HSV-specific CD8 Research Articles

Longitudinal Analysis of Herpes Simplex Virus–Specific CD4+Cell Clonotypes in Infected Tissues and Blood

Genital infection by herpes simplex virus (HSV)-2 offers a unique model for study of the effects of a remitting and exacerbating infection on the survival and persistence of antigen-specific T cells. We used complementarity-determining region 3 (CDR3) length analysis to examine the complete T cell receptor (TCR) beta -chain repertoire in skin-lesion biopsy samples from subjects with genital herpes. We found that herpetic skin lesions consistently demonstrated oligoclonal CDR3 DNA length distribution, indicating the presence of T cell expansions. Sequence analysis of representative HSV-specific lesional CD4(+) cell clones and TCR beta -variable (TCRBV) sequencing confirmed that the oligoclonal expansions were largely related to HSV-specific T cell proliferation. To assess the persistence of HSV-specific CD4(+) cells that localize to genital lesions, we developed a sensitive and highly specific clonal tracking technique using a combination of TCRBV-specific polymerase chain reaction, followed by liquid hybridization with clonotype-specific probes. Two different patterns of clonal persistence were observed. Some long-lasting clones appear to home to different epithelia, such as skin and genital mucosa, and to circulate in the peripheral blood, whereas others detected in lesions were absent or very rare in the peripheral blood.

Homing in on the Cellular Immune Response to HSV‐2 in Humans*

Genital herpes simplex infections are generally limited to epithelia and neurons. Vaccines have had activity in herpes simplex virus (HSV)-seronegative women only. Understanding how HSV-specific T cells traffic to infected sites may assist in vaccine design. Herpes simplex virus epitopes recognized by HSV-specific CD8 T cells were identified and used to make fluorescent human leukocyte antigen (HLA)-peptide tetramers. Molecules related to lymphocyte rolling adhesion were studied by flow cytometry and cell binding. HSV-specific CD4 T cells identified ex vivo by cytokine accumulation or activation marker expression, or detected in vitro by 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) dilution, were similarly investigated. Herpes simplex virus-specific T cells are 10- to 100-fold more prevalent in lesional skin compared with blood and greatly enriched in lesions compared with normal skin. Diverse viral antigens are recognized by HSV-specific T cells. Functionally active E-selectin ligand, and cutaneous lymphocyte antigen (CLA), are expressed by circulating HSV-2-specific CD8 cells. CD4 cells display lower levels of CLA that are dramatically up-regulated upon re-stimulation with antigen. Herpes simplex virus-2-specific CD8 and CD4 T cells differ in constitutive expression of skin homing molecules. Vaccines designed to induce proper homing are postulated to have increased efficacy.

The dual role of CD8 + T lymphocytes in the development of stress-induced herpes simplex encephalitis

Despite the generally restrictive nature of the blood–brain barrier (BBB), circulating lymphocytes can infiltrate into the central nervous system (CNS) during a variety of disease states. Although the contributions of these lymphocytes to CNS-associated disease have been identified in some viral models, the factors which govern this infiltration following herpes simplex virus (HSV) infection remain to be elucidated. We have developed a murine model of HSV encephalitis (HSE) to define the relationship among psychological stress, the recruitment of HSV-specific T cells into the CNS, and the development of HSE. Naive mice, as well as mice that had been vaccinated with a recombinant vaccinia virus (rVVESgB498–505) that elicits the generation of HSV-1 gB498–505-specific CD8 + T cells, were infected intranasally (i.n.) with HSV-1 McIntyre. Beginning one day prior to HSV-1 infection and continuing for a total of 9 days, naive and vaccinated mice were exposed to a well-established stressor, restraint stress. Naive, stressed mice exhibited increased symptoms of HSE and HSE-associated mortality as compared to non-stressed controls. A concomitant increase in CD4 + and CD8 + T cells in the brain was observed throughout the infection, with CD8 + T cells outnumbering CD4 + T cells. The development of HSE in these naive, stressed mice was accompanied by a delayed infiltration of gB498–505-specific CD8 + T cells after HSV spread into the brain. In contrast, both stressed and non-stressed rVVESgB498–505-vaccinated mice possessed gB498–505-specific CD8 + T cells prior to HSV challenge and were protected against HSE despite having detectable HSV-1 DNA in the brain. Together, these findings suggest that a delayed infiltration of CD8 + T cells into the brain may promote HSE in naive mice, while the presence of HSV-specific CD8 + T cells in the brain prior to HSV challenge is protective, possibly by limiting HSV replication and spread within the CNS.

Essential roles of perforin in antigen-specific cytotoxicity mediated by human CD4+ T lymphocytes: analysis using the combination of hereditary perforin-deficient effector cells and Fas-deficient target cells.

Although the cytotoxic mechanisms of murine CTLs have been investigated extensively using various mutant and knockout mice, those of human CTLs, especially CD4+ CTLs, are still obscure. To clarify the roles of perforin in Ag-specific cytotoxicity mediated by human CD4+ CTLs, alloantigen-specific and HSV-specific human CD4+ T lymphocyte bulk lines and clones were established from a patient with hereditary perforin deficiency and her healthy father, and their cytotoxic activities were investigated. Alloantigen-specific CD4+ T lymphocytes expressing perforin exerted cytotoxicity against Fas-negative as well as Fas-positive allogeneic B lymphoblastoid cell lines established from members of a family with hereditary Fas deficiency. Perforin-deficient, but not perforin-expressing, CD4+ T lymphocytes failed to show strong cytotoxicity against HSV-infected autologous B lymphoblastoid cells. Perforin-deficient CD4+ T lymphocytes could exert relatively low level cytotoxicity against allogeneic IFN-gamma-treated keratinocytes. Although cytotoxicity mediated by perforin-expressing CD4+ CTLs was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-mediated cytotoxic pathway, cytotoxicity against IFN-gamma-treated keratinocytes mediated by perforin-deficient CD4+ T lymphocytes was inhibited only partially by concanamycin A, but was inhibited significantly by antagonistic anti-Fas Ab and anti-Fas ligand Ab. The combination of perforin-deficient effector T lymphocytes and Fas-negative target cells used in the present study provides a novel experimental system for studying the detailed mechanisms of human CTL-mediated cytotoxicity. The present data demonstrate that perforin-negative CD4+ CTLs can exert cytotoxicity against Fas-sensitive target cells; however, perforin plays essential roles in Ag-specific cytotoxicity mediated by human CD4+ as well as CD8+ CTLs.

Vaginal Submucosal Dendritic Cells, but Not Langerhans Cells, Induce Protective Th1 Responses to Herpes Simplex Virus-2

Herpes simplex virus (HSV) type 2 infection occurs primarily at the genital mucosal surfaces and is a leading cause of ulcerative lesions. Despite the availability of animal models for HSV-2 infection, little is known regarding the mechanism of immune induction within the vaginal mucosa. Here, we examined the cell types responsible for the initiation of protective Th1 immunity to HSV-2. Intravaginal inoculation of HSV-2 led to a rapid recruitment of submucosal dendritic cells (DCs) to the infected epithelium. Subsequently, CD11c+ DCs harboring viral peptides in the context of MHC class II molecules emerged in the draining lymph nodes and were found to be responsible for the stimulation of IFNγ secretion from HSV-specific CD4+ T cells. Other antigen-presenting cells including B cells and macrophages did not present viral peptides to T cells in the draining lymph nodes. Next, we assessed the relative contribution to immune generation by the Langerhans cells in the vaginal epithelium, the submucosal CD11b+ DCs, and the CD8α+ lymph node DCs. Analysis of these DC populations from the draining lymph nodes revealed that only the CD11b+ submucosal DCs, but not Langerhans cell–derived or CD8α+ DCs, presented viral antigens to CD4+ T cells and induced IFNγ secretion. These results demonstrate a previously unanticipated role for submucosal DCs in the generation of protective Th1 immune responses to HSV-2 in the vaginal mucosa, and suggest their importance in immunity to other sexually transmitted diseases.

Herpes simplex virus (HSV)-associated erythema multiforme (HAEM): A viral disease with an autoimmune component.

Erythema multiforme (EM) is a clinical conundrum the name of which reflects the broad morphological spectrum of the lesions. Molecular and immunologic evidence that herpes simplex virus (HSV) causes a subset of EM lesions [herpes-associated EM (HAEM)] is reviewed, and new data are presented which suggest that autoreactive T-cells triggered by virus infection play an important role in HAEM pathogenesis. Disease development begins with viral DNA fragmentation and the transport of the DNA fragments to distant skin sites by peripheral blood mononuclear cells (PBMCs). HSV genes within DNA fragments deposited on the skin [notably DNA polymerase (Pol)] are expressed, leading to recruitment of HSV-specific CD4+ Th1 cells that respond to viral antigens with production of interferon-gamma (IFN-gamma). This step initiates an inflammatory cascade that includes expression of IFN-gamma induced genes, increased sequestration of circulating leukocytes, monocytes and natural killer (NK) cells, and the recruitment of autoreactive T-cells generated by molecular mimicry or the release of cellular antigens from lysed cells. The PBMCs that pick up the HSV DNA [viz. macrophages or CD34+ Langerhans cells (LC) precursors], their ability to process it, the viral proteins expressed in the skin and the presence of epitopes shared with cellular proteins may determine whether a specific HSV episode is followed by HAEM development. Drug-associated EM (DIEM) is a mechanistically distinct EM subset that involves expression of tumor necrosis factor alpha (TNF-alpha) in lesional skin. It is our thesis that the polymerase chain reaction (PCR) assay for HSV DNA detection in lesional skin and staining with antibodies to IFN-gamma and TNF-alpha, are important criteria for the diagnosis of skin eruptions and improved patient management.

Expression of cutaneous lymphocyte-associated antigen by CD8+ T cells specific for a skin-tropic virus

Virus-specific CD8+ T cells traffic to infected tissues to promote clearance of infection. We used herpes simplex virus type 2 (HSV-2) as a model system to investigate CD8+ T cell trafficking to the skin in humans. Using human leukocyte antigen (HLA) class I tetramers, we observed that HSV-specific CD8+ T cells in the peripheral blood expressed high levels of cutaneous lymphocyte-associated antigen (CLA). In contrast, CD8+ T cells specific for non–skin-tropic herpesviruses lacked CLA expression. CLA-positive HSV-2–specific CD8+ T cells had the characteristics of central memory cells, expressing CCR7, CD62L, and CD28, and they proliferated briskly in response to antigen. CLA is related to a functional E-selectin ligand, and both E-selectin and CLA-positive cells were detected in HSV-2–infected skin. HSV-2–specific T cells adhered to cells transfected with E-selectin. A higher proportion of HSV-specific CD8+ T cells recovered from herpes lesions express CLA compared with blood, consistent with a role for CLA in skin homing. To our knowledge, this is the first report of expression of tissue-specific adhesion-associated molecules by virus-specific CD8+ T cells. The evaluation of vaccines for skin and mucosal pathogens should include study of the induction of appropriate tissue-specific homing molecules.

Expression of cutaneous lymphocyte-associated antigen by CD8(+) T cells specific for a skin-tropic virus.

Virus-specific CD8(+) T cells traffic to infected tissues to promote clearance of infection. We used herpes simplex virus type 2 (HSV-2) as a model system to investigate CD8(+) T cell trafficking to the skin in humans. Using human leukocyte antigen (HLA) class I tetramers, we observed that HSV-specific CD8(+) T cells in the peripheral blood expressed high levels of cutaneous lymphocyte-associated antigen (CLA). In contrast, CD8(+) T cells specific for non-skin-tropic herpesviruses lacked CLA expression. CLA-positive HSV-2-specific CD8(+) T cells had the characteristics of central memory cells, expressing CCR7, CD62L, and CD28, and they proliferated briskly in response to antigen. CLA is related to a functional E-selectin ligand, and both E-selectin and CLA-positive cells were detected in HSV-2-infected skin. HSV-2-specific T cells adhered to cells transfected with E-selectin. A higher proportion of HSV-specific CD8(+) T cells recovered from herpes lesions express CLA compared with blood, consistent with a role for CLA in skin homing. To our knowledge, this is the first report of expression of tissue-specific adhesion-associated molecules by virus-specific CD8(+) T cells. The evaluation of vaccines for skin and mucosal pathogens should include study of the induction of appropriate tissue-specific homing molecules.

Development of HSV-specific CD4+ Th1 responses and CD8+ cytotoxic T lymphocytes with antiviral activity by vaccination with the HSV-2 mutant ICP10ΔPK

A growth compromised herpes simplex virus type 2 (HSV-2) mutant which is deleted in the PK domain of the large subunit of ribonucleotide reductase (ICP10ΔPK) protects from HSV-2 challenge in the mouse and guinea pig cutaneous and vaginal models and reduces the incidence and frequency of recurrent disease (Vaccine (17) (1999) 1951; Vaccine (19) (2001) 1879). The present studies were designed to identify the immune responses induced by ICP10ΔPK and define the component responsible for protective activity. We found that ICP10ΔPK elicits a predominant HSV-specific T helper type 1 (Th1) response, as evidenced by: (1) higher levels of HSV-specific IgG2a (Th1) than IgG1 (Th2) isotypes and (2) higher numbers of CD4+ IFN-γ than IL-10 secreting T cells in popliteal lymph nodes. This Th1 response pattern was associated with a significant increase in the levels of IL-12 produced by dendritic cells from ICP10ΔPK than HSV-2 immunized animals. Lymph node cells (LNCs) from ICP10ΔPK immunized mice had significantly higher levels of HSV-2 specific cytolytic activity than LNCs from mice immunized with HSV-2 and it was mediated by CD8+ T cells. CD8+ CTL were not seen in LNCs from HSV-2 immunized mice. In adoptive transfer experiments, CD8+ T cells and, to a lower extent, CD4+ T cells from ICP10ΔPK immunized mice inhibited HSV-2 replication, suggesting that they are involved in the protective immunity induced by ICP10ΔPK vaccination.

Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons.

We recently demonstrated that CD8(+) T cells could block herpes simplex virus type 1 (HSV-1) reactivation from latency in ex vivo trigeminal ganglion (TG) cultures without destroying the infected neurons. Here we establish that CD8(+) T-cell prevention of HSV-1 reactivation from latency is mediated at least in part by gamma interferon (IFN-gamma). We demonstrate that IFN-gamma was produced in ex vivo cultures of dissociated latently infected TG by CD8(+) T cells that were present in the TG at the time of excision. Depletion of CD8(+) T cells or neutralization of IFN-gamma significantly enhanced the rate of HSV-1 reactivation from latency in TG cultures. When TG cultures were treated with acyclovir for 4 days to insure uniform latency, supplementation with recombinant IFN-gamma blocked HSV-1 reactivation in 80% of cultures when endogenous CD8(+) T cells were present and significantly reduced and delayed HSV-1 reactivation when CD8(+) T cells or CD45(+) cells were depleted from the TG cultures. The effectiveness of recombinant IFN-gamma in blocking HSV-1 reactivation was lost when its addition to TG cultures was delayed by more than 24 h after acyclovir removal. We propose that when the intrinsic ability of neurons to inhibit HSV-1 gene expression is compromised, HSV-specific CD8(+) T cells are rapidly mobilized to produce IFN-gamma and perhaps other antiviral cytokines that block the viral replication cycle and maintain the viral genome in a latent state.

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

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-gamma. 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.

Tegument-specific, virus-reactive CD4 T cells localize to the cornea in herpes simplex virus interstitial keratitis in humans.

Herpes stromal keratitis (HSK) is a prevalent and frequently vision-threatening disease associated with herpes simplex virus type 1 (HSV-1) infection. In mice, HSK progression occurs after viral clearance and requires T cells and neutrophils. One model implicates Th1-like CD4 T cells with cross-reactivity between the HSV-1 protein UL6 and a corneal autoantigen. HSK can be prevented by establishing specific immunological tolerance. However, HSK can also occur in T-cell receptor-transgenic X SCID mice lacking HSV-specific T cells. To study the pathogenesis of HSK in the natural host species, we measured local HSV-specific T-cell responses in HSK corneas removed at transplant surgery (n = 5) or control corneas (n = 2). HSV-1 DNA was detected by PCR in two specimens. HSV-specific CD4 T cells were enriched in three of the five HSK specimens and were not detectable in the control specimens. Reactivity with peptide epitopes within the tegument proteins UL21 and UL49 was documented. Responses to HSV-1 UL6 were not detected. Diverse HLA DR and DP alleles restricted these local responses. Most clones secreted gamma interferon, but not interleukin-5, in response to antigen. HSV-specific CD8 cells were also recovered. Some clones had cytotoxic-T-lymphocyte activity. The diverse specificities and HLA-restricting alleles of local virus-specific T cells in HSK are consistent with their contribution to HSK by a proinflammatory effect.

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.

Dual Role of B Cells in Mediating Innate and Acquired Immunity to Herpes Simplex Virus Infections

μ-Immunoglobulin chain gene targeted B-cell-deficient mice of susceptible BALB/c strain and resistant C57B1/6 strain are up to 100- to 1000-fold more susceptible to cutaneous infection by herpes simplex virus (HSV) than the respective control wild type mice. The effect of the lack of B cells on immunity to HSV infections was analyzed and B cells were found to play a dual role in affecting both innate and acquired immune responses. Natural antibodies (IgM isotype), reactive with HSV have an anti-viral effect in the innate control of primary cutaneous HSV infection. B cells can also function as antigen-presenting cells for the stimulation of HSV-specific CD4+ T-cell responses. Consequently, CD4+ T cells and interferon-γ responses were found to be significantly impaired in HSV-infected B-cell-deficient mice compared to that seen in control mice. No significant differences were found in natural-killer-cell- or HSV-specific CD8+ T-cell activity between control and B-cell-deficient mice. Our results imply a role for B cell in mediating innate and CD4+ T-cell-specific immunity in determining susceptibility to primary HSV infections.

Frequencies of memory T cells specific for varicella-zoster virus, herpes simplex virus, and cytomegalovirus by intracellular detection of cytokine expression.

Memory T cells specific for varicella-zoster virus (VZV), herpes simplex virus (HSV), and human cytomegalovirus (HCMV) were compared in immune adults by intracellular cytokine (ICC) detection. The mean percentages of CD4+ T cells were 0.11% for VZV and 0.22% for HSV by interferon (IFN)-gamma production; the frequency for HCMV was significantly higher at 1.21%. Percentages of VZV-, HSV-, and HCMV-specific CD4+ T cells were similar by use of tumor necrosis factor (TNF)-alpha. HCMV-stimulated CD8+ T cells produced IFN-gamma (1.11%) and TNF-alpha (1.71%); VZV- and HSV-specific CD8+ T cells were not detectable. VZV CD4+ T cell numbers were similar in young adults with natural or vaccine-induced immunity. VZV CD4+ T cells were significantly less frequent in older adults. Secondary varicella immunization did not increase VZV-specific CD4+ T cell frequencies by ICC assay. Numbers of memory T cells specific for herpesviruses may vary with sites of viral latency and with host age.

Fas-Independent Cytotoxicity Mediated by Human CD4+ CTL Directed Against Herpes Simplex Virus-Infected Cells

The present study was undertaken to clarify the mechanisms of cytotoxicity mediated by virus-specific human CD4+ CTLs using the lymphocytes of family members with a Fas gene mutation. CD4+ CTL bulk lines and clones directed against HSV-infected cells were established from lymphocytes of a patient with a homozygous Fas gene mutation and of the patient's mother. HSV-specific CD4+ CTLs generated from lymphocytes of the patient and her mother exerted cytotoxicity against HSV-infected cells from the patient (Fas-/-) and from her mother (Fas+/-) to almost the same degree in an HLA class II-restricted manner. mRNAs for the major mediators of CTL cytotoxicity, Fas ligand, perforin, and granzyme B, were detected in these CD4+ CTLs using the RT-PCR and flow cytometry. The cytotoxicity of the HSV-specific CD4+ CTLs appeared to be Ca2+-dependent and was almost completely inhibited by concanamycin A, a potent inhibitor of the perforin-based cytotoxic pathway. Although the Fas/Fas ligand system has been reported to be the most important mechanism for CD4+ CTL-mediated cytotoxicity in the murine system, the present findings strongly suggest that granule exocytosis, not the Fas/Fas ligand system, is the main pathway for the cytotoxicity mediated by HSV-specific human CD4+ CTLs.

Phenotypic Characteristics Associated with the Acquisition of HSV-Specific CD8 T-Lymphocyte-Mediated Cytolytic Function in Vitro

Class I MHC-restricted, HSV-1-specific CD8+ cytolytic T lymphocyte (CTL) function is rarely detected in lymphocytes isolated directly from the lymph node draining the site of infection. However, culture in vitro for 24 to 72 h in the absence of exogenous antigen results in the development of easily detectable levels of HSV-1-specific CTL effectors. The inability to detect virus-specific CTL in HSV-1-infected mice is not well understood. However, since the in vitro culture of HSV-1-immune lymphocytes results in the transition to CTL function, studies of the changes occurring to the CD8+ T cell subpopulation may provide important insights into the development of virus-specific CTL. Therefore, the phenotypic changes taking place in the CD8+ population of T cells from draining popliteal lymph nodes of HSV-1-infected C57BL/6 (B6) mice were investigated, focusing on changes in the expression of cell surface markers associated with T lymphocyte activation. The results demonstrate an increase in the percentage of CD8+ T cells expressing the activation markers CD44 and CD25 in parallel with the acquisition of HSV-specific CTL effector function. Cytolytic function was found exclusively within the CD8+ CD44hi CD25hi fraction of cells in culture, but, surprisingly, was not detectable in CD8+ CD44hi CD25lo T cells. This suggested that the acquisition of high levels of the high-affinity IL-2 receptor was closely linked to cytolytic function and may define an important developmental stage in the transition from noncytolytic to cytolytic effector cell. In support of this, CD8+ CD25hi T cells isolated from the regional lymph node exhibited direct ex vivo cytolytic function, indicating that cytolytic effector cells were present in the lymph node, but must emigrate rapidly after attaining this level of differentiation.

Immunization with a single major histocompatibility complex class I-restricted cytotoxic T-lymphocyte recognition epitope of herpes simplex virus type 2 confers protective immunity.

We have evaluated the potential of conferring protective immunity to herpes simplex virus type 2 (HSV-2) by selectively inducing an HSV-specific CD8(+) cytotoxic T-lymphocyte (CTL) response directed against a single major histocompatibility complex class I-restricted CTL recognition epitope. We generated a recombinant vaccinia virus (rVV-ES-gB498-505) which expresses the H-2Kb-restricted, HSV-1/2-cross-reactive CTL recognition epitope, HSV glycoprotein B residues 498 to 505 (SSIEFARL) (gB498-505), fused to the adenovirus type 5 E3/19K endoplasmic reticulum insertion sequence (ES). Mucosal immunization of C57BL/6 mice with this recombinant vaccinia virus induced both a primary CTL response in the draining lymph nodes and a splenic memory CTL response directed against HSV gB498-505. To determine the ability of the gB498-505-specific memory CTL response to provide protection from HSV infection, immunized mice were challenged with a lethal dose of HSV-2 strain 186 by the intranasal (i.n.) route. Development of the gB498-505-specific CTL response conferred resistance in 60 to 75% of mice challenged with a lethal dose of HSV-2 and significantly reduced the levels of infectious virus in the brains and trigeminal ganglia of challenged mice. Finally, i.n. immunization of C57BL/6 mice with either a recombinant influenza virus or a recombinant vaccinia virus expressing HSV gB498-505 without the ES was also demonstrated to induce an HSV-specific CTL response and provide protection from HSV infection. This finding confirms that the induction of an HSV-specific CTL response directed against a single epitope is sufficient for conferring protective immunity to HSV. Our findings support the role of CD8(+) T cells in the control of HSV infection of the central nervous system and suggest the potential importance of eliciting HSV-specific mucosal CD8(+) CTL in HSV vaccine design.

Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by CD4 T Cells Infiltrating Human Genital Herpes Lesions

The local cellular immune response to herpes simplex virus (HSV) is important in the control of recurrent HSV infection. The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity, inhibition of viral growth, lymphokine secretion, and support of humoral and CD8 responses. The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells recognizing antigens encoded within map units 0. 67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions. Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase, encoded by genes UL49 and UL50, respectively, are T-cell antigens. Separate epitopes in VP22 were defined for T-cell clones from each of three patients. Reactivity with the tegument protein encoded by UL21 was identified for an additional patient. Three new epitopes were identified in VP16, a tegument protein associated with VP22. Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells. These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines.

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.