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 U_L49 and U_L50, 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 U_L21 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.     

动物生殖器疱疹的实验治疗研究

探讨观察中药MAG抗HSV效果.建立实验动物HSV感染模型,分组进行局部治疗与局部、全身联合治疗对比研究.局部、全身联合治疗有效.中药MAG对家兔阴道局部黏膜无刺激作用和毒性反应,对HSV有一定的直接灭活性作用.     

Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.     

Prevalence of Herpes Simplex Virus Type 1- and 2- Specific Antibodies among the Acute,Recurrent, and Provoked Types of Female Genital Herpes

Sixty-eight sera from the acute, recurrent, and provoked types of female genital herpes were compared for the seroprevalence of herpes simplex virus (HSV) types 1 and 2 by immunodot assay using HSV glycoprotein G. In the HSV-1-isolated patients, no HSV-2 antibodies were detected, whereas in the HSV-2-isolated patients, HSV-1 seroprevalence was 9% for the acute type, 89% for the provoked type (P< 0.005), and 55% for the recurrent type (P<0.05). The natural history of female genital herpes and the possible protective role of pre-existing antibodies in preventing the acquisition or clinical manifestation of a subsequent HSV infection are discussed.     

Immunogenicity and Efficacy of Intramuscular Replication-Defective and Subunit Vaccines against Herpes Simplex Virus Type 2 in the Mouse Genital Model

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted virus that is highly prevalent worldwide, causing a range of symptoms that result in significant healthcare costs and human suffering. ACAM529 is a replication-defective vaccine candidate prepared by growing the previously described dl5-29 on a cell line appropriate for GMP manufacturing. This vaccine, when administered subcutaneously, was previously shown to protect mice from a lethal vaginal HSV-2 challenge and to afford better protection than adjuvanted glycoprotein D (gD) in guinea pigs. Here we show that ACAM529 given via the intramuscular route affords significantly greater immunogenicity and protection in comparison with subcutaneous administration in the mouse vaginal HSV-2 challenge model. Further, we describe a side-by-side comparison of intramuscular ACAM529 with a gD vaccine across a range of challenge virus doses. While differences in protection against death are not significant, ACAM529 protects significantly better against mucosal infection, reducing peak challenge virus shedding at the highest challenge dose by over 500-fold versus 5-fold for gD. Over 27% (11/40) of ACAM529-immunized animals were protected from viral shedding while 2.5% (1/40) were protected by the gD vaccine. Similarly, 35% (7/20) of mice vaccinated with ACAM529 were protected from infection of their dorsal root ganglia while none of the gD-vaccinated mice were protected. These results indicate that measuring infection of the vaginal mucosa and of dorsal root ganglia over a range of challenge doses is more sensitive than evaluating survival at a single challenge dose as a means of directly comparing vaccine efficacy in the mouse vaginal challenge model. The data also support further investigation of ACAM529 for prophylaxis in human subjects.     

Diagnosis of Genital Herpes by Polymerase Chain Reaction Amplification

A new polymerase chain reaction (PCR) method employing type-specific primers and probes was applied to 114 clinical specimens obtained from 58 female patients with genital lesions or who had a history of genital herpes. Ten and 15 specimens, respectively, were positive for herpes simplex virus (HSV)-1 and HSV-2 by cell culture. All of 10 culture-confirmed HSV-1 cases and 11 of 15 (73%) culture-confirmed HSV-2 cases were identified by PCR. Although there were several cases with discrepancy between cell culture and PCR for HSV-2, the results suggest that this PCR procedure could be applied to clinical specimens from the female genital tract.     

Genetic Relatedness of Type 1 and Type 2 Herpes Simplex Viruses

The extent of homology between herpes simplex virus(1) and(2) (HSV-1 and HSV-2) deoxyribonucleic acid (DNA) was measured in two ways: (i) by determination of the relative rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA immobilized on filters and (ii) by determination of the rate of hybridization of labeled HSV-1 and HSV-2 DNA to excess unlabeled HSV-1 or HSV-2 DNA in solution. Approximately 40% of HSV-1 and HSV-2 DNA is homologous at hybridization temperatures 25 C below the melting temperature (T(m)) of HSV DNA (liquid-filter annealing). Lowering the temperature to 34 C below the T(m) increased the extent of homology to 46% (liquid annealing). The extent of base-pairing in HSV-1-HSV-2 heteroduplex DNA was determined by thermal chromatography on hydroxyapatite. Heteroduplexes of HSV-1 and HSV-2 DNA eluted in a single peak whose midpoint (Te(50)) was 10 C below that of the homoduplex. Conspicuously absent were heteroduplexes that eluted at more than 15 C below the Te(50) of the homoduplex. The data indicate the existence of a variable region of DNA (54%) with very little, if any, homology and an invariable region (46%) with relatively good (85%) matching of base pairs.     

Effects of Genital Ulcer Disease and Herpes Simplex Virus Type 2 on the Efficacy of Male Circumcision for HIV Prevention: Analyses from the Rakai Trials

Background

Randomized trials show that male circumcision (MC) reduces the incidence of HIV and herpes simplex virus type 2 (HSV-2) infections, and symptomatic genital ulcer disease (GUD). We assessed the role of GUD and HSV-2 in the protection against HIV afforded by MC.

Methods and Findings

HIV-uninfected men were randomized to immediate (n = 2,756) or delayed MC (n = 2,775) in two randomized trials in Rakai, Uganda. GUD symptoms, HSV-2 status, and HIV acquisition were determined at enrollment and at 6, 12, and 24 mo of follow up. Ulcer etiology was assessed by PCR. We estimated the prevalence and prevalence risk ratios (PRRs) of GUD in circumcised versus uncircumcised men and assessed the effects of HSV-2 serostatus as a risk-modifying factor for GUD. We estimated the proportion of the effect of MC on HIV acquisition that was mediated by symptomatic GUD, and by HSV-2 infection. Circumcision significantly reduced symptomatic GUD in HSV-2-seronegative men (PRR = 0.51, 95% [confidence interval] CI 0.43–0.74), HSV-2-seropositive men (PRR = 0.66, 95% CI 0.51–0.69), and in HSV-2 seroconverters (PRR = 0.48, 95% CI 0.30–0.79). The proportion of acute ulcers due to HSV-2 detected by PCR was 48.0% in circumcised men and 39.3% in uncircumcised men (χ² p = 0.62). Circumcision reduced the risk of HIV acquisition in HSV-2 seronegative men (incidence rate ratio [IRR] = 0.34, 95% CI 0.15–0.81), and potentially in HSV-2 seroconverters (IRR = 0.56, 95% CI 0.19–1.57; not significant), but not in men with prevalent HSV-2 at enrollment (IRR = 0.89, 95% CI 0.49–1.60). The proportion of reduced HIV acquisition in circumcised men mediated by reductions in symptomatic GUD was 11.2% (95% CI 5.0–38.0), and the proportion mediated by reduced HSV-2 incidence was 8.6% (95% CI −1.2 to 77.1).

Conclusions

Circumcision reduced GUD irrespective of HSV-2 status, but this reduction played only a modest role in the protective effect of circumcision on HIV acquisition.

NIH Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00425984","term_id":"NCT00425984"}}NCT00425984

Gates Foundation Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00124878","term_id":"NCT00124878"}}NCT00124878 Please see later in the article for the Editors'' Summary     

In Vivo Modulation of Vaccine-Induced Immune Responses toward a Th1 Phenotype Increases Potency and Vaccine Effectiveness in a Herpes Simplex Virus Type 2 Mouse Model

Several vaccines have been investigated experimentally in the herpes simplex virus type 2 (HSV-2) model system. While it is believed that CD4⁺-T-cell responses are important for protection in general, the correlates of protection from HSV-2 infection are still under investigation. Recently, the use of molecular adjuvants to drive vaccine responses induced by DNA vaccines has been reported in a number of experimental systems. We sought to take advantage of this immunization model to gain insight into the correlates of immune protection in the HSV-2 mouse model system and to further explore DNA vaccine technology. To investigate whether the Th1- or Th2-type immune responses are more important for protection from HSV-2 infection, we codelivered the DNA expression construct encoding the HSV-2 gD protein with the gene plasmids encoding the Th1-type (interleukin-2 [IL-2], IL-12, IL-15, and IL-18) and Th2-type (IL-4 and IL-10) cytokines in an effort to drive immunity induced by vaccination. We then analyzed the modulatory effects of the vaccine on the resulting immune phenotype and on the mortality and the morbidity of the immunized animals following a lethal challenge with HSV-2. We observed that Th1 cytokine gene coadministration not only enhanced the survival rate but also reduced the frequency and severity of herpetic lesions following intravaginal HSV challenge. On the other hand, coinjection with Th2 cytokine genes increased the rate of mortality and morbidity of the challenged mice. Moreover, of the Th1-type cytokine genes tested, IL-12 was a particularly potent adjuvant for the gD DNA vaccination.     

Antiviral Activity of Trappin-2 and Elafin In Vitro and In Vivo against Genital Herpes

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.     

The Genome Sequence of Herpes Simplex Virus Type 2

The genomic DNA sequence of herpes simplex virus type 2 (HSV-2) strain HG52 was determined as 154,746 bp with a G+C content of 70.4%. A total of 74 genes encoding distinct proteins was identified; three of these were each present in two copies, within major repeat elements of the genome. The HSV-2 gene set corresponds closely with that of HSV-1, and the HSV-2 sequence prompted several local revisions to the published HSV-1 sequence (D. J. McGeoch, M. A. Dalrymple, A. J. Davison, A. Dolan, M. C. Frame, D. McNab, L. J. Perry, J. E. Scott, and P. Taylor, J. Gen. Virol. 69:1531–1574, 1988). No compelling evidence for the existence of any additional protein-coding genes in HSV-2 was identified.The complete 152-kbp genomic DNA sequence of herpes simplex virus type 1 (HSV-1) was published in 1988 (56) and since then has been very widely employed in a great range of research on HSV-1. Additionally, results from this most studied member of the family Herpesviridae have fed powerfully into research on other herpesviruses. In contrast, although a substantial number of individual gene sequences have been determined for the other HSV serotype, HSV-2, the complete genome sequence for this virus has not been available hitherto. In this paper we report the sequence of the genome of HSV-2, strain HG52.At a gross level the 155-kbp genome of HSV-2 is viewed as consisting of two extended regions of unique sequence (U_L and U_S), each of which is bounded by a pair of inverted repeat elements (TR_L-IR_L and IR_S-TR_S) (17, 66) (Fig. ​(Fig.1).1). There is a directly repeated sequence of some 254 bp at the genome termini (the a sequence), with one or more copies in the opposing orientation (the a′ sequence) at the internal joint between IR_L and IR_S (21). U_L plus its flanking repeats is termed the long (L) region, and U_S with its flanking repeats is termed the short (S) region. In individual molecules of HSV-2 DNA, the L and S components may be linked with each in either orientation, so that DNA preparations contain four sequence-orientation isomers, one of which is defined as the prototype (66). The sequences of the terminal and internal copies of R_L and of R_S are considered to be indistinguishable. Open in a separate windowFIG. 1Overall organization of the genome of HSV-2. The linear double-stranded DNA is represented, with the scale at the top. The unique portions of the genome (U_L and U_S) are shown as heavy solid lines, and the major repeat elements (TR_L, IR_L, IR_S, and TR_S) are shown as open boxes. For each pair of repeats the two copies are in opposing orientations. As indicated, TR_L, U_L, and IR_L are regarded as comprising the L region, and IR_S, U_S, and TR_S are regarded as comprising the S region. Plasmid-cloned fragments used for sequence determination are indicated at the bottom: BamHI and HindIII fragments are indicated by B and H, respectively, followed by individual fragment designations in lowercase; KH and HK indicate KpnI/HindIII fragments as described in the text.This paper presents properties of the HSV-2 DNA sequence and our present understanding of its content of protein-coding genes and other elements. We are also interested in comparative analysis of the HSV-1 and HSV-2 genomes to examine processes of molecular evolution which have occurred since the two species diverged, and we intend to pursue this topic in a separate paper.     

Preparation of Type 2 Herpes Simplex Virus Complement-Fixing Antigen

Comparable complement-fixing antigens of type 1 and type 2 herpes simplex virus were produced by extraction of infected African green monkey cells with 0.85% NaCl which was buffered at pH 9.0 with 0.05 m glycine-NaOH. The optimal antigen dilutions were higher in titrations against hyperimmune animal sera than in titrations against human sera. Complement-fixing antibody to type 2 herpes antigen was detected in 5 of 17 sera from healthy humans.     

Herpes Simplex Virus 2 UL45 Is a Type II Membrane Protein

In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45.     

Evaluating HIV Prevention Programs: Herpes Simplex Virus Type 2 Antibodies as Biomarker for Sexual Risk Behavior in Young Adults in Resource-Poor Countries

Background

Measuring effectiveness of HIV prevention interventions is challenged by bias when using self-reported knowledge, attitude or behavior change. HIV incidence is an objective marker to measure effectiveness of HIV prevention interventions, however, because new infection rates are relatively low, prevention studies require large sample sizes. Herpes simplex virus type 2 (HSV-2) is similarly transmitted and more prevalent and could thus serve as a proxy marker for sexual risk behavior and therefore HIV infection.

Methods

HSV-2 antibodies were assessed in a sub-study of 70,000 students participating in an education intervention in Western Province, Kenya. Feasibility of testing for HSV-2 antibodies was assessed comparing two methods using Fisher’s exact test. Three hundred and ninety four students (aged 18 to 22 years) were randomly chosen from the cohort and tested for HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis. Out of these, 139 students were tested for HSV-2 with ELISA and surveyed for sexual risk behavior and 89 students were additionally tested for HSV-2 with a point-of-contact (POC) test.

Results

Prevalence rates were 0.5%, 1.8%, 0.3% and 2.3% for HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis, respectively. Prevalence of HSV-2 antibodies was 3.4 % as measured by POC test (n=89) and 14.4 % by ELISA (n=139). Specificity of the POC test compared with ELISA was 100%, and the sensitivity only 23.1%. Associations between self-reported sexual behavior and HSV-2 serostatus could not be shown.

Conclusions

Associations between self-reported sexual risk behavior and HSV-2 serostatus could not be shown, probably due to social bias in interviews since its transmission is clearly linked. HSV-2 antibody testing is feasible in resource-poor settings and shows higher prevalence rates than other sexually transmitted diseases thus representing a potential biomarker for evaluation of HIV prevention interventions.     

Inhibition of Herpes Simplex Virus Type 2 Replication by Thymidine

Replication of herpes simplex virus type 2 (HSV-2) was impeded in KB cells which were blocked in their capacity to synthesize DNA by 2 mM thymidine (TdR). The degree of inhibition was dependent upon the concentration of TdR. In marked contrast, HSV-1 is able to replicate under these conditions. The failure of HSV-2 to replicate is probably due to the inhibition of viral DNA synthesis; there was a marked reduction in the rate of DNA synthesis as well as the total amount of HSV-2 DNA made in the presence of 2 mM TdR. We postulated that the effect of TdR on viral replication occurs at the level of ribonucleotide reductase in a manner similar to KB cells. However, unlike KB cells, an altered ribonucleotide reductase activity, highly resistant to thymidine triphosphate inhibition, was found in extracts of HSV-2-infected KB cells. This activity was present in HSV-2-infected cells incubated in the presence or absence of TdR. Ribonucleotide reductase activity in extracts of HSV-1-infected KB cells showed a similar resistance to thymidine triphosphate inhibition. These results suggest that the effect of TdR on HSV-2 replication occurs at a stage of DNA synthesis other than reduction of cytidine nucleotides to deoxycytidine nucleotides.