Effects of DNA immunization formulated with bupivacaine in murine and guinea pig models of genital herpes simplex virus infection

The immunogenicity and efficacy of a herpes simplex virus type 2 glycoprotein D (gD2) DNA vaccine formulated with bupivacaine was evaluated using murine and guinea pig models of genital herpes. Animals received three doses of 100 microg of gD2 plasmid or control plasmid intramuscularly prior to intravaginal challenge with HSV-2. Immunization induced HSV ELISA and neutralizing antibody in serum and ELISA antibody in the vaginal secretions of all animals evaluated. Following intravaginal HSV-2 challenge, vaginal viral replication was reduced in both models with peak reductions of greater than 99%. Immunization also decreased the number of animals developing any clinical disease (p < 0.001) and the severity of the acute disease (total lesion score 6.4 versus 0.6 in guinea pigs, p < 0.001). Further recurrent lesion days were reduced from 14.5 to 4.9 days in immunized guinea pigs (p < 0.001). DNA immunization with gD2 + bupivacaine was effective in reducing clinical disease and viral replication in both guinea pigs and mice.     

A Vaxfectin-adjuvanted HSV-2 plasmid DNA vaccine is effective for prophylactic and therapeutic use in the guinea pig model of genital herpes

Here we describe studies in the guinea pig model of genital herpes to evaluate a novel plasmid DNA (pDNA) vaccine encoding the HSV-2 glycoprotein D and UL46 and UL47 genes encoding tegument proteins VP11/12 and VP 13/14 (gD2/UL46/UL47), formulated with a cationic lipid-based adjuvant Vaxfectin^®. Prophylactic immunization with Vaxfectin^®-gD2/UL46/UL47 significantly reduced viral replication in the genital tract, provided complete protection against both primary and recurrent genital skin disease following intravaginal HSV-2 challenge, and significantly reduced latent HSV-2 DNA in the dorsal root ganglia compared to controls. We also examined the impact of therapeutic immunization of HSV-2 infected animals. Here, Vaxfectin^®-gD2/UL46/UL47 immunization significantly reduced both the frequency of recurrent disease and viral shedding into the genital tract compared to controls. This novel adjuvanted pDNA vaccine has demonstrated both prophylactic and therapeutic efficacy in the guinea pig model of genital herpes and warrants further development.     

Reduction of recurrent HSV disease using imiquimod alone or combined with a glycoprotein vaccine

The aim of this study is to evaluate the effects of an immune modulator, imiquimod, given alone or in combination with an HSV vaccine on HSV immune responses and as immunotherapy of a genital recurrence model. After recovery from primary genital HSV infection, animals were randomized to placebo, 21 days of imiquimod plus a placebo vaccine, or 21 days of imiquimod plus an HSV-2 glycoprotein vaccine. Placebo or HSV vaccine was given in the footpad on days 16 and 37 after HSV-2 genital inoculation. Daily imiquimod or placebo was given subcutaneously in the shoulder on days 16 through 37. Genital recurrences were monitored and HSV specific NK activity, IL-2 response and ELISA antibody were assayed. For the entire 15 week observation period, imiquimod alone reduced recurrences 62.6%, while addition of HSV vaccine to imiquimod reduced recurrences 80.6% compared to placebo/placebo. The duration of significant recurrence reduction was more notable with the addition of vaccine. Imiquimod alone significantly reduced the weekly HSV recurrent disease in the first 10 weeks (53-94% reduction, mean 75.9%), as did imiquimod plus HSV-vaccine (71-98% reduction, mean 89.5%). In weeks 10-15, imiquimod alone reduced recurrences significantly only in week 10 (20-53% reduction, mean 33%), whereas the addition of vaccine extended the significant recurrence reduction to 14 weeks (44-71% reduction, mean 56.8%). The recurrence reduction is correlated to an increased HSV-induced in vitro IL-2 response and NK activity against HSV targets in treated groups. Both imiquimod and the imiquimod/vaccine combination significantly reduced genital HSV recurrences, but the combination extended the duration and extent of protection for recurrences compared to imiquimod alone. Enhanced HSV specific immune responses correlated to the protection.     

Therapeutic HSV-2 vaccine decreases recurrent virus shedding and recurrent genital herpes disease

BackgroundGenital herpes simplex virus (HSV) type 2 is a common persistent infection that frequently reactivates to cause recurrent lesions and recurrent viral shedding which is incompletely controlled by antiviral therapy. GEN-003 is a candidate therapeutic vaccine containing 2 HSV-2 proteins, gD2 and ICP4, and Matrix-M2 adjuvant (M2).MethodsHSV-2 seropositive persons with genital herpes were randomized into three dose cohorts of Gen-003 (60 µg antigen/50 µg M2, 60 µg/75 µg M2 or Placebo). Three intramuscular doses 21 days apart of GEN-003 or placebo were administered. Participants obtained genital area swabs twice-daily for HSV-2 detection and monitored genital lesions for 12 months. The rates of virus shedding and lesion rates before vaccination were compared to 3 defined periods after vaccination; Days 43–71, Month 6 and Month 12.ResultsGEN-003 at a dose of 60 µg each antigen/50 µg M2 reduced HSV shedding immediately after dosing with a rate ratio of 0.58, compared to 0.75 for the GEN-003 60 µg/75 µg M2 and 1.06 for placebo. Lesion rates, recurrence rates, and duration of recurrences were also reduced. Reactogenicity was higher with the 75 µg M2 dose than the 50 µg M2 dose, specifically for pain, tenderness, malaise and fatigue. Antibody and cellular immune responses were stimulated by both doses and persisted to 12 months.ConclusionsGEN-003 vaccine manufactured with a scalable process gave results similar to those observed in prior clinical trials. GEN-003 had an acceptable safety profile and stimulated both humoral and cellular immune responses. The 60 µg antigen/50 µg M2 provided the maximal effect on virologic and clinical measures and warrants further development. (Funded by Genocea; ClinicalTrials.gov number NCT02515175).     

Vaccine prevents genital herpes in subgroup of women

The herpes simplex virus (HSV) type-2 vaccine studied here prevented genital herpes only in women who were seronegative for HSV-1 and HSV-2 at baseline. Ten of these women would need to be vaccinated to prevent 1 case of genital herpes. The vaccine did not prevent infection with HSV-2 in these women. It did not prevent genital herpes in women with other HSV serologic status or in men. The usefulness of this vaccine is limited by the small subgroup in which it is efficacious. Determining which women fall into this subgroup could prove costly. It is possible that asymptomatic infected persons may spread HSV more readily. Emphasis on the use of condoms and antiviral agents should still be the first line in preventing the spread of genital herpes.     

Effects of herpes simplex virus type 2 glycoprotein vaccines and CLDC adjuvant on genital herpes infection in the guinea pig

Genital herpes simplex virus (HSV) infections are common but results from vaccine trials with HSV-2 glycoprotein D (gD) have been disappointing. We therefore compared a similar HSV gD2 vaccine, to a further truncated gD2 vaccine, to a vaccine with gD2 plus gB2 and gH2/gL2 and to a vaccine with only gB2 and gH2/gL2 in a guinea pig model of genital herpes. All vaccines were administered with cationic liposome-DNA complexes (CLDC) as an adjuvant. All vaccines significantly decreased the severity of acute genital disease and vaginal virus replication compared to the placebo group. The majority of animals in all groups developed at least one episode of recurrent disease but the frequency of recurrent disease was significantly reduced by each vaccine compared to placebo. No vaccine was significantly more protective than gD2 alone for any of the parameters described above. No vaccine decreased recurrent virus shedding. When protection against acute infection of dorsal root ganglia and the spinal cord was evaluated all vaccines decreased the per cent of animal with detectable virus and the quantity of virus but again no vaccine was significantly more protective than another. Improvements in HSV-2 vaccines may require inclusion of more T cell targets, more potent adjuvants or live virus vaccines.     

A vaccine containing highly purified virus particles in adjuvant provides high level protection against genital infection and disease in guinea pigs challenged intravaginally with homologous and heterologous strains of herpes simplex virus type 2

Infection with Herpes Simplex Viruses (HSVs) represents a significant health burden worldwide with HSV-1 and HSV-2 causing genital disease and HSV-2 contributing to human immunodeficiency virus acquisition. Despite great need, there is currently no licensed vaccine against HSV. In this report, we evaluated the protective efficacy of a vaccine containing highly purified, inactivated HSV-2 particles (with and without additional recombinant glycoprotein D) formulated with a monophosphoryl lipid A/Alhydrogel adjuvant in a guinea pig HSV genital model. The key results from 3 independent studies were: (1) vaccination consistently provided significant 3–3.5 Log10 reductions in vaginal HSV-2 titers on day 2 postchallenge; (2) following homologous or heterologous challenge with two U.S. isolates, all vaccine groups showed complete protection against lesion formation, significant 3 Log10 reductions in day 2 virus shedding, enhanced virus clearance, significant reductions in HSV-2 DNA within ganglia, and no detectable shedding (<2 PFU) or latent viral DNA in some immunized animals; (3) following challenge with a third heterologous strain, vaccination provided complete protection against primary and recurrent lesions, significant reductions in primary virus shedding, a 50% reduction in recurrent shedding days, and undetectable latent virus in the ganglia and spinal cords of most animals; and (4) adding glycoprotein D provided no enhanced protection relative to that elicited by the inactivated HSV-2 particles alone. Together, these data provide strong support for further development of this exceedingly protective and highly feasible vaccine candidate for human trials.     

Effect of immunization with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) plus the immune enhancer GPI-0100 on infection with HSV-1 or HSV-2

These studies were performed to determine the effects of GPI-0100, a semi synthetic Quillaja Saponin analog, formulated with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) on immunity to HSV. SKH-1 hairless mice, used as a model of herpes labialis, inoculated with HSV-1 results in facial lesions, virus replication and mortality. Mortality rates, lesion scores and viral titers were significantly reduced in SKH-1 mice immunized with gD/GPI-0100 prior to cutaneous inoculation with HSV-1 and the protective effects were greater than those using the standard alum adjuvant. Genital HSV-2 infections in guinea pigs were also utilized to determine if gD combined with GPI-0100 was protective against infection, disease severity and viral shedding. Guinea pigs immunized with HSV-1 gD with or without GPI-0100 had significantly reduced area under the curve lesion scores, but infection rates and virus shedding was not altered. When Tween 40 was added to gD and GPI-0100, mean peak lesion scores were also significantly reduced. The results obtained in a genital HSV-2 infection of guinea pigs did not indicate enhanced protection or reduced virus shedding following immunization with GPI-0100 and gD. There was, however, a significant improvement in clinical herpetic genital disease with the combination of gD plus the immune enhancer GPI-0100.     

Immunity induced by DNA immunization with herpes simplex virus type 2 glycoproteins B and C

The complete sequence of herpes simplex virus type 2 (HSV-2) glycoproteins B and C (gB & gC) were cloned into plasmid expression vectors and evaluated in murine and guinea pig genital HSV-2 models. Balb/c mice were immunized with either pgB-2 or pgC-2 plasmids intramuscularly (IM) or intradermally (ID). The vaccines induced HSV-2-specific neutralizing and ELISA IgG antibody, but little or no enhancement of viral clearance from the vagina was detected following intravaginal challenge. Immunization of guinea pigs with pgB-2 or pgC-2 induced ELISA IgG antibody; however, antibody titers were approximately one log(10) unit lower than that seen in HSV-2 convalescent sera. IM immunization of guinea pigs with either plasmid also did not decrease vaginal viral shedding following vaginal challenge, but the severity of the acute disease and the subsequent number of recurrent lesion days were reduced in animals immunized with pgB-2. Lastly, IM immunization of latently infected guinea pigs with a combined gB-2 and gC-2 plasmid vaccine significantly reduced the number of subsequent HSV-2 recurrences. DNA vectors expressing gB-2 or gC-2 were both immunogenic, although the gB-2 plasmid induced higher titers of antibody and significantly reduced primary and recurrent herpetic disease in the guinea pig model. These results also suggest that immunotherapy with plasmid expression vectors may be effective against recurrent genital HSV-2 disease.     

The adjuvant CLDC increases protection of a herpes simplex type 2 glycoprotein D vaccine in guinea pigs

Herpes simplex virus (HSV) infections are common but there is no vaccine available. We evaluated cationic liposome–DNA complexes (CLDC) as an adjuvant for an HSV gD2 vaccine and compared it to an MPL/Alum adjuvant in a guinea pig model of genital herpes. The addition of CLDC to the gD2 vaccine significantly decreased acute and recurrent disease and most importantly the number of days with recurrent virus shedding compared to gD2 alone. Reductions in these outcomes were also detected when gD2 + CLDC was compared to gD2 + MPL/Alum. When the vaccine and adjuvants were evaluated as therapeutic vaccines, they were ineffective. CLDC enhanced protection compared to MPL/Alum and is the first vaccine to reduce recurrent virus shedding, a key to decreasing the spread of HSV-2.     

Safety and immunogenicity of long HSV-2 peptides complexed with rhHsc70 in HSV-2 seropositive persons

HSV-2, the primary causative agent of genital herpes, establishes latency in sensory ganglia and reactivates causing recurrent lesions and viral shedding. Induction or expansion of CD4⁺ and CD8⁺ T cell responses are expected to be important for a successful therapeutic vaccine against HSV-2. A candidate vaccine consisting of 32 synthetic 35mer HSV-2 peptides non-covalently complexed with recombinant human Hsc70 protein (named HerpV, formerly AG-707) was tested for safety and immunogenicity in a Phase I study. These peptides are derived from 22 HSV-2 proteins representative of all phases of viral replication. Thirty-five HSV-2 infected participants were randomized and treated in one of four groups: HerpV + QS-21 (saponin adjuvant), HerpV, QS-21, or vehicle. The vaccine was well tolerated and safe. All seven participants with evaluable samples who were administered HerpV with QS-21 demonstrated a statistically significant CD4⁺ T cell response to HSV-2 antigens, and the majority of such participants demonstrated a statistically significant CD8⁺ T cell response as well. To our knowledge, this is the first candidate vaccine against HSV-2 to demonstrate a broad CD4⁺ and CD8⁺ T cell response in HSV-2⁺ participants, and the first HSP-based vaccine to show immune responses against viral antigens in humans.     

Cross protective efficacy of the Non-Neurotropic live attenuated herpes simplex virus type 1 vaccine VC-2 is enhanced by intradermal vaccination and deletion of glycoprotein G

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 respectively) cause life-long latent infections resulting in recurrent orofacial and genital blisters or sores. Ensued disease can be painful and may lead to significant mental anguish of infected individuals. Currently, there are no FDA-approved vaccines for either prophylactic or therapeutic use, and recent clinical trials of subunit vaccines failed to achieve endpoints goals. Development of a safe live-attenuated herpes simplex vaccine may provide the antigenic breadth to ultimately protect individuals from acquiring HSV disease. We have previously shown that prophylactic use of the non-neurotropic live attenuated HSV-1 vaccine, VC-2, provides potent and durable protection from genital HSV-2 disease in the guinea pig model. Here, we investigated the effects of intradermal administration as well as the deletion of the viral glycoprotein G (gG) on the efficacy of prophylactic vaccination. Vaccination with either VC-2, VC-2 gG null, or gD2 MPL/Alum offered robust protection from acute disease regardless of route of vaccination. However, both the VC-2 gG-null and the ID vaccination route were more effective compared to the parent VC2 administered by the IM route. Specifically, the VC-2 gG-null administered ID, reduced HSV-2 vaginal replication on day 2 and day 4 as well as mean recurrent lesion scores more effectively than VC2 administered IM. Most importantly, only VC-2 gG null IM and VC-2 ID significantly reduced the frequency of recurrent shedding, the most likely source for virus transmission. Similarly, while all vaccinated groups demonstrated a significant reduction in the number of animals testing PCR-positive for HSV-2 in their dorsal root ganglia following challenge only VC2 ID vaccinated animals demonstrated a significant reduction in DRG viral load. All vaccinations induced neutralizing antibodies to HSV-2 MS when compared to unvaccinated guinea pigs. Therefore, further investigation of VC-2 gG null delivered ID is warranted.     

Inactivated HSV-2 in MPL/alum adjuvant provides nearly complete protection against genital infection and shedding following long term challenge and rechallenge

Herpes Simplex Virus Type 2 (HSV-2) infection can result in life-long recurrent genital disease, asymptomatic virus shedding, and transmission. No vaccine to date has shown significant protection clinically. Here, we used a mouse model of genital HSV-2 infection to test the efficacy of a vaccine consisting of whole, formalin-inactivated HSV-2 (FI-HSV2) formulated with monophosphoryl lipid A (MPL) and alum adjuvants. Vaccine components were administered alone or as a prime-boost immunization together with DNA vaccines encoding a truncated glycoprotein D2 (gD2t) and two conserved HSV-2 genes necessary for virus replication, UL5 (DNA helicase) and UL30 (DNA polymerase). Our results show: (1) compared with mock immunized controls, mice immunized with FI-HSV2 plus MPL/alum consistently showed protection against disease burden and total viral shedding while the mice immunized with gD2t protein with MPL/alum did not; (2) protection against genital disease and viral replication correlated with the type of boost in a prime-boost immunization with little advantage afforded by a DNA prime; (3) intramuscular (i.m.) immunization with FI-HSV2 in MPL/Alhydrogel adjuvant provided nearly complete protection against vaginal HSV-2 shedding after a lethal intravaginal (i.vag.) short-term challenge and long-term rechallenge; (4) single formulation immunization with DNA vaccines, FI-HSV2, and MPL in an aluminum phosphate (Adju-Phos) adjuvant did not increase protection relative to FI-HSV2/MPL/Adju-Phos alone; and (5) addition of MPL/alum to the FI-HSV2 was required for optimal protection against disease, viral replication, and latent virus load in the dorsal root ganglia (DRG). Most notably, an optimized vaccine formulation of FI-HSV2 MPL/Alhydrogel given i.m. completely protected against detectable vaginal HSV-2 shedding in the majority of animals and HSV-2 latent DNA in the DRG of all animals.     

Evaluation of AD472, a live attenuated recombinant herpes simplex virus type 2 vaccine in guinea pigs

An attenuated recombinant herpes simplex virus type 2 (HSV-2), designated as AD472, was constructed by deleting both copies of the gamma(1)34.5 gene, UL55-56, UL43.5, and the US10-12 region from HSV-2 strain G. This virus was engineered to be a safe and effective live attenuated HSV-2 vaccine and was tested in the guinea pig model of genital herpes to evaluate its ability to protect from disease upon challenge with the wild type (wt) virus, HSV-2 (G). AD472 administered intramuscularly did not prevent infection or virus replication in the vaginal tract, but did reduce both lesion development and severity in a dose-dependent manner in guinea pigs challenged with the wt virus. Frequency of reactivation from latency was low compared with that of the parent virus, HSV-2 (G). Immunization with AD472 at doses of 1x10(5)PFU generally precluded colonization of the ganglia or establishment of latency by the challenge virus. Results presented here support the concept of a rationally engineered live attenuated vaccine for the prevention of the genital disease associated with infection by HSV-2.     

The challenges and opportunities for the development of a T-cell epitope-based herpes simplex vaccine

Herpes simplex virus type 1 and type 2 (HSV-1 & HSV-2) infections have been prevalent since the ancient Greek times. To this day, they still affect a staggering number of over a billion individuals worldwide. HSV-1 infections are predominant than HSV-2 infections and cause potentially blinding ocular herpes, oro-facial herpes and encephalitis. HSV-2 infections cause painful genital herpes, encephalitis, and death in newborns. While prophylactic and therapeutic HSV vaccines remain urgently needed for centuries, their development has been difficult. During the most recent National Institute of Health (NIH) workshop titled “Next Generation Herpes Simplex Virus Vaccines: The Challenges and Opportunities”, basic researchers, funding agencies, and pharmaceutical representatives gathered: (i) to assess the status of herpes vaccine research; and (ii) to identify the gaps and propose alternative approaches in developing a safe and efficient herpes vaccine. One “common denominator” among previously failed clinical herpes vaccine trials is that they either used a whole virus or a whole viral protein, which contain both “pathogenic symptomatic” and “protective asymptomatic” antigens and epitopes. In this report, we continue to advocate developing “asymptomatic” epitope-based sub-unit vaccine strategies that selectively incorporate “protective asymptomatic” epitopes which: (i) are exclusively recognized by effector memory CD4⁺ and CD8⁺ T cells (T_EM cells) from “naturally” protected seropositive asymptomatic individuals; and (ii) protect human leukocyte antigen (HLA) transgenic animal models of ocular and genital herpes. We review the role of animal models in herpes vaccine development and discuss their current status, challenges, and prospects.     

A trivalent gC2/gD2/gE2 vaccine for herpes simplex virus generates antibody responses that block immune evasion domains on gC2 better than natural infection

Vaccines for prevention and treatment of genital herpes are high public health priorities. Our approach towards vaccine development is to focus on blocking virus entry mediated by herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) and to prevent the virus from evading complement and antibody attack by blocking the immune evasion domains on HSV-2 glycoproteins C (gC2) and E (gE2), respectively. HSV-2 gC2 and gE2 are expressed on the virion envelope and infected cell surface where they are potential targets of antibodies that bind and block their immune evasion activities. We demonstrate that antibodies produced during natural infection in humans or intravaginal inoculation in guinea pigs bind to gC2 but generally fail to block the immune evasion domains on this glycoprotein. In contrast, immunization of naïve or previously HSV-2-infected guinea pigs with gC2 subunit antigen administered with CpG and alum as adjuvants produces antibodies that block domains involved in immune evasion. These results indicate that immune evasion domains on gC2 are weak antigens during infection, yet when used as vaccine immunogens with adjuvants the antigens produce antibodies that block immune evasion domains.     

Biologic interactions between HSV-2 and HIV-1 and possible implications for HSV vaccine development

Development of a safe and effective vaccine against herpes simplex virus type 2 (HSV-2) has the potential to limit the global burden of HSV-2 infection and disease, including genital ulcer disease and neonatal herpes, and is a global sexual and reproductive health priority. Another important potential benefit of an HSV-2 vaccine would be to decrease HIV infections, as HSV-2 increases the risk of HIV-1 acquisition several-fold. Acute and chronic HSV-2 infection creates ulcerations and draws dendritic cells and activated CD4+ T cells into genital mucosa. These cells are targets for HIV entry and replication. Prophylactic HSV-2 vaccines (to prevent infection) and therapeutic vaccines (to modify or treat existing infections) are currently under development. By preventing or modifying infection, an effective HSV-2 vaccine could limit HSV-associated genital mucosal inflammation and thus HIV risk. However, a vaccine might have competing effects on HIV risk depending on its mechanism of action and cell populations generated in the genital mucosa. In this article, we review biologic interactions between HSV-2 and HIV-1, consider HSV-2 vaccine development in the context of HIV risk, and discuss implications and research needs for future HSV vaccine development.     

Intranasal nanoemulsion-adjuvanted HSV-2 subunit vaccine is effective as a prophylactic and therapeutic vaccine using the guinea pig model of genital herpes

Genital herpes is a sexually transmitted disease representing a major global health concern. Currently, there is no approved vaccine and existing antiviral therapies exhibit limited efficacy. Herein, we describe an intranasal (IN) vaccine comprised of HSV-2 surface glycoproteins gD2 and gB2 formulated in a nanoemulsion adjuvant (NE01-gD2/gB2). Using the HSV-2 genital herpes guinea pig model, we demonstrate that IN NE01-gD2/gB2 induces higher levels of neutralizing antibody compared to a monovalent IN NE01-gD2 vaccine, but less than an intramuscular (IM) Alum/MPL-gD2 vaccine. Following intravaginal (IVag) challenge with HSV-2, the group immunized with IN NE01-gD2/gB2 exhibited significantly reduced acute and recurrent disease scores compared to placebo recipients. Significantly, latent virus was only detected in the dorsal root ganglia of 1 of 12 IN NE01-gD2/gB2-vaccinated animals compared to 11 of 12 placebo recipient. In the therapeutic model, IN NE01-gD2/gB2 immunized guinea pigs exhibited a significant reduction in the recurrent lesions scores (64%, p < 0.01), number of animal days with disease (64%, p < 0.01), number of animals with viral shedding (50%, p < 0.04) and reduction in virus positive vaginal swabs (56%, p < 0.04), These data suggests that the treatment may be effective in treating chronic disease and minimizing virus transmission. These results warrant advancing the development of IN NE01-gD2/gB2 as both a prophylactic and therapeutic vaccine against HSV-2.     

Incidence of herpes neonatorum in Netherlands

OBJECTIVE: Investigation of the incidence of neonatal herpes in the Netherlands between 1992 and 1998. DESIGN: Inventory questionnaire survey. METHODS: All virological laboratories in the Netherlands were sent a questionnaire on the number of culture proven cases of neonatal herpes recorded between 1992 and 1998 and on the type of herpes simplex virus (HSV-1 or HSV-2). The gynaecological and paediatric departments of all university hospitals and of half of the general hospitals were sent questionnaires as well. Gynaecologists were asked how often caesarean section was performed in order to prevent neonatal herpes and how frequently pregnant women were seen with genital herpes. Paediatricians were asked how often they observed neonatal herpes, the type of HSV and the possible transmission route. Based on these data the figures for the whole of the Netherlands were estimated. RESULTS: The incidence of neonatal herpes in the Netherlands in the period 1992 to 1998 was 2.4 per 100,000 neonates. HSV-1 was the cause of neonatal herpes in 73%, HSV-2 in 9%, and in 18% of the cases the type of infection was not recorded. The number of pregnant women with genital herpes had increased, but, in agreement with a consensus statement, the gynaecologists hardly performed caesarean sections any more to prevent neonatal herpes (2 per year). CONCLUSIONS: The incidence of neonatal herpes in the Netherlands had not increased. There was no predominant role of HSV type 2 causing neonatal herpes.