genital lesions (9, 12). To investigate anatomic patterns of subclinical HSV reactivation in the genital tract, we prospectively studied HSV-2-seropositive

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persons by dividing their genital tracts into 22 regions, swabbing each region separately on a daily basis, and assaying each sample for HSV DNA using a rapid PCR assay. Biopsy specimens from regions in which HSV DNA was detected were taken within 24 h and analyzed for HSV DNA and evidence of an inflammatory response, allowing spatial and histologic characterization of asymptomatic HSV-2 reactivation in the genital region. (Part of this research was presented at the 48th Annual Infectious Disease Society of America Meeting, Vancouver, British Columbia, Canada, 21 to 24 October 2010.)

Received 10 November 2013 Accepted 10 February 2014 Published ahead of print 19 February 2014 Editor: R. M. Longnecker Address correspondence to Christine Johnston, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /JVI.03285-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.03285-13

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Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4ⴙ and CD8ⴙ T cells were quantified by immunofluorescence, and HSV-specific CD4ⴙ T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8ⴙ T cells compared to control tissue (27 versus 13 cells/mm2, P ⴝ 0.03) and identified HSV-specific CD4ⴙ T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract.

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MATERIALS AND METHODS

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lically available software, ImageJ and CellProfiler (18). Slides were randomly selected for quality control by manual counting. (iii) HSV T cell specificity. Bulk lymphocytes were expanded from skin biopsy specimens using nonspecific mitogenic stimulation (19). Autologous peripheral blood mononuclear cells (PBMCs) labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) were used as antigen-presenting cells (APC) at a 1:1 ratio with expanded bulk skin lymphocyte responders as previously described (20). Brefeldin A was added at 1 h, cells were labeled for CD3 and CD4, and intracellular gamma interferon (IFN-␥), tumor necrosis factor alpha (TNF-␣), and interleukin-2 (IL-2) were measured at 6 h as previously described (21). A FACSantoflow cytometer (BD Biosciences) with FlowJo software (Tree Star) was used for determinations performed by gating on lymphocyte forward and side scatter, excluding CFSE-positive cells. HSV-2 ORFeome. For biopsy specimens from participants who had HSV-specific CD4⫹ T cells, we used a complete CD4⫹ T cell HSV-2 protein set, termed the HSV-2 open reading frame (ORF)eome, to test for proliferative responses to individual HSV proteins, as described for HSV-1 (21). Briefly, bulk expanded CD4⫹ lymphocytes, autologous irradiated PBMCs used as APC, and recombinant HSV-2 proteins were plated in duplicate in 96-well U-bottom plates. The primers used to generate the HSV-2 ORFeome are described in Table S1 in the supplemental material. As described previously for HSV-1 (21), some HSV-2 ORFs were cloned in fragments and data are presented as positive for the protein fragments for which specific proliferation was observed. [3H]thymidine incorporation was measured after 3 days to detect cell proliferation. The criterion for positivity for each antigen was set as the mean plus 2.1 times the standard deviation of the [3H]thymidine incorporation for 54 negativecontrol antigens (21). In one participant tested with both the HSV-1 and HSV-2 ORFeomes, CD4⫹ cells in both lesion and asymptomatic biopsy specimens showed reactivity to VP16. Fragments of VP16 of HSV-1 were expressed as described previously (22) and tested individually; overlapping peptides from within the positive protein fragments were used to characterize the peptide epitope using the standard [3H]thymidine incorporation assay. A Qdot655-labeled DRB4*0101 oligomer was made with the optimal VP16 peptide, and bulk responder skin origin cells were stained with anti-CD4 MAb and the HSV-DR4*0101 tetramer as previously described (23). Statistical analysis. Participants with genital swabs collected over the course of more than 1 day were included in the analysis. Rates of HSV shedding (defined as the number of samples with HSV detected per number collected) were calculated overall, per participant, and per day. Swabs collected from foreskin in uncircumcised men were excluded from all analyses, except during assessment of associations at concurrent sites. A shedding episode was defined as any consecutive period of shedding that included no more than one consecutive negative or one consecutive missed swab; episodes were considered to be of known duration if they were preceded and followed by 2 days without shedding (24). Most episodes were of unknown duration due to gaps in the swabbing schedule; in these cases, interval censoring was used to estimate the duration of continuous shedding, assuming an underlying Weibull hazard (25). Episodes were calculated in two ways: as shedding during consecutive days from any site and as shedding from the same site during consecutive days. Linear mixed-effects regression analysis was used to examine the association between the maximum quantity of virus detected on a given day and the number of sites with HSV detected. To evaluate the potential associations of previous and concurrent shedding with the risk of shedding at a given site, we created a site-level data set that includes one row for each site for each day. A generalized linear mixed model with a Poisson link estimated the probability of HSV detection and the association with (i) having sites positive the previous day, (ii) having not collected the previous day, (iii) having nonadjacent sites concurrently positive, (iv) having adjacent sites concurrently positive, and (v) gender. Adjacent sites were defined as two spatial regions sharing any portion of their edges. The analysis

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Participants and procedures. We recruited healthy HSV-2-seropositive adults, ages 18 to 65, at the University of Washington (UW) Virology Research Clinic. Persons who were HSV-2 seropositive by the UW HSV type-specific immunoblot assay (13) were eligible. Pregnant women, those with HIV antibodies (Ab), or those who were taking immunosuppressive medications were excluded, as were persons with coagulopathy or a history of keloid formation or recurrent cellulitis. HLA typing was performed at the Puget Sound Blood Center. The protocol was approved by the UW Human Subjects Division. Written informed consent was obtained from each participant prior to initiating study procedures. Genital swabbing. At enrollment, a digital image of the genital tract was used to create a genital map that was divided into 22 sections for women and circumcised men and into 26 sections for uncircumcised men. Participants were seen daily, Monday through Friday, for 4 weeks. After genital examination, Dacron swabs moistened with sterile water were rubbed on the genital mucosa corresponding to each section of the grid, avoiding touching other sites. Each swab was placed in 1⫻ PCR buffer for HSV PCR (14). Participants were queried about genital symptoms and sexual activity during each visit. If a lesion was present, additional samples from the lesion were obtained and a biopsy was performed, if possible. To map shedding among female participants, we created a shapefile comprising the 22-region grid of our female genital map. We used ArcGIS Version 9.0 (ESRI, Redlands, CA) to create the shapefiles and Adobe illustrator CS4 (Adobe Systems Incorporated, San Jose, CA) to later combine each participant’s daily maps into a complete participantlevel shedding time line. Swabs at nongenital sites (thigh, abdomen, or arm) were collected as negative controls. Each examination room was thoroughly cleaned with Virex and bleach daily. In addition, examination rooms were swabbed at 12 potentially contaminated sites, including examination tables, faucets, door handles, and countertops. Genital biopsies. Genital biopsies were performed on keratinized genital tissue. The clitoral hood, labia minora, urethra, and penile shaft were excluded from biopsy. Genital tissue was cleaned with chlorhexidine and anesthetized with 1% lidocaine. A 3-mm-diameter punch biopsy specimen was obtained and placed in tissue culture media. The biopsy specimens were sectioned for immunohistochemistry and cell culture. For sites with documented shedding in the absence of symptoms or lesions on careful examination (defined as sites of asymptomatic shedding), biopsies were performed within 24 h of obtaining a positive PCR result. Control biopsies were performed on the contralateral genital skin or the inner arm. Biopsies were performed on a genital lesion in the first 24 to 48 h after lesions were noted by participants. Participants without a genital lesion documented during the intensive sampling period were followed for up to 1 year after enrollment. Digital images were obtained when biopsies were performed. Laboratory methods. (i) HSV PCR. A validated rapid quantitative real-time HSV PCR assay with primers corresponding to the glycoprotein B gene was performed on all specimens, with results available within 8 h of specimen collection (15). DNA extraction was performed using a QiaAmp 96 blood kit. Positive and negative controls were included with samples collected from the clinic; experiments were performed in a blind manner by laboratory technicians with respect to control samples. Samples with ⱖ2.2 log10 copies/ml were considered positive (16). For every 5 samples, a negative control was coprocessed in the PCR laboratory, and each DNA extraction plate had one positive control. (ii) Immunofluorescence. Biopsy tissue was snap-frozen and stored at ⫺80°C in optimal cutting temperature (OCT) media. Frozen tissue was sectioned into 7-␮m-thick slices and fixed and permeabilized in acetone. Tissue was stained with primary monoclonal Ab (MAb) specific for human CD4 (Dako) and CD8 (BD Biosciences) and counterstained with DAPI (4=,6-diamidino-2-phenylindole; Fluka) as previously described (17). Tyramide signal amplification was used for antibody detection (Invitrogen). CD4⫹ and CD8⫹ cells were autocounted (per slide) using pub-

Anatomic Characterization of Genital HSV-2 Shedding

approximated based on reference 37. T, thoracic; L, lumbar; S, sacral. (D) Example of male grid. Grid outlines in red in panels B and D reflect sites that were not considered to be safe for a genital biopsy procedure.

included a random term for individual and site. A similar model analysis was performed to assess the influence of bilateral shedding. Sensitivity analysis was performed using ⬎2.7 log10 copies/ml as a cutoff for the linear mixed-effects model and the generalized linear mixed model. The Wilcoxon signed-rank test was used to test for a difference in CD4⫹ and CD8⫹ T cell concentrations between paired-lesion, asymptomatic, and control biopsy specimens. Two-sided P ⬍ 0.05 results were considered statistically significant.

RESULTS

Participants and genital HSV shedding. We enrolled 29 HSV-2seropositive persons. One provided a single day of swabbing only and was excluded from all analyses. Of the remaining 28 participants, 21 (75%) were women and 17 (61%) were white. Five (71%) of the 7 men were circumcised. The median age was 42 years (range, 20 to 65). Twenty-five (89%) participants had symptomatic genital HSV-2 infection, and 3 (11%) had no history of known genital HSV-2 infection. Thirteen (46%) participants were both HSV-1 and HSV-2 seropositive; 15 (54%) were only HSV-2 seropositive. Among the participants with symptomatic infection, the median time since HSV acquisition was 13 years (range, ⬍1 to 35 years). Twenty-five (89%) participants completed the entire 28-day intensive swabbing period (median, 28 days; range, 11 to 36) (Fig. 1A). We created anatomical grids for women (Fig. 1B) and men (Fig. 1D) on the basis of the neuronal innervation patterns of the

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external genitalia (shown for women in Fig. 1C). The grid area included innervation from multiple branches of sacral ganglia (Fig. 1C). We collected 11,603 genital swabs on 528 days, with a median of 440 swabs (range, 198 to 462 swabs) per person. Eleven (40%) participants had lesions on a total of 77 (15%) days during the follow-up period. The remaining 17 (60%) participants were asymptomatic. At least 1 day of shedding during the follow-up period was detected in 23 (82%) of the participants; no HSV shedding was detected in 5 (18%) female participants. Overall, HSV was detected on 123 (23%) days and from 868 (7%) swabs, rates similar to those seen in previously studied cohorts of HSV-2-seropositive persons sampled once daily (24). Of 123 days with shedding, 48 (39%) were collected on days on which participants had lesions and 75 (61%) were during asymptomatic periods. Of swabs with HSV detected, 573 (66%) were collected on days with lesions and 295 (34%) were collected on days without lesions. Overall and subclinical rates of shedding did not differ between men and women (data not shown). Multifocal anatomic distribution of reactivation. Of the 23 participants with HSV genital shedding, 18 (78%) had HSV shedding detected on both sides of the genital tract during the study period. HSV was detected bilaterally on 83 (67%) days with shedding. Of all days with shedding, 46 (37%) days had two or more contiguous positive regions. In individual participants, we observed distinct anatomic HSV-2 shedding patterns over time, with

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FIG 1 (A) Study design and participant flow. M–F, Monday to Friday. (B) Example of female grid. (C) Innervation of female genitalia. Innervation patterns were

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in each region is shown according to the heat map (see legend). (A to C) Focal and widespread asymptomatic shedding in three women. (D and E) Widespread genital shedding in the presence of a lesion in women. (D) A lesion was present on days 1 to 7 and 11 to 21 as indicated by the outlined sites. (E) A lesion was present on days 1 to 7 and 16 to 20 and on day 31 as indicated by the outlined sites. (F) Shedding patterns in men (L ⫽ left, R ⫽ right, Dor ⫽ dorsal, Pen ⫽ penis, Ven ⫽ ventral). (G) A lesion was present on days 22 to 25, as indicated by the outlined sites.

some days having focal sites positive or multiple sites positive in the absence of lesions (Fig. 2A to C) and widespread shedding involving the entire genital tract in the presence of lesions (Fig. 2D and E). For example, the participant whose results are represented in Fig. 2A had shedding located at focal sites on days 1 to 4 and again on days 18 and 19. The participant corresponding to Fig. 2B had focal shedding on days 1 to 4, 16 and 17, and 29 to 31. Widespread shedding in the presence of a lesion is shown in Fig. 2D; there was shedding throughout the genital region on day 2 and day 18, and a similar pattern is seen in Fig. 2E for days 16, 17, and 31. In men, similar patterns of focal shedding (Fig. 2F, days 10, 12, 22, and 23; Fig. 2G, days 5 and 9) and widespread shedding (Fig. 2G, days 22 to 25) were demonstrated. Additional shedding patterns for the remaining participants are shown in Fig. S1 and S2 in the supplemental material. Both focal and widespread shedding patterns were observed in 10 (36%) participants. Shedding in individual participants was detected at some time during the observation period at a median of 12 sites (range, 0 to 22); 4 (14%) participants had shedding detected at all 22 sites. A median of 5 sites (range, 1 to 22) were positive on the days when any HSV was detected. A median of 2 (range, 1 to 22) sites were positive on HSV

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detection days without the presence of lesions. In the presence of a lesion, shedding was often disseminated throughout the genital tract, with a median of 13 sites with HSV detected (range, 1 to 22), with a gradual decline in the number of sites with shedding as the lesion healed over time (Fig. 2D, days 11 to 19; Fig. 2E, days 1 to 3 and 16 to 19). Of 12 participants with lesions, 8 (67%) had lesions at two or more sites, and 7 (58%) had lesions at bilateral sites. Quantity and duration of shedding episodes. As shown in Fig. 2, participants had several episodes of focal shedding in different regions during the observation period. Overall, we observed 44 episodes of shedding. However, considering each of the 22 sites separately, there were 454 shedding episodes. Overall, the median number of consecutive days with HSV detected at the same site was 3.3 and with HSV detected at any site was 4.1. In the absence of lesions, HSV was detected for a median of 2.3 consecutive days at the same site and 3.1 days at any site. In the presence of lesions, shedding at the same site was often prolonged, with a median of 4.0 consecutive days of shedding at the same site and 6.1 days of shedding at any site. Over all swabs with HSV detected, the median quantity of HSV detected was 3.3 log10 copies/ml (range, 2.2 to 8.3 log10 copies/ml)

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FIG 2 Shedding patterns from individual participants collected over the swabbing period. The time line indicates the days of study. The quantity of virus shed

Anatomic Characterization of Genital HSV-2 Shedding

of sites with HSV detected on single days. Light gray hollow dots ⫽ days without lesions; black dots ⫽ days with lesions. The lines indicate the regression slope calculated using linear mixed effects (P ⬍ 0.001).

(Fig. 3A). The median per-day maximum quantities of HSV detected were 2.8 log10 copies/ml (range, 2.2 to 7.3 log10 copies/ml) without lesions and 5.9 log10 copies/ml (range, 2.2 to 8.3 log10 copies/ml) with lesions. We observed a significant positive association between the median quantity of virus detected and the number of sites positive on any given day (Fig. 3B). For each additional site positive on a given day, the median day-level copy numbers were 0.09 log10 copies/ml greater in the absence of lesions (P ⬍ 0.001) and 0.06 log10 copies/ml greater in the presence of lesions (P ⬍ 0.001). Predictors of shedding. The finding that shedding was found simultaneously at multiple anatomic genital sites could be explained by (i) contiguous viral spread through epithelial cells; (ii) contamination through infected fluids; or (iii) simultaneous, multifocal reactivation within sacral ganglia innervating the genital tract. Shedding at an adjacent site could be consistent with all three possibilities, while shedding at nonadjacent sites is most consistent with simultaneous shedding from more than one ganglion. Of 892 positive swabs, 34 (3.8%) were collected on a day with only one site positive, 58 (6.5%) were collected when shedding was present at nonadjacent sites only, and 800 (90%) were collected with concurrent adjacent shedding. The shedding rate increased 60% when the results from the prior day were positive (risk ratio [RR] ⫽ 1.6, 95% confidence interval [CI] ⫽ 1.3 to 1.9, P ⬍ 0.0001). The risk ratio for having any nonadjacent site positive concurrently was 11.0 (95% CI ⫽ 7.1 to 17, P ⬍ 0.0001) and for having an adjacent site positive was 91 (95% CI ⫽ 63 to 131, P ⬍ 0.0001). In another model, we looked at the risk ratio for concurrent shedding on the same or the opposite side and found that the shedding risk increased when shedding was present the day prior (RR ⫽ 1.9, 95% CI ⫽ 1.5 to 2.3), when shedding was present on the opposite side concurrently (RR ⫽ 13.0, 95% CI ⫽ 7.7 to 22.0, P ⬍ 0.0001), and when shedding was present on the same side concurrently (RR ⫽ 57, 95% CI ⫽ 40 to 83, P ⬍ 0.001). Therefore, shedding at an adjacent site or on the same side was associated with a very high risk of concurrent shedding at a given site, but shedding at nonadjacent sites or at a contralateral site was also associated with an increased risk of shedding, consistent with simultaneous ganglionic reactivation. To evaluate whether self-contamination contributed to the results, control swabs were obtained from the leg or arm. Of 283 swabs collected from these sites, 12 (4%) were positive, with me-

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dian of 2.6 log10 copies/ml (range, 2.2 to 3.0). To assess environmental contamination, we swabbed examination rooms: of the 60 swabs obtained, 2 (3%) were positive, with viral loads of 2.27 and 2.36 log10 copies/ml, respectively. These data suggest that environmental contamination or self-contamination was rare and low level but that contamination could account for some of our findings. We performed a sensitivity analysis in which we used a cutoff of 2.7 log10 copies/ml. This lowered the overall frequency of shedding from 123 days (23%) to 92 days (17%). We found that the results of the spatial analyses and the association between the median HSV quantity detected and the number of sites with HSV detected were, however, unchanged. In addition, since high quantities of virus are associated with lesions, which may be more likely to contaminate other areas, we performed an analysis restricted only to non-lesion days, and the results were similar (RR with nonadjacent site positive ⫽ 6.9, 95% CI ⫽ 3.3 to 14.1; RR with adjacent site[s] positive ⫽ 94, 95% CI ⫽ 58 to 151). Biopsy specimens from sites with asymptomatic shedding. We hypothesized that sites of asymptomatic reactivation would be associated with a host immune response. We obtained genital biopsy specimens from 15 persons at sites of asymptomatic shedding within 24 h of HSV shedding; control skin biopsy specimens were also obtained (Fig. 1A). We did not observe lesions prior to biopsies at asymptomatic sites of shedding in any case. The median quantity of HSV DNA detected prompting the biopsy was 3.4 log10 copies/ml (range, 2.1 to 6.7 log10 copies/ml). Nine (60%) biopsy specimens were negative by surface HSV PCR on the day of biopsy, indicating rapid clearance of the shedding episode (Table 1). One (6.7%) biopsy specimen had HSV DNA detected within its tissue. Ten participants also had a lesion biopsy performed (Fig. 1A). High densities of CD4⫹ and CD8⫹ cells were detected in the tissue of several biopsy specimens of asymptomatic areas compared to control samples (Fig. 4A). The focal distribution of CD4⫹ T cells was observed in the dermis, with some areas showing intense infiltration of CD4⫹ T cells (Fig. 4A) whereas other areas of the same biopsy specimen lacked detectable CD4⫹ T cells. Asymptomatic biopsy specimens had higher CD8⫹ T cell density than did controls (median, 27 cells/mm2 versus 13 cells/mm2, P ⫽ 0.03). However, we found no significant difference in overall CD4⫹ T cell density between asymptomatic and control biopsy specimens (20 cells/mm2 versus 10 cells/mm2, P ⫽ 0.52) (Fig. 4B), despite the

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FIG 3 (A) Histogram of quantity of HSV detected in swabs n ⫽ 868). (B) Association between median log10 copies of HSV DNA detected/ml and the number

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TABLE 1 Quantity of HSV detected before and on day of biopsy for asymptomatic specimens Amount of HSV detected (log10 copies/ml) On day of biopsy

In biopsy specimen

Region of HSV detection and biopsya

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

2.70 2.11 4.10 2.50 4.72 2.62 3.20 5.68 4.70 3.73 3.09 2.95 2.71 3.80 3.63

0.00 0.00 3.00 0.00 4.75 0.00 2.20 2.85 3.00 3.03 0.00 0.00 0.00 0.00 0.00

0.00 0.00 0.00 0.00 0.00 0.00 2.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

1 7 15 15 10 15 21 18 15 22 10 11 16 21 10

a

See Fig. 1 and its legend for explanation of region numbering.

focal enrichment of CD4⫹ T cells seen in the tissue of some asymptomatic biopsy specimens (Fig. 4A). As expected, lesion biopsy specimens had the highest concentrations of CD8⫹ cells (median, 396 cells/mm2 versus 27 cells/mm2, P ⫽ 0.016) and CD4⫹ T cells (median, 698 cells/mm2 versus 20 cells/mm2, P ⫽ 0.016) compared to asymptomatic biopsy specimens (Fig. 4C) and control biopsy specimens (data not shown). To determine if CD4⫹ T cells present in asymptomatic and lesion biopsy specimens were HSV-2 specific, we performed intracellular cytokine cytometry (ICC) to assess IFN-␥, TNF-␣, and IL-2 production in response to whole HSV antigen (UV-treated HSV-2). Representative findings from a single participant are shown in Fig. 5. Among 8 asymptomatic biopsy specimens with sufficient cellular expansion, 3 (38%) had HSV-2-specific CD4⫹ T cells present. By comparison, in 4 (66%) of 6 lesion biopsy specimens, HSV-specific CD4⫹ T cells were present, and HSV-specific CD4⫹ T cells were not detected in control biopsy specimens. Antigen and epitope mapping. To map the antigen specificity of the CD4⫹ T cells in genital biopsy specimens, we used the HSV-2 ORFeome, a tool which can be used to study proliferative responses to all HSV-2 open reading frames and which allows dissection of antigen-specific T cell responses (21). We observed reactivity to several HSV-2 antigens (range, 3 to 18) in lesion biopsy specimens in three of three participants who had HSV-specific cells detected from genital biopsy specimens (see Table S2 in the supplemental material). In one of three participants, the asymptomatic biopsy specimen also had HSV-specific T cells. In this participant, T cells from both the lesion biopsy specimen (Fig. 6A) and the asymptomatic biopsy specimen (Fig. 6B) reacted to VP16 (gene UL48), a late tegument protein that transactivates HSV gene transcription and may be required for initiating replication from latency (26). The asymptomatic biopsy specimen produced IFN-␥ corresponding to whole HSV antigen (Fig. 6C). We finely mapped the epitope in the asymptomatic biopsy specimen to UL48/VP16 amino acids (aa) 187 to 199. After creating Qdot655-conjugated HSV-2-specific DRB4 multimers, as previ-

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DISCUSSION

Our intensive study to characterize spatiotemporal patterns of genital HSV-2 shedding and the immune response to asymptomatic HSV shedding episodes showed (i) widespread viral shedding throughout the genital tract, suggesting that reactivation of HSV-2 often occurred simultaneously from distinct regions of the genital tract, and (ii) that asymptomatic reactivations were associated with increased focal concentrations of CD8⫹ T cells and the detection of HSV-2-specific T cells at the site of subclinical shedding in nearly 40% of biopsy specimens, providing evidence of robust host-pathogen interactions. In one case,we found that an asymptomatic shedding-site biopsy specimen contained HSV-2specific CD4⫹ T cells targeting a specific viral epitope, confirming HSV-2 immunologic specificity in areas of asymptomatic shedding. Our findings are consistent with the body of literature showing that HSV frequently reactivates (6, 24). We previously observed a high correlation between shedding duration and peak quantity of virus detected during a shedding episode (27). In this study, we added a spatial component that showed a positive correlation between increasing virus quantity and larger anatomic area with shedding. Mathematical models have predicted this relationship, as low-copy-number shedding episodes are associated with few infected cells (8). However, we were surprised to find evidence of widespread shedding, regardless of the presence of lesions. This was most dramatic with the high frequency of contralateral shedding, suggesting that HSV-2 infection may be present in multiple anatomic areas: areas often innervated by different sacral ganglia. These findings are consistent with previous findings in the guinea pig model of vaginal HSV-2 infection as well as with studies of human sacral ganglia which indicate HSV-2 latency is widespread. Bernstein and Stanberry performed a detailed analysis of primary vaginal HSV-2 infection in the guinea pig and found that HSV-2 was detected from ipsilateral dorsal root ganglia on days 2 and 3 postinfection and from contralateral dorsal root ganglia on days 4 and 5 and that the majority (9/12) of animals shed virus on the contralateral genital skin from the initially infected ganglion (28), suggesting that HSV-2 infection can spread throughout neuronal tissues. Although data on the distribution of HSV-2 in human sacral ganglia are limited, Obara et al. described PCR and in situ hybridization evidence of widespread sacral ganglion infection in three persons coinfected with HSV-1 and HSV-2, with HSV-2 detected in 21 of 23 sacral ganglia and HSV-1 in 22 of 23 ganglia (29), and concluded that both HSV-1 infection and HSV-2 infection are latent bilaterally throughout the sacral ganglia. Croen et al. also detected evidence of HSV-2 infection in the sacral ganglia of 5 persons at autopsy using in situ hybridization; HSV-2 was detected in two or more sacral ganglia in 3 of 5 persons (30). Shedding at an adjacent site increased the risk of site-specific shedding nearly 100-fold, consistent with either viral spread through epithelial viral replication or spread through contami-

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Participant

On day prior to biopsy

ously reported for HLA class I reagents (9), we showed that this participant had CD4⫹ HSV-specific T cells reactive to this epitope at the site of asymptomatic genital shedding (Fig. 6D) and also in blood (data not shown). These results demonstrate that HSVspecific CD4⫹ T cells can be recovered from sites of asymptomatic genital HSV shedding, suggesting that asymptomatic shedding episodes are associated with a targeted host immune response.

Anatomic Characterization of Genital HSV-2 Shedding

from 2 participants who had biopsy specimens collected from an asymptomatic site, lesion site, and control skin is shown. Magnification, ⫻20. Scale bar, 100 ␮m. (B and C) CD4 (B) and CD8 (C) T cell densities in asymptomatic, lesion, and control biopsy specimens. Results are from computerized counting of individual slides. Each dot represents counts from a single participant. Median quantities are indicated by the solid bar; interquartile ranges are indicated by dotted lines. P values for pairwise comparisons are indicated above the panels.

nated fluids from adjacent sites. While this form of spread or contamination would be reasonably expected over a small anatomic area, it is difficult to envision spread or contamination that would include all 22 sites, as was seen on many days in the presence of lesions. This suggests a pattern of diffuse reactivation. The finding that the risk of shedding was increased 10-fold when only nonadjacent sites were positive supports this hypothesis and is corroborated by evidence of an inflammatory response in tissue biopsy specimens from areas of reactivation. Determining whether these shedding patterns reflect either a simultaneous loss of control of latency at multiple neurons or a defect in local immunologic control resulting in increased peripheral viral replication will require further study. From a transmission standpoint, the linkages between shedding episode duration, virus quantity, and localization of shedding are likely to be critical. Still, it is difficult to determine if the primary driver of shedding duration is quantity (which might reflect the amount of virus released from neurons), space

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(which might reflect the number of neurons releasing virus), or time (which might reflect mucosal replication). Our participants shed on a median of 12 sites throughout this short study, suggesting that it may be important to inform patients that shedding, and therefore the possibility of transmission, is not confined to the site of genital herpes recurrences. This widespread genital shedding even in the absence of symptoms may explain the partial efficacy of condoms in preventing HSV acquisition (31), as viral shedding frequently occurs in areas not protected by condoms. That CD8⫹ T cells were significantly more numerous in asymptomatic biopsy specimens than in control biopsy specimens suggests that asymptomatic HSV shedding is associated with a targeted immunologic response in tissue and offers additional evidence that observed shedding patterns are not an artifact of contamination. We recently described a CD8-␣␣⫹ T cell population at the dermis-epidermis junction that functions in immune surveillance and early containment at sites of previous genital HSV-2

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FIG 4 (A) CD4 and CD8 staining in asymptomatic, lesion, and control biopsy specimens. CD4 (green) and CD8 (red) immunofluorescence staining of samples

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Downloaded from http://jvi.asm.org/ on June 5, 2014 by OAKLAND UNIV FIG 5 Representative intracellular cytokine cytometry using bulk expanded lymphocytes stimulated with UV–HSV-2 demonstrates that asymptomatic biopsy

specimens contain HSV-2-specific T cells. Cells produce gamma interferon (IFN-␥), IL-2, and TNF-␣. Percentages of positive cells gated on live CD3⫹, CD8⫺, and CD4⫹ cells are provided.

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Anatomic Characterization of Genital HSV-2 Shedding

lesions (12). We are conducting ongoing studies to determine associations between the quantity of CD8⫹ T cells and the rate of HSV shedding at anatomically defined sites. At asymptomatic biopsy sites, we observed HSV-2-specific CD4⫹ T cells that had an antiviral and proliferative phenotype, producing IFN-␥, TNF-␣, and IL-2 in response to HSV-2 antigen. We previously reported that HSV-2-specific CD4⫹ T cells can persist at sites of healed HSV-2 lesions for several weeks (32). It is possible, then, that the HSV-2-specific responses described in this report represent memory T cells that were holdovers from previous cycles of HSV-2 replication rather than T cells responsive to the current shedding episode. Alternatively, HSV-specific CD4⫹ T

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cells may have migrated from the circulation to the skin in response to the viral shedding detected the day prior to the biopsy, which would be consistent with mouse models of herpes showing that CD4⫹ T cells rapidly traffic to mucosal sites after antigen stimulation (33). It is critical to determine whether it is tissueresident memory or circulating CD4⫹ T cells that contain HSV, particularly for therapeutic vaccine development. Both incident and prevalent HSV-2 infection are associated with an increased risk of HIV acquisition (3), and CD4⫹ T cells found at the sites of HSV lesions bear the HIV coreceptors CCR5 and CXCR4 (17, 34). We found HSV-specific CD4⫹ T cells that produce proinflammatory cytokines in response to HSV stimula-

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FIG 6 HSV-specific T cells are found in lesion and asymptomatic biopsy specimens. (A) T cells expanded from a lesion biopsy specimen showed strong proliferative responses to full-length VP16 (gene UL48), UL37, and UL26. (B) T cells expanded from an asymptomatic biopsy specimen from the same participant showed a strong proliferative response to full-length VP16 (gene UL48). In panels A and B, data represent averages of the results of 2 reads. Red bars represent ORFs that met the criterion for positivity in 2 replicates; pink bars represent ORFs that were positive in only one replicate. (C) CD4⫹ T cells from the asymptomatic biopsy specimen produce IFN-␥ in response to whole HSV-2 antigen. (D) The VP16 (gene UL48) epitope was mapped to aa 187 to 199, and a Qdot655 oligomer containing DRB4*0101, and peptide was created. Cells expanded from the asymptomatic biopsy specimen show strong reactivity to the DRB4*0101-VP16 aa 187 to 199 multimers.

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ACKNOWLEDGMENTS We are indebted to the dedicated participants and clinicians (Michael Remington, Steve Kuntz, Negusse Ocbamichael, and Michael Stern) who made this study possible. C.J. has received research support from Aicuris GmbH. D.M.K. is listed as a coinventor on patents describing T cell responses to HSV-2 and is a consultant to Agenus Inc. A.W. has received research funding from Gilead, Agenus, and Genocea and has been a consultant for Aicuris. L.C. is on the scientific advisory board for and holds stock (⬍1% of company) in Immune Design Corp. and is a coinventor listed on several patents involving potential HSV vaccine development. This work was supported by the following grants: National Institutes

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of Health K23 AI079394 (C.J.), K24 AI071113 (A.W.), P01 AI30731 (C.J., A.M., D.M.K., L.C., A.W.), R56 AI093746 (J.Z.), R01 AI042528 (L.C., J.Z.), and R01 AI 094019 (D.M.K.).

REFERENCES 1. Looker KJ, Garnett GP, Schmid GP. 2008. An estimate of the global prevalence and incidence of herpes simplex virus type 2 infection. Bull. World Health Organ. 86(10):805– 812, A. http://dx.doi.org/10.2471/BLT .07.046128. 2. Paz-Bailey G, Rahman M, Chen C, Ballard R, Moffat HJ, Kenyon T, Kilmarx PH, Totten PA, Astete S, Boily MC, Ryan C. 2005. Changes in the etiology of sexually transmitted diseases in Botswana between 1993 and 2002: implications for the clinical management of genital ulcer disease. Clin. Infect. Dis. 41:1304 –1312. http://dx.doi.org/10.1086/496979. 3. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. 2006. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS 20:73– 83. http://dx.doi.org/10.1097/01.aids.0000198081.09337.a7. 4. Corey L, Adams HG, Brown ZA, Holmes KK. 1983. Genital herpes simplex virus infections: clinical manifestations, course, and complications. Ann. Intern. Med. 98:958 –972. http://dx.doi.org/10.7326/0003 -4819-98-6-958. 5. Wald A, Zeh J, Selke S, Warren T, Ryncarz AJ, Ashley R, Krieger JN, Corey L. 2000. Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. N. Engl. J. Med. 342:844 – 850. http://dx.doi.org/10.1056/NEJM200003233421203. 6. Wald A, Zeh J, Selke S, Ashley RL, Corey L. 1995. Virologic characteristics of subclinical and symptomatic genital herpes infections. N. Engl. J. Med. 333:770 –775. http://dx.doi.org/10.1056/NEJM199509213331205. 7. Tata S, Johnston C, Huang ML, Selke S, Magaret A, Corey L, Wald A. 2010. Overlapping reactivations of herpes simplex virus type 2 in the genital and perianal mucosa. J. Infect. Dis. 201:499 –504. http://dx.doi.org/10 .1086/650302. 8. Schiffer JT, Abu-Raddad L, Mark KE, Zhu J, Selke S, Magaret A, Wald A, Corey L. 2009. Frequent release of low amounts of herpes simplex virus from neurons: results of a mathematical model. Sci. Transl. Med. 1:7ra16. http://dx.doi.org/10.1126/scitranslmed.3000193. 9. Zhu J, Koelle DM, Cao J, Vazquez J, Huang ML, Hladik F, Wald A, Corey L. 2007. Virus-specific CD8⫹ T cells accumulate near sensory nerve endings in genital skin during subclinical HSV-2 reactivation. J. Exp. Med. 204:595– 603. http://dx.doi.org/10.1084/jem.20061792. 10. Crespi CM, Cumberland WG, Wald A, Corey L, Blower S. 2007. Longitudinal study of herpes simplex virus type 2 infection using viral dynamic modelling. Sex. Transm. Infect. 83:359 –364. http://dx.doi.org /10.1136/sti.2006.022020. 11. Schiffer JT, Abu-Raddad L, Mark KE, Zhu J, Selke S, Koelle DM, Wald A, Corey L. 2010. Mucosal host immune response predicts the severity and duration of herpes simplex virus-2 genital tract shedding episodes. Proc. Natl. Acad. Sci. U. S. A. 107:18973–18978. http://dx.doi.org/10.1073 /pnas.1006614107. 12. Zhu J, Peng T, Johnston C, Phasouk K, Kask AS, Klock A, Jin L, Diem K, Koelle DM, Wald A, Robins H, Corey L. 2013. Immune surveillance by CD8␣␣⫹ skin-resident T cells in human herpes virus infection. Nature 497:494 – 497. http://dx.doi.org/10.1038/nature12110. 13. Ashley RL, Militoni J, Lee F, Nahmias A, Corey L. 1988. Comparison of Western blot (immunoblot) and glycoprotein G-specific immunodot enzyme assay for detecting antibodies to herpes simplex virus types 1 and 2 in human sera. J. Clin. Microbiol. 26:662– 667. 14. Jerome KR, Huang ML, Wald A, Selke S, Corey L. 2002. Quantitative stability of DNA after extended storage of clinical specimens as determined by real-time PCR. J. Clin. Microbiol. 40:2609 –2611. http://dx.doi .org/10.1128/JCM.40.7.2609-2611.2002. 15. Gardella C, Huang ML, Wald A, Magaret A, Selke S, Morrow R, Corey L. 2010. Rapid polymerase chain reaction assay to detect herpes simplex virus in the genital tract of women in labor. Obstet. Gynecol. 115:1209 – 1216. http://dx.doi.org/10.1097/AOG.0b013e3181e01415. 16. Magaret AS, Wald A, Huang ML, Selke S, Corey L. 2007. Optimizing PCR positivity criterion for detection of herpes simplex virus DNA on skin and mucosa. J. Clin. Microbiol. 45:1618 –1620. http://dx.doi.org/10.1128 /JCM.01405-06. 17. Zhu J, Hladik F, Woodward A, Klock A, Peng T, Johnston C, Remington M, Magaret A, Koelle DM, Wald A, Corey L. 2009. Persistence of

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tion at asymptomatic shedding sites in nearly 40% of asymptomatic biopsy specimens; this supports the hypothesis that subclinical shedding episodes may augment the inflammatory milieu that increases HIV susceptibility. Since rapidly cleared episodes of subclinical shedding are the most common form of HSV reactivation (35), this immunologic response may contribute substantially to increased HIV risk. HSV-specific CD4⫹ T cells require further study to learn if they also express HIV coreceptors. While we were unable to assess the anatomic distribution of short episodes of shedding, we hypothesize that more-frequent sampling would demonstrate additional short episodes of anatomically defined shedding, as has been described with mixed anogenital sampling (35). This was the first study to collect samples in defined geographic areas. Each patient in our study had over 400 separate collections over a 30-day period. Our study was directed at obtaining samples carefully from each region, hence requiring office visits for each collection. Weekend and holidays interrupted sampling, leaving the true length of some shedding episodes unknown. Strengths of our study include excellent adherence, especially in light of the complex protocol and time demands on participants, and careful acquisition of clinical, virologic, and histopathologic data. The HSV-2 shedding frequency on a per-day basis is similar to that described in several previous cohorts with HSV-2 infection (24, 36), suggesting that the population studied in this report is similar to the large number of persons sampled in a less-detailed fashion. In addition, the duration of shedding episodes is similar to what has been reported in other once-daily sampling studies (27). Use of the rapid HSV PCR assay allowed us to capture asymptomatic shedding episodes and obtain biopsy specimens at sites with shedding within 24 h. Our sampling grid may have been too coarse to allow biopsies to capture all areas of focal HSV shedding; a finer grid could have improved our ability to target small areas of HSV shedding. While the study team worked to minimize contamination during sample collection, it is possible that participants selfcontaminated different regions through daily activities such as dressing and toilet use. However, our sensitivity analysis suggests that low-level contamination would not alter our findings indicating that reactivation of HSV-2 occurred simultaneously at multiple regions of the genital tract in immunocompetent men and women. This report demonstrates our ability to precisely define the spatial localization of HSV reactivation from tissue at sites of asymptomatic HSV shedding in the human host. Our findings reveal that HSV genital infection is widespread throughout the genital tract and that genital HSV shedding is associated with an HSV-specific inflammatory response in the genital skin, even in the absence of lesions.

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