Immunology in the Clinic Review Series; focus on host responses: T cell responses to herpes simplex viruses.

Herpes virus infections are chronic and co-exist with acquired immune responses that generally prevent severe damage to the host, while allowing periodic shedding of virus and maintenance of its transmission in the community. Herpes simplex viruses type 1 and 2 (HSV-1, HSV-2) are typical in this regard and are representative of the viral subfamily Alphaherpesvirinae, which has a tropism for neuronal and epithelial cells. This review will emphasize recent progress in decoding the physiologically important CD8(+) and CD4(+) T cell responses to HSV in humans. The expanding data set is discussed in the context of the search for an effective HSV vaccine as therapy for existing infections and to prevent new infections.

HLA-DQB1 codon 57 is critical for peptide binding and recognition.

The association of specific HLA-DQ alleles with autoimmunity is correlated with discrete polymorphisms in the HLA-DQ sequence that are localized within sites suitable for peptide recognition. The polymorphism at residue 57 of the DQB1 polypeptide is of particular interest since it may play a major structural role in the formation of a salt bridge structure at one end of the peptide-binding cleft of the DQ molecules. This polymorphism at residue 57 is a recurrent feature of HLA-DQ evolution, occurring in multiple distinct allelic families, which implies a functional selection for maintaining variation at this position in the class II molecule. We directly tested the amino acid polymorphism at this site as a determinant for peptide binding and for antigen-specific T cell stimulation. We found that a single Ala-->Asp amino acid 57 substitution in an HLA-DQ3.2 molecule regulated binding of an HSV-2 VP-16-derived peptide. A complementary single-residue substitution in the peptide abolished its binding to DQ3.2 and converted it to a peptide that can bind to DQ3.1 and DQ3.3 Asp-57-positive MHC molecules. These binding studies were paralleled by specific T cell recognition of the class II-peptide complex, in which the substituted peptide abolished T cell reactivity, which was directed to the DQ3.2-peptide complex, whereas the same T cell clone recognized the substituted peptide presented by DQ3.3, a class II restriction element differing from DQ3.2 only at residue 57. This structural and functional complementarity for residue 57 and a specific peptide residue identifies this interaction as a key controlling determinant of restricted recognition in HLA-DQ-specific immune response.

Multicenter study of QuantiFERON®-TB Gold Plus in patients with active tuberculosis.

SETTING: QuantiFERON®-TB Gold Plus (QFT-Plus), recently approved for use in the United States, is a new-generation QuantiFERON assay that differs from its predecessors in that it uses an additional antigen tube containing peptides to elicit both CD8+ and CD4+ T-lymphocyte responses. OBJECTIVE: To assess the sensitivity of QFT-Plus compared with QuantiFERON®-TB Gold In-Tube (QFT-GIT) in participants with active TB. DESIGN: Adult patients with active TB at three US and two Japanese sites were eligible for this study if they had culture-confirmed TB and were either untreated or had received 14 days of anti-tuberculosis treatment. RESULTS: We enrolled 164 participants, nine of whom had indeterminate results. Excluding indeterminate values, there were 150 QFT-GIT-positive results among 159 tests and 146 QFT-Plus-positive results among 157 tests, with sensitivities of respectively 94.3% (95%CI 89.5-97.4) and 93.02% (95%CI 87.8-96.5%). The estimated sensitivities for the two tests were not significantly different (P = 0.16). Overall test agreement was 98.7%, with a κ statistic of 0.89 (95%CI 0.75-1.00). CONCLUSION: In this multisite study, we found that QFT-Plus had similar sensitivity to QFT-GIT in adult patients with active TB.

Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

Herpes simplex virus infection of human fibroblasts and keratinocytes inhibits recognition by cloned CD8+ cytotoxic T lymphocytes.

CD8+ cytotoxic T lymphocytes (CTL) clones with specificity for herpes simplex virus (HSV) were derived from two donors with genital HSV-2 infection. These CTL clones specifically lysed HSV-infected autologous B lymphoblastoid cells, but not HSV-infected fibroblasts. Exogenous peptide loading sensitized both cell types to lysis by an HSV-specific CTL clone of known specificity. HSV infection rendered fibroblasts refractory to peptide sensitization. HSV infection also rendered fibroblasts and keratinocytes insensitive to lysis by allospecific CD8+ CTL clones. Lysis of B lymphoblastoid cells in this system was only slightly reduced by HSV infection. Reduction of fibroblast allospecific lysis was dose and time dependent and was blocked by acyclovir, indicating the involvement of a late HSV gene product. HSV caused a reduction of fibroblast cell surface HLA class I antigen, at least in part due to reduction of synthesis of heavy chain-beta 2 microglobulin heterodimers. These results suggest that HSV-induced blockade of antigen presentation by cutaneous cells to CD8+ CTL may be a mechanism by which HSV limits or evades the immune response of the host.

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.

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

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.

Enrichment of herpes simplex virus type 2 (HSV-2) reactive mucosal T cells in the human female genital tract.

Local mucosal cellular immunity is critical in providing protection from HSV-2. To characterize and quantify HSV-2-reactive mucosal T cells, lymphocytes were isolated from endocervical cytobrush and biopsy specimens from 17 HSV-2-infected women and examined ex vivo for the expression of markers associated with maturation and tissue residency and for functional T-cell responses to HSV-2. Compared with their circulating counterparts, cervix-derived CD4+ and CD8+ T cells were predominantly effector memory T cells (CCR7-/CD45RA-) and the majority expressed CD69, a marker of tissue residency. Co-expression of CD103, another marker of tissue residency, was highest on cervix-derived CD8+ T cells. Functional HSV-2 reactive CD4+ and CD8+ T-cell responses were detected in cervical samples and a median of 17% co-expressed CD103. HSV-2-reactive CD4+ T cells co-expressed IL-2 and were significantly enriched in the cervix compared with blood. This first direct ex vivo documentation of local enrichment of HSV-2-reactive T cells in the human female genital mucosa is consistent with the presence of antigen-specific tissue-resident memory T cells. Ex vivo analysis of these T cells may uncover tissue-specific mechanisms of local control of HSV-2 to assist the development of vaccine strategies that target protective T cells to sites of HSV-2 infection.

Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition.

Peptides derived from the HSV-2 VP16 protein were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide VP16 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., VP16 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.

Preferential presentation of herpes simplex virus T-cell antigen by HLA DQA1*0501/DQB1*0201 in comparison to HLA DQA1*0201/DQB1*0201.

The HLA DQA1 locus is polymorphic. Haplotypes containing HLA DQA1*0501, but not HLA DQA1*0201, together with HLA DQB1*0201 are associated with Grave's disease and celiac sprue. In this report, we demonstrate a functional correlate of DQA1 polymorphism. T cells infiltrating a herpes simplex virus (HSV) lesion from a HLA DQ 2,7 individual yielded a virus-specific CD4+ clone restricted by DQ2. Presentation of viral peptide and protein segregated with DQA1 allele, because cell lines bearing DQA1*0501/DQB1*0201 heterodimers presented antigen in proliferation and cytotoxicity assays much more efficiently than cell lines bearing DQA1*0201/DQB1*0201. Binding of viral peptide to cell lines bearing DQA1*0201, in comparison to DQA1*0501, was only moderately reduced and may not explain this effect. Truncation and substitution analyses of peptide binding and T-cell activation were performed to determine which viral peptide residues contacting TCR might therefore be presented in an altered conformation by DQA1*0201/DQB1*0201. Residues 432, 435, 437, 438, and 440 (position P1, P4, P6, P7, and P9) contributed to DQ2 binding, whereas residues 431, 433, 434, and 436 (positions P 1, P2, P3, and P5) contributed to TCR contact. Differential presentation of peptide by HLA DQ2 heterodimers varying at the DQA1 locus may have relevance to host defense and the pathogenesis of HLA DQ2-associated autoimmune diseases.

Prevalence of human herpesvirus-8 salivary shedding in HIV increases with CD4 count.

Human herpesvirus-8 (HHV-8) is the etiologic agent of Kaposi's sarcoma (KS), which occurs in epidemic form in human immunodeficiency virus(HIV)-infected individuals. Saliva is the only mucosal fluid in which infectious HHV-8 has been identified, although factors associated with HHV-8 salivary shedding remain unclear. Our study performed PCR analysis for HHV-8 DNA in saliva (and other body fluids) in 66 HIV- and HHV-8-co-infected women without KS so that we could examine predictors for HHV-8 DNA detection. CD4 count was the most significant predictor of HHV-8 salivary shedding, with increased prevalence of HHV-8 salivary DNA at higher CD4 counts. The odds of salivary HHV8 shedding at CD4 counts > = 350 cells/microL was 63 times the odds of shedding at CD4 < 350 (95%CI, 1.3-3078), with an increase in effect size when the analysis was restricted to those with a CD4 nadir > 200. Analysis of these data suggests an increased potential for HHV-8 transmission early in HIV infection, with implications for HHV-8 prevention.

Development of a low-dose fast-dissolving tablet formulation of Newcastle disease vaccine for low-cost backyard poultry immunisation.

The immunisation of backyard poultry is critical for maintaining healthy flocks to provide nutrition and income for low-resource farmers worldwide. A vaccine presentation for flocks of less than 50 birds could make it more affordable and accessible, increasing uptake and impact. Fast-dissolving tablets (FDT) of Newcastle disease virus (NDV) vaccine were produced by freeze drying the LaSota NDV strain combined with excipients into tablets containing a small number of doses and packaged in polymer blister sheets. The NDV-FDT vaccine maintained virus stability for more than six months at 4°C, based on plaque assay and egg infectivity dose data. Stability was further confirmed in a challenge study, where the tablet vaccine elicited a strong immune response and provided 100 per cent protection to vaccinated chickens infected with a virulent strain of NDV. The vaccine tablet can be diluted in water (no needle or syringe required) and administered either in drinking water or with a dropper via an intraocular and/or intransal route. Results indicate that FDTs containing a small number of doses are a feasible presentation for backyard poultry farmers. The compact packaging of the FDTs will also provide cost savings in storing and distributing the vaccine in the cold chain.

Herpes simplex virus: the importance of asymptomatic shedding.

Herpes simplex virus (HSV) is frequently shed after infection of the genital or perianal area. HSV shedding, as determined by culture, occurs on about 3% of days for immunocompetent women and men, and more for persons with HIV infection or if measured by polymerase chain reaction (PCR). Most horizontal and vertical transmission of HSV occurs during unrecognized or asymptomatic shedding, and the majority of HSV-2-infected persons are unaware of their infection. Many persons with 'asymptomatic' HSV-2 infection can learn to recognize genital signs and symptoms as recurrences of HSV-2 infection. However, some shedding episodes remain truly asymptomatic even after patient education. Antiviral therapy dramatically reduces asymptomatic shedding, and trials to evaluate its effect on HSV transmission are underway.

Antigen-specific T cells localize to the uterine cervix in women with genital herpes simplex virus type 2 infection.

Genital reinfection with herpes simplex virus type 2 (HSV-2) is uncommon in humans. The mechanism of acquired immunity is unknown. Because the cervix is a site of HSV exposure, we measured antigen-specific T cell responses to HSV in cervical lymphocytes during both lesional and nonlesional time periods. Cells were expanded without secondary in vitro stimulation with antigen. Proliferative and cytotoxic responses to HSV were detectable in specimens from most subjects. Limiting dilution assays showed a high frequency of antigen-specific cells. Cytotoxic T cell responses included both CD4 and CD8 components. Responses were present both during and between symptomatic infection episodes and persisted during suppressive antiviral therapy. Natural infection with HSV-2 is associated with a persistent cervical mucosal cellular immune response. This local response may possibly assist in limiting the clinical consequences of secondary HSV-2 infection, whether due to endogenous reactivation or exogenous reinfection.

Asymptomatic reactivation of herpes simplex virus in women after the first episode of genital herpes.

OBJECTIVE: To determine frequency, anatomic site, and host factors associated with asymptomatic shedding of herpes simplex virus after initial episodes of genital herpes. DESIGN: Cohort study with follow-up for a median of 63 weeks. SETTING: Referral clinic. PATIENTS: Women (306) with first episode of herpes; 43 had primary herpes simplex virus type 1, and 227 and 36 had primary and nonprimary herpes simplex virus type 2, respectively. MEASUREMENTS: Cultures were obtained for herpes simplex virus every 4 to 6 weeks at times in which genital lesions and symptoms were not present. MAIN RESULTS: Asymptomatic shedding was detected among 11.9%, 18.3%, and 22.9% of women with primary herpes simplex virus type 1, primary HSV type 2, and nonprimary HSV type 2, respectively. Among patients with type 2 infection, previous type 1 antibody was associated with a lower rate of asymptomatic vulvar shedding. Asymptomatic cervical shedding was 3 times more frequent during the first three months after resolution of primary type 2 disease than during later time periods. In contrast, the rate of symptomatic recurrent herpes did not change over time. CONCLUSIONS: Asymptomatic genital herpes simplex type 2 is more common than type 1. Asymptomatic genital shedding occurs more often during the first 3 months after acquisition of primary type 2 disease than during later periods. Patients with HSV type 2 should be advised of this high early rate of asymptomatic shedding and of potential transmission to sexual partners.

High frequency of CD8+ cytotoxic T-lymphocyte precursors specific for herpes simplex viruses in persons with genital herpes.

Herpes simplex virus (HSV)-specific CD8+ cytotoxic T lymphocytes (CTL) have rarely been detected in humans, presumably because of virus-induced mechanisms that downregulate major histocompatibility complex class I expression. We have developed a method that has allowed us to consistently demonstrate HSV-specific CD8+ precursor CTL (pCTL) from HSV type 1- and 2-seropositive persons. Major histocompatibility complex-restricted HSV-specific CD8+ pCTL were found in 10 consecutively tested HSV type 1- and 2-seropositive subjects at frequencies ranging from 1 in 21,000 to 1 in 300 (median, 1 in 6,000) versus a pCTL frequency of 1 in 100,000 in HSV-seronegative donors. These results suggest that CD8+ CTL are important effector cells in resolving HSV lesions.

Long term persistence of herpes simplex virus-specific CD8+ CTL in persons with frequently recurring genital herpes.

Herpes simplex virus (HSV) establishes a lifelong infection in humans. Reactivation of latent virus occurs intermittently so that the immune system is frequently exposed to viral Ag, providing an opportunity to evaluate memory T cells to a persistent human pathogen. We studied the persistence of genital herpes lesion-derived HSV-specific CD8+ CTL from three immunocompetent individuals with frequently recurring genital HSV-2 infection. All CTL clones were HSV-2 type specific and only one to three unique clonotypes were identified from any single biopsy specimen. The TCRBV genes utilized by these clonotypes were sequenced, and clonotype-specific probes were used to longitudinally track these clonotypes in PBMC and genital lesions. CTL clonotypes were consistently detected in PBMC and lesions for at least 2 and up to 7 years, and identical clonotypes infiltrated herpes lesions spaced as long as 7.5 years apart. Moreover, these clones were functionally lytic in vivo over these time periods. Additionally, CTL clones killed target cells infected with autologous viral isolates obtained 6.5 years after CTL clones were established, suggesting that selective pressure by these CTL did not result in the mutation of CTL epitopes. Thus, HSV recurs in the face of persistent CD8+ CTL with no evidence of clonal exhaustion or mutation of CTL epitopes as mechanisms of viral persistence.

Emergence of ganciclovir-resistant cytomegalovirus disease among recipients of solid-organ transplants.

BACKGROUND: Concerns have been raised about emergence of ganciclovir resistance as a result of the advent of both routine oral ganciclovir prophylaxis and highly potent immunosuppression. We retrospectively assessed the occurrence of ganciclovir-resistant cytomegalovirus disease among transplant recipients who had received oral ganciclovir prophylaxis and highly potent immunosuppression. METHODS: We studied 240 recipients of liver, kidney, or pancreas transplants. Antiviral susceptibility testing of blood cytomegaloviral isolates was done when patients failed to respond to intravenous ganciclovir treatment for symptomatic cytomegalovirus infection. Portions of the UL97 gene associated with ganciclovir resistance were sequenced in cytomegalovirus isolates with phenotypic resistance to ganciclovir. FINDINGS: Ganciclovir-resistant cytomegalovirus disease developed in five (7%) of 67 seronegative recipients of cytomegalovirus-seropositive organs (D+/R-) compared with none of 173 seropositive recipients (p=0.002). Among the 25 (10.4%) patients who developed cytomegalovirus disease within 1 year after transplantation, five had ganciclovir-resistant cytomegalovirus disease. Among D+/R-transplant recipients, ganciclovir-resistant cytomegalovirus disease was more common among the group receiving the most potent immunosuppression--ie, recipients of kidney and pancreas or pancreas alone (four of 19) compared with all other transplant recipients (one of 48, p=0.02). Ganciclovir-resistant cytomegalovirus disease was diagnosed at a median of 10 months after transplantation (range 7-12) after lengthened exposure to ganciclovir, was associated with previously described mutations of the UL97 gene, and led to serious clinical complications. INTERPRETATION: Ganciclovir-resistant cytomegalovirus is an important cause of late morbidity among D+/R- transplant recipients who have had lengthened exposure to ganciclovir and have received highly potent immunosuppression. Strategies to reduce this complication, especially among D+/R- patients, are warranted.