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The host enzyme heparanase (HPSE) facilitates the release of herpes simplex virus type 2 (HSV-2) from target cells by cleaving the viral attachment receptor heparan sulfate (HS) from infected cell surfaces. HPSE 2, an isoform of HPSE, binds to but does not possess the enzymatic activity needed to cleave cell surface HS. Our study demonstrates that HSV-2 infection significantly elevates HPSE 2 protein levels, impacting two distinct stages of viral replication. We show that higher HPSE 2 negatively affects HSV-2 replication which may be through the regulation of cell surface HS. By acting as a competitive inhibitor of HPSE, HPSE 2 may be interfering with HPSE’s interactions with HS. We demonstrate that the enhanced expression of HPSE 2, either via viral infection or plasmid transfection, reduces HPSE’s ability to cleave HS, thereby hindering viral egress. Conversely, low HPSE 2 levels achieved through siRNA transfection allow HPSE to cleave more HS, reducing viral entry. Altogether, we propose a hypothetical model in which the modulation of HPSE 2 impedes HSV-2 replication by regulating HS availability on the cell surface. This dual role of HPSE 2 in viral replication and potential tumor suppression underscores its significance in cellular processes and viral pathogenesis.

Herpes simplex virus type 2 (HSV-2) is a leading cause of genital ulcer disease and a major risk factor for acquisition and transmission of HIV. Frequent recurrent genital lesions and concerns about transmitting infection to intimate partners affect the quality of life of infected individuals. Therapeutic vaccines are urgently needed to reduce the frequency of genital lesions and transmission. S-540956 is a novel vaccine adjuvant that contains CpG oligonucleotide ODN2006 annealed to its complementary sequence and conjugated to a lipid that targets the adjuvant to lymph nodes. Our primary goal was to compare S-540956 administered with HSV-2 glycoprotein D (gD2) with no treatment in a guinea pig model of recurrent genital herpes (studies 1 and 2). Our secondary goals were to compare S-540956 with oligonucleotide ODN2006 (study1) or glucopyranosyl lipid A in a stable oil-in-water nano-emulsion (GLA-SE) (study 2). gD2/S-540956 reduced the number of days with recurrent genital lesions by 56%, vaginal shedding of HSV-2 DNA by 49%, and both combined by 54% compared to PBS, and was more efficacious than the two other adjuvants. Our results indicate that S-540956 has great potential as an adjuvant for a therapeutic vaccine for genital herpes, and merits further evaluation with the addition of potent T cell immunogens.

The toxicity of mRNA-lipid nanoparticle (LNP) vaccines depends on the total mRNA-LNP dose. We established that the maximum tolerated dose of our trivalent mRNA-LNP genital herpes vaccine was 10 μg/immunization in mice. We then evaluated one of the mRNAs, gD2 mRNA-LNP, to determine how much of the 10 μg total dose to assign to this immunogen. We immunized mice with 0.3, 1.0, 3.0, or 10 μg of gD2 mRNA-LNP and measured serum IgG ELISA, neutralizing antibodies, and antibodies to six crucial gD2 epitopes involved in virus entry and spread. Antibodies to crucial gD2 epitopes peaked at 1 μg, while ELISA and neutralizing titers continued to increase at higher doses. The epitope results suggested no immunologic benefit above 1 μg of gD2 mRNA-LNP, while ELISA and neutralizing titers indicated higher doses may be useful. We challenged the gD2 mRNA-immunized mice intravaginally with HSV-2. The 1-μg dose provided total protection, confirming the epitope studies, and supported assigning less than one-third of the trivalent vaccine maximum dose of 10 μg to gD2 mRNA-LNP. Epitope mapping as performed in mice can also be accomplished in phase 1 human trials to help select the optimum dose of each immunogen in a multivalent vaccine.

Deaths from herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are rare. A major exception is perinatally acquired HSV-1 or HSV-2 infection where the neonatal death rate is substantial. Fatal HSV infection also occurs occasionally in pregnant women. The goal of this review is to enumerate the reports that describe dual deaths of both a pregnant woman and her newborn from a herpesvirus infection. A total of 15 reports were found in the medical literature, of which five described pregnant women with HSV encephalitis and 10 described women with disseminated HSV infection. When the virus was typed, most cases of dual mother/newborn deaths were caused by HSV-2. Of interest, in two situations caused by HSV-1, the pregnant woman probably acquired her primary HSV-1 infection from one of her children and not by sexual transmission. Complete genomic sequencing was performed on one set of HSV-1 isolates collected from mother (blood) and newborn (blood and skin). The mother’s strain and the newborn’s skin strain were 98.9% identical. When the newborn’s two strains were compared, they were 97.4% identical. Only one mother was tested by the HerpeSelect IgG antibody kit. During the nine days of her undiagnosed disseminated infection preceding her death, her serology was negative. In summary, although dual mother/newborn deaths from HSV infection are rare, they continue to be reported as recently as 2017.

The contribution of virus components to liberation of herpes simplex virus type 2 (HSV-2) progeny virions from the surface of infected cells is poorly understood. We report that the HSV-2 mutant deficient in the expression of a mucin-like membrane-associated glycoprotein G (mgG) exhibited defect in the release of progeny virions from infected cells manifested by ~2 orders of magnitude decreased amount of infectious virus in a culture medium as compared to native HSV-2. Electron microscopy revealed that the mgG deficient virions were produced in infected cells and present at the cell surface. These virions could be forcibly liberated to a nearly native HSV-2 level by the treatment of cells with glycosaminoglycan (GAG)-mimicking oligosaccharides. Comparative assessment of the interaction of mutant and native virions with surface-immobilized chondroitin sulfate GAG chains revealed that while the mutant virions associated with GAGs ~fourfold more extensively, the lateral mobility of bound virions was much poorer than that of native virions. These data indicate that the mgG of HSV-2 balances the virus interaction with GAG chains, a feature critical to prevent trapping of the progeny virions at the surface of infected cells.

Herpes simplex virus type 2 (HSV-2) is the causative pathogen of genital herpes and is closely associated with the occurrence of cervical cancer and human immunodeficiency virus (HIV) infection. The absence of an effective vaccine and the emergence of drug resistance to commonly used nucleoside analogs emphasize the urgent need for alternative antivirals against HSV-2. Recently, ABMA [1-adamantyl (5-bromo-2-methoxybenzyl) amine] has been demonstrated to be an inhibitor of several pathogens exploiting host-vesicle transport, which also participates in the HSV-2 lifecycle. Here, we showed that ABMA inhibited HSV-2-induced cytopathic effects and plaque formation with 50% effective concentrations of 1.66 and 1.08 μM, respectively. We also preliminarily demonstrated in a time of compound addition assay that ABMA exerted a dual antiviral mechanism by impairing virus entry, as well as the late stages of the HSV-2 lifecycle. Furthermore, in vivo studies showed that ABMA protected BALB/c mice from intravaginal HSV-2 challenge with an improved survival rate of 50% at 5 mg/kg (8.33% for the untreated virus infected control). Consequently, our study has identified ABMA as an effective inhibitor of HSV-2, both in vitro and in vivo, for the first time and presents an alternative to nucleoside analogs for HSV-2 infection treatment.

The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.