Vaccine induced HSV 1 antibodies fail to correlate with protection against HSV 2 in guinea pigs

Abstract Guinea pigs immunised with HSV 1 subunit vaccine were bled, and subsequently challenged intravaginally with HSV 2. The clinical response to virus challenge was quantified, and correlations were sought between clinical score and virus-specific serum antibody titre in functional and binding assays (ELISA, neutralization, complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity). No significant relationships were found, and it was concluded that reactivity in the serological assays chosen did not correlate with protection against HSV 2 genital infection in vaccinated female guinea pigs.     

Immunogenicity,Protective Efficacy,and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.     

Recombinant interleukin 2 as an adjuvant for vaccine-induced protection. Immunization of guinea pigs with herpes simplex virus subunit vaccines

We determined that recombinant interleukin 2 (rIL-2) administered in conjunction with herpes simplex virus (HSV) crude extract or recombinant glycoprotein D subunit vaccine enhances the protective effect of either antigen preparation against HSV type 2 genital infection in guinea pigs. Animals that received the vaccine accompanied by rIL-2 had an incidence of infection, assessed by detection of clinical lesions and/or viral shedding, that varied between 0 and 43% significantly lower than the incidence of 63 to 100% in guinea pigs submitted to the same immunization schedule without rIL-2. Animals that escaped acute infection failed to develop recurrent disease. In addition, severity of acute infection was decreased by rIL-2 co-administration as well as by increasing the number of vaccine doses. We also studied the immune response of the guinea pigs to vaccination and the mechanism of protection. Both enzyme-linked immunosorbent assay titers of antibodies to HSV type 2 and specific antigen stimulation of lymphocytes measured by proliferation and interferon production did not significantly differ among the immunization groups. However, specific cellular cytotoxicity was enhanced by rIL-2 co-administration and was positively correlated with protection. This suggests that rIL-2 may become an important adjuvant in active immunization programs using subunit vaccines, particularly against diseases in which cellular cytotoxicity is a major defense mechanism.     

Anti-CeHV1 antibodies of two cynomolgus macaques cross-react with HSV2 but not HSV1 antigens in ELISA

BACKGROUND: The aim of the study was to compare the cross-reactivity of macaque anti-CeHV1 antibodies with type 1 and type 2 human herpes simplex viruses (HSV1 and HSV2). METHODS: We studied the serum of 344 animals which had been tested either positive (n = 39) or negative (n = 305) for the presence of CeHV1 antibodies by expert laboratories. Macaque serums were studied by means of two ELISA: one based on HSV1 antigen-coated wells, the other on polystyrene beads coated with HSV1 and HSV2 antigens in approximately equal proportions. RESULTS: In the serum of two animals originating from Vietnam, we found anti-CeHV1 antibodies cross-reacting with HSV2 but not with HSV1 antigens. For the serum with the highest titer, inhibition by soluble antigens confirmed the high affinity of the antibodies for HSV2 antigens. CONCLUSIONS: Tests using HSV1 and HSV2 in a combined way are better suited to macaque screening than tests using only HSV1 antigens.     

Herpes simplex virus (HSV)-specific proliferative and cytotoxic T-cell responses in humans immunized with an HSV type 2 glycoprotein subunit vaccine.

Studies were undertaken to determine whether immunization of humans with a herpes simplex virus type 2 (HSV-2) glycoprotein-subunit vaccine would result in the priming of both HSV-specific proliferating cells and cytotoxic T cells. Peripheral blood lymphocytes (PBL) from all eight vaccines studied responded by proliferating after stimulation with HSV-2, HSV-1, and glycoprotein gB-1. The PBL of five of these eight vaccines proliferated following stimulation with gD-2, whereas stimulation with gD-1 resulted in relatively low or no proliferative responses. T-cell clones were generated from HSV-2-stimulated PBL of three vaccinees who demonstrated strong proliferative responses to HSV-1 and HSV-2. Of 12 clones studied in lymphoproliferative assays, 9 were found to be cross-reactive for HSV-1 and HSV-2. Of the approximately 90 T-cell clones isolated, 14 demonstrated HSV-specific cytotoxic activity. Radioimmunoprecipitation-polyacrylamide gel electrophoresis analyses confirmed that the vaccinees had antibodies only to HSV glycoproteins, not to proteins which are absent in the subunit vaccine, indicating that these vaccinees had not become infected with HSV. Immunization of humans with an HSV-2 glycoprotein-subunit vaccine thus results in the priming of T cells that proliferate in response to stimulation with HSV and its glycoproteins and T cells that have cytotoxic activity against HSV-infected cells. Such HSV-specific memory T cells were detected as late as 2 years following the last boost with the subunit vaccine.     

Lymphocyte stimulation by herpes simplex virus (HSV) antigens. 1. Culture conditions, cell fractionation and presentation of HSV antigens.

Optimal culture conditions for rabbit lymphocytes were established. Inclusion of 2-mercaptoethanol in the medium greatly enhanced responses. Lymphocytes from the blood of HSV-immunized rabbits responded specifically in vitro to heat-killed (56 degree, 60 min) or UV-inactivated HSV preparations. A UV dose of 13,392 ergs/mm2 was the most suitable inactivating dose. Oral intramucosal injection was the most effective way of generating blood lymphocyte responses to HSV, but animals immunized in tradermally or in tramuscularly with HSV in Freund's adjuvant also produced antigen-reactive cells after five or more immunizations. There were differences in the shape of the dose-response curves to various HSV1 mutants which were probably due to differences in the production of "stimulatory" and inhibitory antigens. Appreciable lymphocyte stimulation could be obtained with tissue culture fluid and enveloped virus antigens. Lymphocytes from HSV1-immune animals were also responsive to HSV2 antigens.     

Routine diagnosis of herpes simplex virus (HSV) encephalitis by an internal DNA controlled HSV PCR and an IgG-capture assay for intrathecal synthesis of HSV antibodies

Background: The development of antiviral therapy increases the need for rapid, sensitive and reliable methods or combination of methods for diagnosis and monitoring herpes simplex encephalitis, HSE.Objectives: Evaluation of diagnostic performance of three successively developed HSV PCR assays when combined with a new capture ELISA for HSV intrathecal antibody production (ITT).Study design: During a 3.6 year period a total of 4.206 CSF and serum samples from about 4.140 hospitalized patients with a tentative diagnosis of HSE were analyzed by a new ELISA for ITT. 1.962 CSF samples were examined also by PCR. Clinical signs and symptoms and additional tests were obtained on all ITT and/or PCR positive patients. In 1993 the PCR was a double PCR. In 1994 the PCR was a single PCR with internal inhibition control. Positive samples were confirmed by a different confirmative PCR to increase the specificity. From 1995 the PCR was as in 1994 but samples were no longer divided in the serology routine laboratory.Results: A total of 33 HSE cases was found (incidence 1.8 HSE per million people). All patients were treated with aciclovir. Three patients died, 9 patients had primary infection, 2 patients had HSE previously, and 2 patients relapsed. Only 11 patients recovered satisfactory. Of all 37 positive ITT 7 were unlikely positive. False positive PCR was seen in 1993 and 1994, due to sample-to-sample contamination during division of samples, but was not seen since 1995 when this procedure was changed. The test results depended on the state of the disease. Thus, the sensitivity, specificity, PPV and NPV for ITT were highest when performed more than 1 week after debut of symptoms whereas these values were highest using PCR within the first week.Conclusion: Routine PCR diagnosis of HSE type 1 and 2 is a highly sensitive and specific method that should be performed together with serological ITT to cover the whole time span from debut of symptoms to several weeks after hospitalization.     

HSV neutralization by the microbicidal candidate C5A

Genital herpes is a major risk factor in acquiring human immunodeficiency virus type-1 (HIV-1) infection and is caused by both Herpes Simplex virus type 1 (HSV-1) and HSV-2. The amphipathic peptide C5A, derived from the non-structural hepatitis C virus (HCV) protein 5A, was shown to prevent HIV-1 infection but neither influenza nor vesicular stomatitis virus infections. Here we investigated the antiviral function of C5A on HSV infections. C5A efficiently inhibited both HSV-1 and HSV-2 infection in epithelial cells in vitro as well as in an ex vivo epidermal infection model. C5A destabilized the integrity of the viral HSV membrane. Furthermore, drug resistant HSV strains were inhibited by this peptide. Notably, C5A-mediated neutralization of HSV-1 prevented HIV-1 transmission. An in vitro HIV-1 transmigration assay was developed using primary genital epithelial cells and HSV infection increased HIV-1 transmigration. Treatment with C5A abolished HIV-1 transmigration by preventing HSV infection and by preserving the integrity of the genital epithelium that was severely compromised by HSV infection. In conclusion, this study demonstrates that C5A represents a multipurpose microbicide candidate, which neutralizes both HIV-1 and HSV, and which may interfere with HIV-1 transmission through the genital epithelium.     

Rapid detection of HSV from cytologic specimens collected into ThinPrep fixative

OBJECTIVE: Herpes simplex virus (HSV) infection is associated with substantial morbidity and mortality in neonates. A diagnosis of HSV on cervical cytologic studies could lead to a cesarean section, with an increase in the risk of maternal morbidity. The identification of viral lesions in sexually active women has medical and social implications. There have been reports of false positive diagnoses of HSV in patients with altered endocervical cells and with cervical intraepithelial neoplasia 3. We evaluated a polymerase chain reaction (PCR)-based assay to detect HSV-1 and HSV-2 in routinely collected cervical cytology specimens in ThinPrep fixative (Cytyc Corp., Marlborough, Massachussets, U.S.A.). STUDY DESIGN: DNA was extracted from five cases that demonstrated cytologic changes suggestive of an HSV infection. PCR amplification with subsequent gel electrophoresis was performed to detect the presence of HSV. RESULTS: HSV DNA was detected in three of five cases that were cytologically diagnosed as suspicious or strongly suspicious for HSV infection. CONCLUSION: The combination of the ThinPrep liquid-based method for cervical cytology with PCR allows prompt confirmation of the diagnosis of HSV without sacrificing the diagnostic morphology on the slide.     

Herpes simplex virus-infected human fibroblasts are resistant to and inhibit cytotoxic T-lymphocyte activity.

We examined the ability of human anti-herpes simplex virus (HSV) cytotoxic T lymphocytes (CTL) to lyse autologous human fibroblasts infected with HSV. In contrast to HSV-infected human Epstein-Barr virus-transformed B cells (LCL), which were lysed by HLA-restricted anti-HSV CTL, autologous fibroblasts infected with HSV were resistant to lysis. This resistance was not due to a lack of infectivity or production of HSV proteins since greater than 90% of the cells were infected and expressed abundant levels of viral proteins. HSV-infected human fibroblasts were also tested for susceptibility to lysis by alloantigen-specific CTL. Although allogeneic LCL and uninfected allogeneic fibroblasts were killed, human fibroblasts infected with HSV demonstrated a time-dependent resistance to lysis by alloantigen-specific CTL. HSV-infected human fibroblasts were not resistant to all forms of cell-mediated cytotoxicity since they were sensitive to antibody-dependent cellular cytotoxicity. Although one may suspect that the resistance of HSV-infected human fibroblasts to anti-HSV CTL and alloantigen-specific CTL-mediated lysis was due to a lack of major histocompatibility complex expression, Confer et al. (Proc. Natl. Acad. Sci. USA 87:3609-3613, 1990) previously demonstrated that incubation of human natural killer and lymphokine-activated killer cells with monolayers of human fibroblasts infected with HSV "disarmed" the killers in that they were unable to lyse sensitive target cells. We extend their results and show that incubation of anti-HSV CTL or alloantigen-specific CTL with uninfected fibroblasts did not affect their lytic activity, whereas CTL incubated with HSV-infected fibroblasts for 2 to 6 h rendered the CTL incapable of lysing their normally sensitive target cells. Indeed, human fibroblasts infected for merely 2 h with HSV were able to profoundly inhibit the cytotoxic activity of alloantigen-specific CTL. Thus, HSV-infected human fibroblasts are not inherently resistant to lysis by anti-HSV CTL or alloantigen-specific CTL, but rather contact of CTL with HSV-infected fibroblasts resulted in inactivation of the CTL. The inactivation of CTL appears to be HSV specific since incubation of alloantigen-specific CTL in sandwich assays with fibroblasts infected with HSV type 1 (HSV-1) or HSV-2 resulted in inactivation, whereas incubation of CTL with fibroblasts infected with adenovirus or vaccinia virus had no effect. Further, although incubation of alloantigen-specific CTL in sandwich assays with HSV-infected fibroblasts resulted in inhibition of CTL activity, exposure of CTL in Transwell cultures to cell-free supernatant from HSV-infected fibroblasts did not mediate this inhibitory effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

18F]FHPG positron emission tomography for detection of herpes simplex virus (HSV) in experimental HSV encephalitis

Herpes simplex virus type 1 (HSV-1) is one of the most common causes of sporadic encephalitis. The initial clinical course of HSV encephalitis (HSE) is highly variable, and the infection may be rapidly fatal. For effective treatment with antiviral medication, an early diagnosis of HSE is crucial. Subtle brain infections with HSV may be causally related to neuropsychiatric disorders such as Alzheimer's dementia. We investigated the feasibility of a noninvasive positron emission tomography (PET) imaging technique using [(18)F]FHPG as a tracer for the detection of HSE. For this purpose, rats received HSV-1 (infected group) or phosphate-buffered saline (control group) by intranasal application, and dynamic PET scans were acquired. In addition, the distribution of tracer accumulation in specific brain areas was studied with phosphor storage imaging. The PET images revealed that the overall brain uptake of [(18)F]FHPG was significantly higher for the infected group than for control animals. Phosphor storage images showed an enhanced accumulation of [(18)F]FHPG in regions known to be affected after intranasal infection with HSV. High-performance liquid chromatography metabolite analysis showed phosphorylated metabolites of [(18)F]FHPG in infected brains, proving that the increased [(18)F]FHPG uptake in infected brains was due to HSV thymidine kinase-mediated trapping. Freeze lesion experiments showed that damage to the blood-brain barrier could in principle induce elevated [(18)F]FHPG uptake, but this nonspecific tracer uptake could easily be discriminated from HSE-derived uptake by differences in the tracer kinetics. Our results show that [(18)F]FHPG PET is a promising tool for the detection of HSV encephalitis.     

Immunodominant epitopes in herpes simplex virus type 2 glycoprotein D are recognized by CD4 lymphocytes from both HSV-1 and HSV-2 seropositive subjects

In human recurrent cutaneous herpes simplex, there is a sequential infiltrate of CD4 and then CD8 lymphocytes into lesions. CD4 lymphocytes are the major producers of the key cytokine IFN-gamma in lesions. They recognize mainly structural proteins and especially glycoproteins D and B (gD and gB) when restimulated in vitro. Recent human vaccine trials using recombinant gD showed partial protection of HSV seronegative women against genital herpes disease and also, in placebo recipients, showed protection by prior HSV1 infection. In this study, we have defined immunodominant peptide epitopes recognized by 8 HSV1(+) and/or 16 HSV2(+) patients using (51)Cr-release cytotoxicity and IFN-gamma ELISPOT assays. Using a set of 39 overlapping 20-mer peptides, more than six immunodominant epitopes were defined in gD2 (two to six peptide epitopes were recognized for each subject). Further fine mapping of these responses for 4 of the 20-mers, using a panel of 9 internal 12-mers for each 20-mers, combined with MHC II typing and also direct in vitro binding assay of these peptides to individual DR molecules, showed more than one epitope per 20-mers and promiscuous binding of individual 20-mers and 12-mers to multiple DR types. All four 20-mer peptides were cross-recognized by both HSV1(+)/HSV2(-) and HSV1(-)/HSV2(+) subjects, but the sites of recognition differed within the 20-mers where their sequences were divergent. This work provides a basis for CD4 lymphocyte cross-recognition of gD2 and possibly cross-protection observed in previous clinical studies and in vaccine trials.     

Evidence that neomycin inhibits HSV 1 infection of BHK cells

The effect of neomycin on the Herpes Simplex Virus (HSV) type 1 and 2 infection of baby hamster kidney cells was studied. Neomycin concentrations of 3 mM or more caused a more than 90% inhibition of HSV 1 proliferation, while it had no effect on HSV 2 proliferation, measured as plaque-forming units. Furthermore, neomycin must be present at the time of infection in order to exert full effect, addition 1 hour postinfection was comparable to untreated cells. This indicates that neomycin may specifically interfere with very early stages of HSV 1 infection.     

A test for the relative potency of herpes simplex virus vaccines based upon the female guinea-pig model of HSV 2 genital infection

An ELISA for total herpes simplex virus (HSV) 1 antigen content and a test of immunogenicity based upon the female guinea-pig model of HSV 2 genital infection were applied to two samples from batches of HSV 1 subunit ('Skinner') vaccine. The ELISA was reproducible within an approximately threefold limit of error and batches 1 and 2 were indistinguishable in antigen content. The effects of vaccination in the guinea-pig model were assessed by a statistical analysis of scores derived from the principal clinical signs, vaginal oedema and lesions on the external genitalia. The statistical power of the guinea-pig assay was such that reductions in the severity of illness approaching 40% would be significant (P less than 0.05) on 90% of occasions. The ability to make quantitative estimates of immunogenicity will prove useful in the quality control of HSV vaccine batches which are destined for clinical trials in man.     

Antigenic structure of soluble herpes simplex virus (HSV) glycoprotein D correlates with inhibition of HSV infection.

Soluble forms of herpes simplex virus (HSV) glycoprotein D (gD) block viral penetration. Likewise, most HSV strains are sensitive to gD-mediated interference by cells expressing gD. The mechanism of both forms of gD-mediated inhibition is thought to be at the receptor level. We analyzed the ability of different forms of soluble, truncated gD (gDt) to inhibit infection by different strains of HSV-1 and HSV-2. Strains that were resistant to gD-mediated interference were also resistant to inhibition by gDt, thereby suggesting a link between these two phenomena. Virion gD was the major viral determinant for resistance to inhibition by gDt. An insertion-deletion mutant, gD-1(delta 290-299t), had an enhanced inhibitory activity against most strains tested. The structure and function of gDt proteins derived from the inhibition-resistant viruses rid1 and ANG were analyzed. gD-1(ridlt) and gD-1(ANGt) had a potent inhibitory effect on plaque formation by wild-type strains of HSV but, surprisingly, little or no effect on their parental strains. As measured by quantitative enzyme-linked immunosorbent assay with a diverse panel of monoclonal antibodies, the antigenic structures of gD-1(rid1t) and gD-1(ANGt) were divergent from that of the wild type yet were similar to each other and to that of gD-1 (delta 290-299t). Thus, three different forms of gD have common antigenic changes that correlate with enhanced inhibitory activity against HSV. We conclude that inhibition of HSV infectivity by soluble gD is influenced by the antigenic conformation of the blocking gDt as well as the form of gD in the target virus.     

Detection of herpes simplex and varicella zoster viruses in clinical specimens using direct immunofluorescence and cell culture assays

Patients (33 in toto) with a clinical diagnosis of herpes infections (simplex, zoster or chickenpox) were investigated for the presence of herpes simplex virus (HSV) and varicella zoster virus (VZV) in skin samples, using direct immunofluorescence and cell culture assays. Five patients with nonherpetic vesiculobullous disorders were included as negative controls. Of the 33 patients, nineteen (57.6%) were positive for HSV or VZV and fourteen (42.4%) were negative. Five controls were all negative for HSV or VZV. Of the nineteen positive patients, HSV was isolated from eight (42.1%) patients, by both direct immunofluorescence and cell culture assays. VZV was isolated from eleven (57.9%) patients, eleven (100%) by direct immunofluorescence assay, and six (54.5%) by cell culture assays. HSV was isolated from one patient clinically diagnosed as chickenpox (VZV), but otherwise the positive laboratory results were concordant with the clinical diagnosis. For epidemiological studies, atypical cases and immunocompromised patients the clinical diagnosis should be confirmed in the laboratory.     

Bioluminescence imaging after HSV amplicon vector delivery into brain

BACKGROUND: Firefly luciferase (Fluc) has routinely been used to quantitate and analyze gene expression in vitro by measuring the photons emitted after the addition of ATP and luciferin to a test sample. It is now possible to replace luminometer-based analysis of luciferase activity and measure luciferase activity delivered by viral vectors directly in live animals over time using digital imaging techniques. METHODS: An HSV amplicon vector expressing Fluc cDNA from an inducible promoter was delivered to cells in culture and into the mouse brain. In culture, expression of Fluc was measured after induction in a dose-dependent manner by a biochemical assay, and then confirmed by Western blot analysis and digital imaging. The vectors were then stereotactically injected into the mouse brain and Fluc expression measured non-invasively using bioluminescence imaging. RESULTS: Rapamycin-mediated induction of Fluc from an HSV amplicon vector in culture resulted in dose-dependent expression of Fluc when measured using a luminometer and by digital analysis. In mouse cortex, a single injection of an HSV amplicon vector (2 microl, 1x10(8) transducing units (t.u.)/ml) expressing Fluc from a viral promoter (CMV) was sufficient to detect robust luciferase activity for at least 1 week. Similarly, an HSV amplicon vector expressing Fluc under an inducible promoter was also detectable in the mouse cortex after a single dose (2 microl, 1x10(8) t.u./ml) for up to 5 days, with no detectable signal in the uninduced state. CONCLUSIONS: This HSV amplicon vector-based system allows for fast, non-invasive, semi-quantitative analysis of gene expression in the brain.     

Herpes simplex virus (HSV)-specific human T-cell clones recognize HSV glycoprotein D expressed by a recombinant vaccinia virus.

Human cytotoxic T-cell (CTL) clones that lyse autologous cells infected with herpes simplex virus (HSV) type 1 or 2 were generated by stimulating lymphocytes with a recombinant vaccinia virus (recombinant vaccinia-gD-1 virus) that expresses HSV type 1 glycoprotein D (gD-1). Furthermore, CTL clones generated with HSV type 1 or with cloned gD-1 lysed autologous cells infected with the recombinant vaccinia-gD-1 virus. Our findings thus showed that gD serves as a target antigen for human CTLs and that a recombinant vaccinia-gD virus activates HSV-specific human CTL.     

一次PCR扩增单纯疱疹和巨细胞病毒基因的检测方法

宫内感染是影响优生优育质量的一个重要因素,由病毒引起的宫内感染主要有以下四类病原：ＨＳＶ、ＣＭＶ、ＴＯＸＯ和风疹病毒（ＲＶ）,其中ＨＳＶ和ＣＭＶ的感染率较高。孕期这两种病原的感染会影响胎儿的正常发育,严重的可导致流产甚至胎儿畸形。目前已有针对这两种病...