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Background Previously, our group conducted the Herpevac Trial for Women, a randomized efficacy field trial of type 2 glycoprotein D (gD2) herpes simplex virus (HSV) vaccine adjuvanted with ASO4 in 8323 women. Study participants were selected to be seronegative for HSV-1 and HSV-2. We found that the vaccine was 82% protective against culture-positive HSV-1 genital disease but offered no significant protection against HSV-2 genital disease. Efficacy against HSV-1 was associated with higher levels of antibody to gD2 at enzyme-linked immunosorbent assay (ELISA). Methods To better understand the results of the efficacy study, we measured postvaccination concentrations of neutralizing antibody (nAb) to either HSV-1 and HSV-2 from HSV-infected study participants and matched uninfected controls. Statistical modeling was used to determine whether these responses were correlated with protection against HSV. Results nAbs to either HSV-1 or HSV-2 were correlated with ELISA binding antibodies to gD2. HSV-1 or HSV-2 nAb findings support the observation of protection by higher levels of antibody against HSV-1 infection, but the lack of protection against HSV-2 remains unexplained. Conclusions The protection against HSV-1 infection observed in the Herpevac Trial for Women was associated with nAbs directed against the virus, although the power to assess this was lower in the nAb study compared with the ELISA results owing to smaller sample size. Clinical trials registration NCT00057330. [ABSTRACT FROM AUTHOR]

Background: The glycoprotein D (gD)/AS04 vaccine failed to prevent herpes simplex virus (HSV) 2 in clinical trials. Failure was recapitulated in mice, in which the vaccine elicited neutralizing antibody but not antibody-dependent cell-mediated cytotoxicity (ADCC) responses. Preclinical findings suggest that ADCC is important for protection, but the clinical data are limited. We hypothesized that gD/AS04 and acute HSV-2 infection elicit primarily neutralizing antibodies, whereas ADCC emerges over time.Methods: HSV-specific immunoglobulin G, subclass, function (neutralization, C1q binding and ADCC), and antigenic targets were compared (paired t test or Mann-Whitney U test) at enrollment and after gD/AS04 vaccination, before and after HSV-2 acquisition in vaccine controls, and in an independent cohort with chronic HSV-2 infection.Results: Vaccination elicited only a neutralizing antibody response, whereas acute infection elicited neutralizing and C1q-binding antibodies but not a significant ADCC response. Antibodies to gD were exclusively immunoglobulin G1 and only neutralizing. In contrast, women with chronic HSV-2 infection had significantly greater ADCC responses and targeted a broader range of viral antigens compared with acutely infected or gD/AS04 vaccine recipients (P < .001).Conclusions: Results from gD/AS04 vaccinated or acutely infected women recapitulate murine findings of limited functional antibody responses, supporting the speculation that vaccines that generate polyfunctional and specifically ADCC responses may be required to prevent HSV-2 acquisition and limit recurrences. [ABSTRACT FROM AUTHOR]

Preventive viral vaccine efficacy trials require large-scale sample analysis to quantitate immune responses and their correlation with infection outcomes. Traditional plaque reduction assays measure a functionally important form of humoral immunity, neutralizing antibody titer. These assays, however, are time-consuming and laborious. We previously developed a higher throughput assay of neutralizing antibody to herpes simplex viruses 1 and 2 (Blevins et al., PLOS ONE, 10(12), e0144738) using the enzyme-linked virus inducible system (ELVIS) cell line; this cell line produces β-galactosidase in response to HSV infection. Here, serum samples from recipients of an investigational vaccine in the Herpevac Trial for Women were used to compare the ELVIS cell assay with the lower throughput, traditional plaque reduction assay. We demonstrate that neutralizing antibody titers to HSV-1 or HSV-2 determined using ELVIS cells positively correlate with neutralizing antibody titers determined by traditional plaque reduction assay, thus validating a higher throughput alternative for large-scale sample analysis. [ABSTRACT FROM AUTHOR]

Herpes simplex virus 2 (HSV-2) is a large double-stranded DNA virus that causes genital sores when spread by sexual contact and is a principal cause of viral encephalitis in newborns and infants. Viral glycoproteins enable virion entry into and spread between cells, making glycoproteins a prime target for vaccine development. A truncated glycoprotein D2 (gD2) vaccine candidate, recently tested in the phase 3 Herpevac Trial for Women, did not prevent HSV-2 infection in initially seronegative women. Some women who became infected experienced multiple recurrences during the trial. The HSV US7, US8, and US9 genes encode glycoprotein I (gI), glycoprotein E (gE), and the US9 type II membrane protein, respectively. These proteins participate in viral spread across cell junctions and facilitate anterograde transport of virion components in neurons, prompting us to investigate whether sequence variants in these genes could be associated with frequent recurrence. The nucleotide sequences and dN/dS ratios of the US7-US9 region from viral isolates of individuals who experienced multiple recurrences were compared with those who had had a single episode of disease. No consistent polymorphism(s) distinguished the recurrent isolates. In frequently recurring isolates, the dN/dS ratio of US7 was low while greater variation (higher dN/dS ratio) occurred in US8, suggesting conserved function of the former during reactivation. Phylogenetic reconstruction of the US7-US9 region revealed eight strongly supported clusters within the 55 U.S. HSV-2 strains sampled, which were preserved in a second global phylogeny. Thus, although we have demonstrated evolutionary diversity in the US7-US9 complex, we found no molecular evidence of sequence variation in US7-US9 that distinguishes isolates from subjects with frequently recurrent episodes of disease. [ABSTRACT FROM AUTHOR]

The Herpevac Trial for Women revealed that three dose HSV-2 gD vaccine was 58% protective against culture-positive HSV-1 genital disease, but it was not protective against HSV-2 infection or disease. To determine whether vaccine-induced immune responses had selected for a particular gD sequence in strains infecting vaccine recipients compared with viruses infecting control subjects, genetic sequencing studies were carried out on viruses isolated from subjects infected with HSV-1 or HSV-2. We identified naturally occurring variants among the gD sequences obtained from 83 infected subjects. Unique or low frequency amino acid substitutions in the ectodomain of gD were found in 6 of 39 HSV-1-infected subjects and in 7 of 44 HSV-2-infected subjects. However, no consistent amino acid change was identified in isolates from gD-2 vaccine recipients compared with infected placebo recipients. gC and gE surround and partially shield gD from neutralizing antibody, and gB also participates closely in the viral entry process. Therefore, these genes were sequenced from a number of isolates to assess whether sequence variation may alter protein conformation and influence the virus strain’s capacity to be neutralized by vaccine-induced antibody. gC and gE genes sequenced from HSV-1-infected subjects showed more variability than their HSV-2 counterparts. The gB sequences of HSV-1 oral isolates resembled each other more than they did gB sequences rom genital isolates. Overall, however, comparison of glycoprotein sequences of viral isolates obtained from infected subjects did not reveal any singular selective pressure on the viral cell attachment protein or surrounding glycoproteins due to administration of gD-2 vaccine. [ABSTRACT FROM AUTHOR]

We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency. [ABSTRACT FROM AUTHOR]

The Herpevac Trial evaluated a herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit vaccine to prevent genital herpes. Unexpectedly, the vaccine protected against genital HSV-1 infection but not genital HSV-2 infection. We evaluated sera from 30 women seronegative for HSV-1 and HSV-2 who were immunized with gD2 in the Herpevac Trial. Neutralizing antibody titers to HSV-1 were 3.5-fold higher than those to HSV-2 (P < .001). HSV-2 gC2 and gE2 on the virus blocked neutralization by gD2 antibody, while HSV-1 gC1 and gE1 did not block neutralization by gD2 antibody. The higher neutralizing antibody titers to HSV-1 offer an explanation for the Herpevac results, and shielding neutralizing domains provides a potential mechanism. [ABSTRACT FROM PUBLISHER]

Background. Previously we conducted a double-blind controlled, randomized efficacy field trial of gD-2 HSV vaccine adjuvanted with ASO4 in 8323 women. Subjects had been previously selected to be seronegative for HSV-1 and HSV-2. We found that vaccine was 82% protective against HSV-1 genital disease, but offered no significant protection against HSV-2 genital disease.Methods. To better understand the results of the efficacy study, post-vaccination anti-gD-2 antibody concentrations from all HSV infected subjects and matched uninfected controls were measured. Three models were used to determine whether these responses correlated with protection against HSV infection or disease. Similarly, cellular immune responses from a subset of subjects and matched controls were evaluated for a correlation with HSV protection.Results. Antibodies to gD-2 correlated with protection against HSV-1 infection with higher antibody concentration associated with higher efficacy. Cellular immune responses to gD-2 did not correlate with protection.Conclusions. The protection against HSV-1 infection observed in the Herpevac Trial for Women was associated with antibodies directed against the vaccine.Clinical Trials Registration NCT00057330. [ABSTRACT FROM PUBLISHER]

In this large prospective study of primary herpes simplex virus (HSV) infections in young, HSVseronegative women, we found that the rate of HSV-1 infections was more than twice that for HSV-2, but there were important age and race differences.Background. Herpes simplex virus infections type 1 (HSV-1) and type 2 (HSV-2) are common, but the epidemiology of HSV disease is changing.Methods. HSV-seronegative women, aged 18–30 years, who were in the control arm of the HERPEVAC Trial for Women were followed for 20 months for primary HSV infections.Results. Of the 3438 evaluable participants, 183 became infected with HSV: 127 (3.7%) with HSV-1 and 56 (1.6%) with HSV-2. The rate of infection for HSV-1 (2.5 per 100 person-years) was more than twice that for HSV-2 (1.1 per 100 person-years). Most infections (74% of HSV-1 and 63% of HSV-2) occurred without recognized signs or symptoms of herpes disease. The HSV-2 infection rate was 2.6 times higher in non-Hispanic black participants than in Hispanics and 5.5 times higher than in non-Hispanic whites (P < .001), while the HSV-1 infection rate was 1.7 times higher in non-Hispanic whites than non-Hispanic blacks. Younger participants (18–22 years) were more likely to acquire HSV-1 infections and less likely to develop recognized disease than older participants. Overall, 84% of recognized disease cases were genital. No differences were noted in the clinical manifestations of genital HSV-1 vs genital HSV-2 disease. The clinicians’ assessment that cases were caused by HSV was good when they assessed cases as clinically confirmed or unlikely (validated in 83% and 100% of cases, respectively).Conclusions. HSV-1 is now more common than HSV-2 as a cause of oral and genital mucosal infections in young women, but there are important age and race differences. [ABSTRACT FROM PUBLISHER]

Background: Two previous studies of a herpes simplex virus type 2 (HSV-2) subunit vaccine containing glycoprotein D in HSV-discordant couples revealed 73% and 74% efficacy against genital disease in women who were negative for both HSV type 1 (HSV-1) and HSV-2 antibodies. Efficacy was not observed in men or HSV-1 seropositive women. Methods: We conducted a randomized, double-blind efficacy field trial involving 8323 women 18 to 30 years of age who were negative for antibodies to HSV-1 and HSV-2. At months 0, 1, and 6, some subjects received the investigational vaccine, consisting of 20 μg of glycoprotein D from HSV-2 with alum and 3-O-deacylated monophosphoryl lipid A as an adjuvant; control subjects received the hepatitis A vaccine, at a dose of 720 enzyme-linked immunosorbent assay (ELISA) units. The primary end point was occurrence of genital herpes disease due to either HSV-1 or HSV-2 from month 2 (1 month after dose 2) through month 20. Results: The HSV vaccine was associated with an increased risk of local reactions as compared with the control vaccine, and it elicited ELISA and neutralizing antibodies to HSV-2. Overall, the vaccine was not efficacious; vaccine efficacy was 20% (95% confidence interval [CI], −29 to 50) against genital herpes disease. However, efficacy against HSV-1 genital disease was 58% (95% CI, 12 to 80). Vaccine efficacy against HSV-1 infection (with or without disease) was 35% (95% CI, 13 to 52), but efficacy against HSV-2 infection was not observed (−8%; 95% CI, −59 to 26). Conclusions: In a study population that was representative of the general population of HSV-1– and HSV-2–seronegative women, the investigational vaccine was effective in preventing HSV-1 genital disease and infection but not in preventing HSV-2 disease or infection. (Funded by the National Institute of Allergy and Infectious Diseases and GlaxoSmithKline; ClinicalTrials.gov number, NCT00057330.) [ABSTRACT FROM PUBLISHER]

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates. [ABSTRACT FROM AUTHOR]

Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48 h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation ( r = 0.9499, p < 0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines. [ABSTRACT FROM AUTHOR]

Objectives: Recent evidence suggests that the epidemiology of herpes simplex viruses (HSVs) is changing because fewer HSV- 1 infections are acquired in childhood and increased sexual transmission of HSV-1 is reported. The objective of the study was to assess the seroprevalence of type-specific antibodies to HSV-1 and HSV-2 in the United States. Methods: We used the Western blot antibody screening data from a large phase HI vaccine efficacy trial (Herpevae Trial for Women) to assess the seroprevalence of type-specific antibodies to HSV-1 and HSV-2 in the United States. Results: The antibody status of 29,022 women (>31,000 women interviewed and then had their blood drawn for the HSV testing [29,022 women]) between the ages of 18 and 30 years in the United States revealed that increasing age was associated with increasing seroprevalence to HSV Overall, in asymptomatic women unaware of any HSV infection, HSV-1/-2 status was positive/negative in 45%, negative/positive in 5%, positive/positive in 7%, negative/negative in 38%, and indeterminate in 5%. HSV-1 infections were more common in Hispanic and non-Hispanic black women and in the US northeast and in individuals living in urban areas. HSV-2 was more common in non-Hispanic black women, the US south, and in urban areas. Conclusions: Seronegative status for both HSV-1 and HSV-2 was the second most common finding after positive antibody to HSV-1 but negative antibody to HSV-2. Despite recent changes in genital herpes epidemiology, most women acquired HSV-1 but not HSV-2 infections before 18 years of age. Among participants screened for study participation and who were unaware of any HSV-infection, progressively higher prevalence of the HSV-1 or HSV-2 antibody was observed in older subjects. Many women who test positive for HSV-1 and/or HSV-2 are unaware of their status. [ABSTRACT FROM AUTHOR]