David M. Koelle

Clearance of HSV-2 from recurrent genital lesions correlates with infiltration of HSV-specific cytotoxic T lymphocytes.

The mechanisms involved in host clearance of symptomatic mucocutaneous herpes simplex virus (HSV) infection are unclear. We studied the functional properties of bulk cultures of skin-infiltrating lymphocytes from normal skin and serial biopsies of recurrent genital HSV-2 lesions, and compared HSV-specific and NK responses with viral clearance. HSV-specific CD4+ or CD8+ T cells were rarely detected in lymphocytes cultured from normal skin. The total lymphocyte count and HSV-specific and NK-like effector cell activities were markedly higher in cultures derived from lesional skin. HSV-specific CD4+ proliferative responses and NK-like cytotoxic responses were present at all stages of herpetic lesions, including biopsies early in the disease course. In contrast, cytotoxic T lymphocyte activity was generally low among cells derived from early culture-positive lesions, and increased during lesion evolution. Viral clearance from the lesion site was associated with a high level of local cytolytic activity towards HSV-infected cells. The phenotypes of cells with HSV-specific cytotoxic responses varied between patients, having CD4+ and CD8+ components. Immunotherapeutic approaches to HSV should be directed at improving in vivo cytolytic activity to HSV.

Recent Progress in Herpes Simplex Virus Immunobiology and Vaccine Research

SUMMARY Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause prevalent, chronic infections that have serious outcomes in some individuals. Neonatal herpes may occur when the infant traverses the cervix during maternal genital herpes. Genital herpes is a major risk factor for human immunodeficiency virus type 1 transmission. Considerable efforts have been made to design and test vaccines for HSV, focusing on genital infection with HSV-2. Several protein subunit vaccines based on HSV-2 envelope glycoproteins have reached advanced-phase clinical trials. These antigens were chosen because they are the targets of neutralizing-antibody responses and because they elicit cellular immunity. Encouraging results have been reported in studies of treatment of HSV-seronegative women with a vaccine consisting of truncated glycoprotein D of HSV-2 and a novel adjuvant. Because most sexual HSV transmission occurs during asymptomatic shedding, it is important to evaluate the impact of vaccination on HSV-2 infection, clinically apparent genital herpes, and HSV shedding among vaccine recipients who acquire infection. There are several other attractive formats, including subunit vaccines that target cellular immune responses, live attenuated virus strains, and mutant strains that undergo incomplete lytic replication. HSV vaccines have also been evaluated for the immunotherapy of established HSV infection.

Frequent Detection of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) DNA in Saliva of Human Immunodeficiency Virus-Infected Men: Clinical and Immunologic Correlates

The prevalence, quantity, temporal pattern, and clinical and immunologic correlates of shedding of Kaposis sarcoma (KS)-associated herpesvirus (KSHV; or human herpesvirus [HHV]-8) DNA in saliva were studied. KSHV DNA was detected in saliva from 18 (75%) of 24 human immunodeficiency virus (HIV)-positive patients with KS and from 1 of 1 HIV-negative patient with KS, 3 (15%) of 20 HIV-positive patients without KS, and none of 24 controls. KSHV DNA levels ranged from 10(2.4) to 10(6) copies/mL and were lower than levels for Epstein-Barr virus but comparable to those for HHV-6. Detection of KSHV DNA in saliva was not associated with oral KS or decreased peripheral blood CD4 cell counts. KSHV DNA was not detected in semen. Resistance of KSHV DNA from saliva to DNase treatment was consistent with the presence of virions. These data suggest that KSHV can replicate in the oropharynx and that salivary contact could contribute to KSHV transmission.

Famciclovir for the Suppression of Symptomatic and Asymptomatic Herpes Simplex Virus Reactivation in HIV-Infected Persons: A Double-Blind, Placebo-Controlled Trial

Persistent genital herpes simplex virus (HSV) infection was one of the first opportunistic infections described in persons with AIDS [1]. Although 60% to 80% of the general population has serum antibodies to HSV-1, HSV-2, or both [2-4], up to 95% of HIV-positive persons are seropositive for either HSV-1, HSV-2, or both [5-11]. This makes HSV one of the most common viral infections complicating HIV infection. Anecdotal reports indicate that clinical reactivations usually persist for an extended period, may involve many cutaneous and mucosal sites, and seem to increase with progression of HIV disease [12, 13]. In addition, long-term antiviral therapy is commonly used to suppress frequent HSV reactivations. Although the efficacy of such therapy is well established in immunocompetent persons [14-16], no published trials have quantitated the efficacy of suppressive antiviral therapy on HSV-1 or HSV-2 reactivation in HIV-infected persons. Famciclovir is a nucleoside analogue recently licensed for the treatment of herpes zoster and recurrent genital HSV infection [17]. To determine the efficacy of famciclovir for the suppression of HSV reactivation in HIV-infected persons, we did a double-blind, placebo-controlled, crossover trial in 48 persons with HIV infection. Methods Study Participants and Design Persons infected with both HIV and HSV were recruited into our study through advertisements in local newspapers and referrals from private physicians. The study was approved by the University of Washington institutional review board. We screened 123 persons and enrolled 48. Reasons for exclusion were lack of HSV antibodies (14%), inability to complete or lack of interest in completing 4 consecutive months of daily home culture (80%), and unwillingness to go without therapy during a recurrence of HSV (6%). At enrollment, each participant completed a standardized interview designed to record history of previous HSV reactivation, frequency of previous antiherpesvirus therapy, and extent of HIV disease. We excluded persons if they were younger than 18 years of age, were currently infected or had previously been infected with acyclovir-resistant HSV, had known gastrointestinal disorders affecting absorption, or had received suppressive antiviral therapy with acyclovir in the 6 months before enrollment. Patients were paid

CD8 CTL from Genital Herpes Simplex Lesions: Recognition of Viral Tegument and Immediate Early Proteins and Lysis of Infected Cutaneous Cells

300 for completion of the protocol; those who completed only the first arm of the protocol were paid

Role for HLA class II molecules in HIV-1 suppression and cellular immunity following antiretroviral treatment

150. At study entry, each participant was randomly assigned to receive either famciclovir tablets, 500 mg twice daily, or placebo tablets (identical in appearance to the famciclovir tablets) twice daily, for 8 weeks. This 8-week period was followed by a 7-day washout period, during which no pills were taken, and then by a second 8-week period during which participants received whichever regimen they had not received during the first phase of the trial. We chose this study design because it controlled for the variability between individual persons in CD4 cell count (which can affect the frequency of HSV reactivation [13]) and because a 7-day washout period had been shown not to influence subsequent HSV reactivation [18]. Famciclovir and placebo were dispensed in 28-day supplies (56 pills). During the entire 119-day study period, patients obtained once-daily cultures of the oropharynx, genitals (urethra and penile shaft in men; cervicovaginal area and labia in women), and rectum. Participants returned to the clinic at 28-day intervals so that we could review their daily symptom diaries, monitor compliance, distribute more study medication and culture supplies, and assess safety. All participants were also asked to return to the clinic during episodes of genital herpes. At these visits, we performed genital examinations to confirm the presence of lesions and obtained additional cultures of the lesions. Both participants and investigators were blinded to culture results until the conclusion of the study. Neither open-label oral acyclovir nor topical anti-HSV products for treatment of an HSV recurrence were allowed. Collection of Daily Cultures The methods used to collect swabs for HSV isolation have been described elsewhere [16, 18]. Each participant collected one specimen daily from each of four anatomic sites, using a separate Dacron swab for each culture. Oral-pharyngeal cultures were obtained by inserting a swab into the mouth and vigorously rubbing it along the gum line and over the palate. Men obtained urethral cultures by rubbing a swab over the urethral opening and obtained penile cultures by rubbing a separate swab along the entire ventral and dorsal shaft of the penis. Women obtained labial cultures by rubbing a swab over the entire labia majora and minora. Cervicovaginal cultures were obtained by rubbing a swab over the exocervix and posterior vaginal fornix. Rectal cultures were obtained by inserting the swab approximately 2 to 4 cm into the anus and gently rotating it. Each swab was placed in a separate vial that contained viral transport media, was labeled with site and date, and was stored in the refrigerator. Participants were instructed to obtain the cultures at the same time each day, preferably upon awakening. Cultures were picked up by a courier within 36 hours of collection and were transported to the virology laboratory of the University of Washington, where they were immediately inoculated into tissue culture. Participants also filled out daily diary cards that recorded the site-specific presence or absence of symptoms (pain, tingling, numbness or itching, presence of lesions) and the number of pills taken each day. The cards were collected monthly and reviewed by the study clinician. Unused cards of medication were collected, and pill counts were done to confirm compliance records. Laboratory Studies Serologic and T-Cell Analysis Seropositivity for HIV was confirmed by using standard enzyme-linked immunoassays and Western blot assays. CD4 cell subset analysis was done by using flow cytometry. Tests for antibodies to HSV were performed with Western blot analysis on serum specimens obtained at study entry [11, 19]. Patients were classified into three groups: those who were seropositive for HSV-1 only, those who were seropositive for HSV-2 only, and those who were seropositive for both HSV-1 and HSV-2. Herpes Simplex Virus Culture and Sensitivity Testing For isolation of HSV, each sample was inoculated in triplicate into 48-well microtiter plates that contained human diploid fibroblasts [20]. Each well was examined three times weekly for evidence of cytopathic changes of HSV infection. Cultures that showed cytopathic effects were confirmed and typed by using HSV-specific monoclonal antibodies. The initial culture supernatant from each clinical isolate that was obtained while patients were receiving famciclovir was inoculated into human diploid fibroblast cells [21]. Cell-associated virus (1:200 dilution in MEM [minimal essential media]-10% fetal calf serum) was inoculated in duplicate onto freshly confluent diploid fibroblast cells in 24-well plates. Plates were rocked at 30 tilts per minute for 60 minutes; penciclovir (Smith-Kline Beecham Pharmaceuticals, Philadelphia, Pennsylvania) containing medium was then added at 11 serial log2 dilutions from 40.96 to 0.04 g/mL. Control wells with no penciclovir were also established. When controls without penciclovir demonstrated 4+ cytopathic effect, the Hybriwick kit (Diagnostic Hybrids, Athens, Ohio) was used to measure HSV DNA [22] according to the manufacturers directions. The IC50 value was estimated as the lowest concentration of drug that caused a 50% or greater reduction of mean cycles per minute hybridization of HSV-specific DNA probe to cell lysates. Values for IC50 were calculated with a computer algorithm that used a Michaelis-Menten model and nonlinear least-squares curve fitting. Definition of Terms Total HSV shedding was defined as the total number of days on which HSV was isolated by culture (regardless of anatomic site) divided by the total number of days on which cultures were obtained. Asymptomatic HSV shedding was defined as the total number of days on which a participant reported no symptoms or lesions at an anatomic site from which HSV was isolated divided by the total number of days on which cultures were obtained. Symptomatic HSV shedding was defined as the total number of days on which a lesion or symptom was reported by the participant in the symptom diary at an anatomic site from which HSV was isolated divided by the total number of culture days. A recurrence of genital herpes was defined as lasting from the onset of lesions to the complete healing of lesions. Statistical Analysis Treatment effect was initially evaluated with intention-to-treat analyses that included all randomly assigned participants before crossover. Survival analysis was used to handle data censoring caused by early withdrawals. Time to first isolation of HSV (either HSV-1 or HSV-2) by culture, time to HSV-1 isolation, and time to HSV-2 isolation were examined. Differences in Kaplan-Meier survival curves between the famciclovir and placebo groups in the first treatment period were assessed by using the log-rank test. The relative risk for shedding was estimated by using the Cox proportional-hazards regression model with first-period treatment as the only predictor variable. To determine the effect of famciclovir on reduction of HSV-1 and HSV-2 shedding rates and days with symptoms, we used crossover analysis methods to further analyze participants who successfully completed both arms of the study. Successful completion was defined as more than 28 days with cultures in each treatment arm. Because HSV shedding and symptom rates were not normally distributed, nonparametric tests were used. Wilcoxon rank-sum tests for carryover (residual) and period effects compared the sums and differences, respectively, of rates during placebo and famciclovir administra

HSV-2: in pursuit of a vaccine

HSV-2 causes chronic infections. CD8 CTL may play several protective roles, and stimulation of a CD8 response is a rational element of vaccine design for this pathogen. The viral Ags recognized by CD8 T cells are largely unknown. It has been hypothesized that HSV inhibition of TAP may favor recognition of virion input proteins or viral immediate early proteins. We tested this prediction using HSV-specific CD8 CTL clones obtained from genital HSV-2 lesions. Drug and replication block experiments were consistent with specificity for the above-named classes of viral proteins. Fine specificity was determined by expression cloning using molecular libraries of viral DNA, and peptide epitopes recognized at nanomolar concentrations were identified. Three of four clones recognized the viral tegument proteins encoded by genes UL47 and UL49. These proteins are transferred into the cytoplasm on virus entry. Processing of the tegument Ag-derived epitopes was TAP dependent. The tegument-specific CTL were able to lyse HLA class I-appropriate fibroblasts after short times of infection. Lysis of keratinocytes required longer infection and pretreatment with IFN-γ. Another clone recognized an immediate early protein, ICP0. Lymphocytes specific for these lesion-defined epitopes could be reactivated from the PBMC of additional subjects. These data are consistent with an influence of HSV immune evasion genes upon the selection of proteins recognized by CD8 CTL in lesions. Tegument proteins, identified for the first time as Ags recognized by HSV-specific CD8 CTL, are rational candidate vaccine compounds.

Merkel polyomavirus-specific T cells fluctuate with merkel cell carcinoma burden and express therapeutically targetable PD-1 and Tim-3 exhaustion markers.

HIV-1-infected patients treated early with combination antiretrovirals respond favorably, but not all maintain viral suppression and improved HIV-specific Th function. To understand if genetic factors contribute to this variation, we prospectively evaluated over 18 months 21 early-treated patients stratified by alleles of class II haplotypes. All seven subjects with the DRB1*13-DQB1*06 haplotype, but only 21% of other subjects, maintained virus suppression at every posttreatment measurement. Following HIV-1 p24 antigen stimulation, PBMCs from patients with this haplotype demonstrated higher mean lymphoproliferation and IFN-gamma secretion than did cells from patients with other haplotypes. Two DRB1*13-restricted Gag epitope regions were identified, a promiscuous one that bound its putative restriction element with nanomolar affinity, and another that mapped to a highly conserved region. These findings suggest that class II molecules, particularly the DRB1*13 haplotype, have an important impact on virologic and immunologic responses. The advantage of the haplotype may relate to selection of key HIV-1 Th1 epitopes in highly conserved regions with avid binding to class II molecules. Eliciting responses to the promiscuous epitope region may be beneficial in vaccine strategies.

Standard-dose and high-dose daily antiviral therapy for short episodes of genital HSV-2 reactivation: three randomised, open-label, cross-over trials

Herpes simplex virus type 2 (HSV-2) is one of the most prevalent sexually transmitted infections worldwide. In addition to recurrent genital ulcers, HSV-2 causes neonatal herpes, and it is associated with a 3-fold increased risk for HIV acquisition. Although many HSV-2 vaccines have been studied in animal models, few have reached clinical trials, and those that have been tested in humans were not consistently effective. Here, we review HSV-2 pathogenesis, with a focus on novel understanding of mucosal immunobiology of HSV-2, and vaccine efforts to date, in an attempt to stimulate thinking about future directions for development of effective prophylactic and therapeutic HSV-2 vaccines.

Diversity in the Acute CD8 T Cell Response to Vaccinia Virus in Humans

Purpose: The persistent expression of Merkel cell polyomavirus (MCPyV) oncoproteins in Merkel cell carcinoma (MCC) provides a unique opportunity to characterize immune evasion mechanisms in human cancer. We isolated MCPyV-specific T cells and determined their frequency and functional status. Experimental Design: Multiparameter flow cytometry panels and HLA/peptide tetramers were used to identify and characterize T cells from tumors (n = 7) and blood (n = 18) of patients with MCC and control subjects (n = 10). PD-1 ligand (PD-L1) and CD8 expression within tumors were determined using mRNA profiling (n = 35) and immunohistochemistry (n = 13). Results: MCPyV-specific CD8 T cells were detected directly ex vivo from the blood samples of 7 out of 11 (64%) patients with MCPyV-positive tumors. In contrast, 0 of 10 control subjects had detectable levels of these cells in their blood (P < 0.01). MCPyV-specific T cells in serial blood specimens increased with MCC disease progression and decreased with effective therapy. MCPyV-specific CD8 T cells and MCC-infiltrating lymphocytes expressed higher levels of therapeutically targetable PD-1 and Tim-3 inhibitory receptors compared with T cells specific to other human viruses (P < 0.01). PD-L1 was present in 9 of 13 (69%) MCCs and its expression was correlated with CD8-lymphocyte infiltration. Conclusions: MCC-targeting T cells expand with tumor burden and express high levels of immune checkpoint receptors PD-1 and Tim-3. Reversal of these inhibitory pathways is therefore a promising therapeutic approach for this virus-driven cancer. Clin Cancer Res; 19(19); 5351–60. ©2013 AACR.