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.

Immune evasion is critical for survival of viruses that establish persistent or recurrent infections. However, at the molecular level, little is known about how viruses evade immune attack in vivo. Herpes simplex virus (HSV)-1 glycoprotein gC has two domains that are involved in modulating complement activation; one binds C3, and the other is required for blocking C5 and properdin (P) binding to C3. To evaluate the importance of these regions in vivo, HSV-1 gC mutant viruses were constructed that lacked one or both gC domains and studied in a murine model of infection. Each gC region of complement regulation contributed to virulence; however, the C3 binding domain was far more important, as virus lacking this domain was much less virulent than virus lacking the C5/P inhibitory domain and was as attenuated as virus lacking both domains. Studies in C3 knockout mice and mice reconstituted with C3 confirmed that the gC domains are inhibitors of complement activation, accounting for a 50-fold difference in virulence between mutant and wild-type viruses. We conclude that the C3 binding domain on gC is a major contributor to immune evasion and that this site explains at a molecular level why wild-type virus resists complement attack.

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.

Herpes simplex virus 1 (HSV-1) glycoprotein E (gE) mediates cell-to-cell spread and functions as an IgG Fc receptor (FcγR) that blocks the Fc domain of antibody targeting the virus or infected cell. Efforts to assess the functions of the HSV-1 FcγRin vivohave been hampered by difficulties in preparing an FcγR-negative strain that is relatively intact for spread. Here we report the FcγR and spread phenotypes of NS-gE264, which is a mutant strain that has four amino acids inserted after gE residue 264. The virus is defective in IgG Fc binding yet causes zosteriform disease in the mouse flank model that is only minimally reduced compared with wild-type and the rescue strains. The presence of zosteriform disease suggests that NS-gE264 spread functions are well maintained. The HSV-1 FcγR binds the Fc domain of human, but not murine IgG; therefore, to assess FcγR functionsin vivo, mice were passively immunized with human IgG antibody to HSV. When antibody was inoculated intraperitoneally 20 h prior to infection or shortly after virus reached the dorsal root ganglia, disease severity was significantly reduced in mice infected with NS-gE264, but not in mice infected with wild-type or rescue virus. Studies of C3 knockout mice and natural killer cell-depleted mice demonstrated that the HSV-1 FcγR blocked both IgG Fc-mediated complement activation and antibody-dependent cellular cytotoxicity. Therefore, the HSV-1 FcγR promotes immune evasion from IgG Fc-mediated activities and likely contributes to virulence at times when antibody is present, such as during recurrent infections.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log₁₀) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Herpes simplex virus type 1 (HSV-1) encodes a complement-interacting glycoprotein, gC, and an immunoglobulin G (IgG) Fc binding glycoprotein, gE, that mediate immune evasion by affecting multiple aspects of innate and acquired immunity, including interfering with complement components C1q, C3, C5, and properdin and blocking antibody-dependent cellular cytotoxicity. Previous studies evaluated the individual contributions of gC and gE to immune evasion. Experiments in a murine model that examines the combined effects of gC and gE immune evasion on pathogenesis are now reported. Virulence of wild-type HSV-1 is compared with mutant viruses defective in gC-mediated C3 binding, gE-mediated IgG Fc binding, or both immune evasion activities. Eliminating both activities greatly increased susceptibility of HSV-1 to antibody and complement neutralization in vitro and markedly reduced virulence in vivo as measured by disease scores, virus titers, and mortality. Studies with C3 knockout mice indicated that other activities attributed to these glycoproteins, such as gC-mediated virus attachment to heparan sulfate or gE-mediated cell-to-cell spread, do not account for the reduced virulence of mutant viruses. The results support the importance of gC and gE immune evasion in vivo and suggest potential new targets for prevention and treatment of HSV disease.