The goals of a genital herpes vaccine are to prevent painful genital lesions and reduce or eliminate subclinical infection that risks transmission to partners and newborns. We evaluated a trivalent glycoprotein vaccine containing herpes simplex virus type 2 (HSV-2) entry molecule glycoprotein D (gD2) and two immune evasion molecules: glycoprotein C (gC2), which binds complement C3b, and glycoprotein E (gE2), which blocks immunoglobulin G (IgG) Fc activities. The trivalent vaccine was administered as baculovirus proteins with CpG and alum, or the identical amino acids were expressed using nucleoside-modified mRNA in lipid nanoparticles (LNPs). Both formulations completely prevented genital lesions in mice and guinea pigs. Differences emerged when evaluating subclinical infection. The trivalent protein vaccine prevented dorsal root ganglia infection, and day 2 and 4 vaginal cultures were negative in 23 of 30 (73%) mice compared with 63 of 64 (98%) in the mRNA group (P = 0.0012). In guinea pigs, 5 of 10 (50%) animals in the trivalent subunit protein group had vaginal shedding of HSV-2 DNA on 19 of 210 (9%) days compared with 2 of 10 (20%) animals in the mRNA group that shed HSV-2 DNA on 5 of 210 (2%) days (P = 0.0052). The trivalent mRNA vaccine was superior to trivalent proteins in stimulating ELISA IgG antibodies, neutralizing antibodies, antibodies that bind to crucial gD2 epitopes involved in entry and cell-to-cell spread, CD4⁺ T cell responses, and T follicular helper and germinal center B cell responses. The trivalent nucleoside-modified mRNA-LNP vaccine is a promising candidate for human trials.

HSV-1 causes 50% of first-time genital herpes infections in resource-rich countries and affects 190 million people worldwide. A prophylactic herpes vaccine is needed to protect against genital infections by both HSV-1 and HSV-2. Previously our laboratory developed a trivalent vaccine that targets glycoproteins C, D, and E present on the HSV-2 virion. We reported that this vaccine protects animals from genital disease and recurrent virus shedding following lethal HSV-2 challenge. Importantly the vaccine also generates cross-reactive antibodies that neutralize HSV-1, suggesting it may provide protection against HSV-1 infection. Here we compared the efficacy of this vaccine delivered as protein or nucleoside-modified mRNA immunogens against vaginal HSV-1 infection in mice. Both the protein and mRNA vaccines protected mice from HSV-1 disease; however, the mRNA vaccine provided better protection as measured by lower vaginal virus titers post-infection. In a second experiment, we compared protection provided by the mRNA vaccine against intravaginal challenge with HSV-1 or HSV-2. Vaccinated mice were totally protected against death, genital disease and infection of dorsal root ganglia caused by both viruses, but somewhat better protected against vaginal titers after HSV-2 infection. Overall, in the two experiments, the mRNA vaccine prevented death and genital disease in 54/54 (100%) mice infected with HSV-1 and 20/20 (100%) with HSV-2, and prevented HSV DNA from reaching the dorsal root ganglia, the site of virus latency, in 29/30 (97%) mice infected with HSV-1 and 10/10 (100%) with HSV-2. We consider the HSV-2 trivalent mRNA vaccine to be a promising candidate for clinical trials for prevention of both HSV-1 and HSV-2 genital herpes. Herpes simplex virus type 1 (HSV-1) is an important cause of genital herpes infection, although worldwide herpes simplex virus type 2 (HSV-2) is the most common cause. Herpes infections persist for life and there is no cure. A preventative vaccine is the best approach to reduce new genital herpes infections. An optimal vaccine should protect against both HSV-1 and HSV-2 infection. Our vaccine targets HSV-2 glycoproteins C, D, and E administered either as proteins or mRNA encapsulated in lipid nanoparticles. We compared the vaccine delivered as mRNA or proteins for prevention of HSV-1 genital infection in mice. Both vaccines prevented genital disease but the mRNA vaccine was better at limiting virus replication in the genital tract. We then compared protection by the HSV-2 mRNA vaccine against genital HSV-1 and HSV-2 infection. Mice infected with either virus were totally protected from genital disease. Importantly, in two experiments, the mRNA vaccine prevented HSV invasion of the dorsal root ganglia, the site of virus latency, in 39/40 (97.5%) mice infected with either HSV-1 or HSV-2. We conclude that the HSV-2 trivalent mRNA vaccine provides potent protection against both HSV-1 and HSV-2 genital infection and is a promising vaccine candidate for human trials.

A genital herpes vaccine is urgently needed to prevent pain and suffering, reduce the incidence of neonatal herpes, and decrease the risk of HIV acquisition and transmission that accompanies genital infection. We evaluated a trivalent HSV-2 subunit antigen vaccine administered with CpG and alum in rhesus macaques and guinea pigs. The vaccine contains glycoproteins C, D and E (gC2, gD2, gE2) to block virus entry by gD2 and immune evasion by gC2 and gE2. In rhesus macaques, the trivalent vaccine induced plasma and mucosa neutralizing antibodies, antibodies that block gC2 and gE2 immune evasion activities, and stimulated CD4 T cell responses. After intravaginal challenge, a self-limited vaginal infection of brief duration was detected by histopathology and immunohistochemistry in naïve, but not in trivalent immunized macaques. Vaccine efficacy was evaluated in female guinea pigs. Animals were mock immunized, or immunized with gD2, the trivalent vaccine or the trivalent vaccine followed by a booster dose of gD2 (trivalent + gD2). The trivalent and trivalent + gD2 groups were 97% and 99% efficacious, respectively in preventing genital lesions and both outperformed gD2 alone. As a marker of transmission risk, vaginal swabs were evaluated daily for HSV-2 DNA and replication competent virus between five and seven weeks after challenge. HSV-2 DNA shedding was reduced in all groups compared with mock. Shedding of replication competent virus occurred on fewer days in the trivalent than gD2 immunized animals while the trivalent + gD2 group had no shedding of replication competent virus. Overall, the trivalent group had genital lesions on < 1% days and shedding of replication competent virus on 0.2% days. The vaccine has outstanding potential for prevention of genital herpes in humans. Approximately a half-billion people worldwide are infected with herpes simplex virus type 2 (HSV-2), the virus that causes genital herpes. In some individuals, infection results in painful, recurrent genital ulcers, while in others, the infection remains quiescent. In both settings, infected individuals may transmit virus to their intimate partners. Genital herpes increases the risk that an infected person will acquire HIV if exposed during sexual intercourse. A vaccine for the prevention of genital herpes is a high priority. We describe a vaccine that induces antibodies that block the ability of the virus to enter cells and that prevents the virus from escaping immune attack mediated by antibody and complement. The vaccine contains HSV-2 glycoproteins C, D and E and is immunogenic in non-human primates. The vaccine protects immunized non-human primates against a mild vaginal infection that develops in naïve animals after intravaginal inoculation of virus. Naïve guinea pigs develop severe genital disease, while immunized animals are almost 100% protected after intravaginal infection. The vaccine greatly reduces the number of days during the recurrent phase of infection that animals shed virus in genital secretions, thereby reducing the risk of transmission. We consider this novel vaccine a leading candidate for clinical trials aimed at preventing genital herpes infection in humans.

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to evaluate in future Phase I/II prophylactic human vaccine trials that contain gD2 antigen. Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) mediates virus entry and cell-to-cell spread. We hypothesized that an effective gD2 vaccine needs to block these activities. Neutralizing titers, which assess blocking virus entry, correlated with protection against HSV-2 genital lesions in gD2-immunized guinea pigs. We used a high throughput biosensor assay to evaluate whether gD2-immunized guinea pigs produce antibodies that block the activities of gD2 epitopes involved in virus entry and cell-to-cell spread. We detected a strong correlation between the number of epitopes blocked by immune serum and protection against genital lesions. Passive transfer of monoclonal antibodies in mice infected intravaginally with HSV-2 confirmed the in vivo importance of antibodies to these epitopes. We re-evaluated our prior report of epitope-specific antibody responses in the gD2 Herpevac Trial, a trial that failed to protect subjects against HSV-2 genital infection. We detected antibody responses to significantly fewer epitopes in immunized humans than guinea pigs. We conclude that neutralizing antibodies and antibodies to multiple gD2 epitopes involved in virus entry and cell-to-cell spread represent important immune correlates of protection. Future human trials that contain gD2 as an immunogen need to assess these responses and correct deficiencies prior to embarking on large trials.

Nucleoside-modified mRNA vaccines have gained global attention because of COVID-19. We evaluated a similar vaccine approach for preventing a chronic, latent genital infection rather than an acute respiratory infection. We used animal models to compare an HSV-2 trivalent nucleoside-modified mRNA vaccine with the same antigens prepared as proteins, with an emphasis on antigen-specific memory B cell responses and immune correlates of protection. In guinea pigs, serum neutralizing-antibody titers were higher at 1 month and declined far less by 8 months in mRNA- compared with protein-immunized animals. Both vaccines protected against death and genital lesions when infected 1 month after immunization; however, protection was more durable in the mRNA group compared with the protein group when infected after 8 months, an interval representing greater than 15% of the animal’s lifespan. Serum and vaginal neutralizing-antibody titers correlated with protection against infection, as measured by genital lesions and vaginal virus titers 2 days after infection. In mice, the mRNA vaccine generated more antigen-specific memory B cells than the protein vaccine at early times after immunization that persisted for up to 1 year. High neutralizing titers and robust B cell immune memory likely explain the more durable protection by the HSV-2 mRNA vaccine.