Expression of the herpes simplex virus type 2 latency-associated transcript enhances spontaneous reactivation of genital herpes in latently infected guinea pigs.

The latency-associated transcript (LAT) is the only herpes simplex virus (HSV) gene product detectable in latently infected humans and animals. In this report, we show that a 624-bp deletion in the promoter of the HSV-2 LAT had no discernable effect on viral growth in tissue culture or in acute genital infection of guinea pigs, but impaired LAT accumulation and led to a marked decrease in spontaneous genital recurrences when compared with the behavior of wild-type and rescuant strains. Differences in the ability of the mutant to replicate, or in how readily it established or maintained latency did not account for this finding. Thus, HSV LAT expression facilitates the spontaneous reactivation of latent virus.

The characteristic site-specific reactivation phenotypes of HSV-1 and HSV-2 depend upon the latency-associated transcript region.

After replication at sites of initial inoculation, herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) establish lifelong latent infections of the sensory and autonomic neurons of the ganglia serving those sites. Periodically, the virus reactivates from these neurons, and travels centripetally along the neuronal axon to cause recurrent epithelial infection. The major clinically observed difference between infections with herpes simplex virus type 1 and type 2 is the anatomic site specificity of recurrence. HSV-1 reactivates most efficiently and frequently from trigeminal ganglia, causing recurrent ocular and oral-facial lesions, while HSV-2 reactivates primarily from sacral ganglia causing recurrent genital lesions. An intertypic recombinant virus was constructed and evaluated in animal models of recurrent ocular and genital herpes. Substitution of a 2.8-kbp region from the HSV-1 latency-associated transcript (LAT) for native HSV-2 sequences caused HSV-2 to reactivate with an HSV-1 phenotype in both animal models. The HSV-2 phenotype was restored by replacing the mutated sequences with wild-type HSV-2 LAT-region sequences. These sequences or their products must act specifically in the cellular environments of trigeminal and sacral neurons to promote the reactivation patterns characteristic of each virus.

Effect of adrenal manipulation on glucocorticoid receptors in MM1 hamster melanoma.

Cytosol glucocorticoid receptor of hamster Malignant Melanoma No. 1 (MM1) was characterized by dextran-coated charcoal and sucrose gradient assays. MM1 contains a specific, saturable, high-affinity (Kd 2.5 nM; 73.3 fmol/mg cytosol protein) receptor for glucocorticoid. The receptor sedimented at 7 to 8S in low-salt buffer and 5.5S in 0.4 M KCl and was sensitive to proteolysis by trypsin. Glucocorticoid receptor was inactivated by 40% (NH4)2SO4. Addition of 20 mM molybdate to the homogenizing buffer prevented inactivation. Adrenalectomy increased MM1 receptor content without altering affinity. Chronic exposure of intact or adrenalectomized hamsters to gluco- and high-dose mineralocorticoid significantly decreased the amount of unbound glucocorticoid receptors available for binding. Exposure of intact hamsters to high doses of mineralocorticoid also decreased apparent receptor affinity. These results suggest that hamster MM1 contains a high-affinity cytosol receptor for glucocorticoids similar to that of other glucocorticoid-responsive tissues, which may mediate the action of adrenal steroids on tumor behavior.

Biological behavior of MM1 hamster melanoma.

We have reported evidence recently for a high-affinity receptor for glucocorticoid Malignant Melanoma No. 1 hamster melanoma and suggested that tumor growth was facilitated by adrenal steroids. This report characterizes the behavior of Malignant Melanoma No. 1 following manipulation of the pituitary-adrenal axis in vivo. Bilateral adrenalectomy significantly retarded tumor growth. Hypophysectomy also significantly reduced tumor growth. Silastic implants of hydrocortisone in intact hamsters produced a dose (7 to 28 micrograms/day)-related increase in tumor growth. Implants releasing a low dose (3 micrograms/day) of dexamethasone also increased tumor growth. Chronic exposure of adrenalectomized and intact hamsters to a high dose (125 micrograms/day) of desoxycorticosterone acetate also produced a significant increase over adrenalectomized and sham-adrenalectomized controls. In contrast, chronic administration of adrenocorticotropic hormone and alpha-melanocyte-stimulating hormone to intact hamsters did not significantly alter melanoma growth. These observations support the suggestion that adrenocorticosteroids influence the growth of Malignant Melanoma No. 1 hamster melanoma and provide a model for studying the regulation of growth of a glucocorticoid-positive neoplasm originating outside the reticuloendothelial system.

Herpes simplex virus vaccines as immunotherapeutic agents.

As persistent viruses can escape immune surveillance, chronic or recurrent disease can be a major problem. Only after nearly 60 years of work have recent reproducible data, using herpes simplex virus infection as a model for persistent viral disease, established that vaccine immunotherapy is effective in the treatment of such viral infections.

Gender-specific predictors of genital herpes vaccine acceptance in a college population.

Vaccines represent one promising method for reducing the sexually transmitted disease (STD) epidemic. This study evaluated whether influences on the decision to accept a genital herpes vaccine differed by gender. In all, 518 college students completed a questionnaire on sexual history, health beliefs, and acceptance of a potential genital herpes vaccine. Each predictor variable plus a gender interaction term were analysed in separate logistic regression models. Follow-up analyses were performed by gender for outcomes that displayed significant interactions. Results indicated that a prior history of an STD and increased perception of risk for acquiring genital herpes were significant predictors of vaccine acceptance for men, while younger age and concerns about vaccine safety were significant predictors for women. Endorsement of a vaccine strategy targeting sexually experienced people was an influential factor for both genders, but was a much stronger one for women. Results suggest that gender-specific strategies may be crucial to genital herpes vaccine acceptance.

Photoperiodic control of melanoma growth in hamsters: influence of pinealectomy and melatonin.

Pinealectomy (PX) increased MM1 (melanotic melanoma no. 1) hamster melanoma growth in animals held under a 14-h light, 10-h dark (14:10) photoperiod without altering tumor latency. Hamsters maintained under a 6-h light, 18-h dark (6:18) photoperiod exhibited gonadal collapse, a longer tumor latency, and slower tumor growth rate than animals held under 14:10. PX produced a further increase in tumor latency and a decrease in growth in these animals. In contrast, acute morning injection of low doses (50 micrograms/day) of melatonin or delivery by Silastic capsule (35 micrograms/day) implanted at the time of tumor cell inoculation increased MM1 melanoma growth in hamsters held under 14:10 photocycle, without affecting testicular or adrenal function. Treatment of hamsters 11 weeks before tumor cell inoculation with 14 micrograms/day melatonin via Silastic capsule produced a decrease in serum PRL but no change in tumor growth or testicular or adrenal weights in animals held under 14:10. Treatment of hamsters with 17.7 micrograms/day melatonin (Silastic capsule) 11 weeks before tumor cell inoculation increased testes and adrenal weights as well as serum PRL and androgen levels, but significantly decreased tumor growth in hamsters held under a short daily photoperiod. These results suggest that the photoperiod under which hamsters are maintained dictates the growth rate of MM1 tumors and the effect of PX on tumor behavior. When photoperiod significantly alters gonadal and adrenal function, the quantity, time, and duration of melatonin presentation are all important variables in the effect of melatonin on tumor growth.

The concept of immune-based therapies in chronic viral infections.

Treatment of recurrent herpes simplex virus (HSV) infection with a vaccine was attempted as early as 1938. The concept of immune therapy is based on the premise that vaccine administration may augment host immunity, resulting in better control of the infection. The guinea pig model is useful in the study of recurrent HSV infection for several reasons. This animal model mimics human infection, with self-limited primary vulvovaginitis developing after inoculation with HSV. Despite a full range of host immune responses, the virus persists in a latent state within sensory ganglia. Periodically, the latent virus reactivates to produce recurrent genital infections. Use of the guinea pig model has made it possible to demonstrate that the administration of HSV glycoprotein vaccines to infected animals results in a significant reduction in the frequency and severity of recurrences. Studies demonstrate that a variety of factors influence the effectiveness of immunotherapy. These factors include not only immunogen and dose but also the route of administration, timing of treatment relative to primary infection, and adjuvant formulation. Investigations of HSV immunotherapy in the guinea pig model provide the first controlled experimental data indicating that vaccine administration to an infected host can favorably alter the natural history of a persistent infection.

Evaluation of varicella-zoster antiviral drugs by a nucleic acid hybridization assay.

A simple nucleic acid hybridization assay was developed for the evaluation of antiviral compounds with activity against varicella-zoster virus. Antiviral-induced reduction in varicella-zoster virus DNA, as measured by hybridization with 32P-labeled probes, correlated with drug-induced inhibition of viral cytopathic effect in cell culture. Three compounds were shown to be more potent than acyclovir at inhibiting varicella-zoster virus in vitro.

Drug testing for activity against varicella-zoster virus in hairless guinea pigs.

Inoculation of congenitally hairless guinea pigs with varicella-zoster virus (VZV) (Oka strain) results in a self-limited exanthematous infection analogous to varicella in children. Administration of acyclovir or 6-methoxypurine arabinoside modified the course of infection. This model should facilitate pre-clinical testing of putative anti-VZV agents.

Evaluation of the herpes simplex virus antiviral activity of pyrethrins.

Pyrethrins, complex esters extracted from Chrysanthemum cinerariaefolium, exhibit only minimal in vitro activity against herpes simplex virus (HSV). Employing a guinea pig model of HSV genital infection, no in vivo activity could be demonstrated. Although purported to be an effective remedy for the treatment of genital herpes, we were unable to demonstrate efficacy for either the oral administration of an alcoholic solution of pyrethrins or the topical application of pyrethrins in mineral oil.

Plant products as topical microbicide candidates: assessment of in vitro and in vivo activity against herpes simplex virus type 2.

There is considerable interest in developing topical microbicides; products to be used intravaginally by women for protection against sexually transmitted diseases. Many compounds derived from plants have been shown to have antimicrobial properties. We examined 19 such compounds in vitro by plaque reduction assay to determine their activity against a common sexually transmitted pathogen, herpes simplex virus type 2. Compounds with an ED50 < or = 7.0 mg/ml were tested for efficacy in vivo. Four compounds, carrageenan lambda type IV, cineole, curcumin, and eugenol, provided significant protection (P < 0.05) in a mouse model of intravaginal HSV-2 challenge. Eugenol, which provided the greatest protection in mice was also evaluated using the guinea pig model of genital HSV-2 infection where it also demonstrated significant protection. Based on these results, several plant-derived compounds appear to warrant further evaluation as potential microbicides.

Effect of undecylenic acid as a topical microbicide against genital herpes infection in mice and guinea pigs.

There is increasing interest in the use of topical microbicides to help prevent the spread of sexually transmitted diseases (STD). Undecylenic acid (UA), a monosaturated fatty acid, is the active ingredient in a number of over-the-counter (OTC) antifungal spray powders, that also exhibits in vitro antibacterial and antiviral activity, including herpes simplex virus (HSV) activity. We, therefore, evaluated UA as a topical microbicide against genital HSV infection using the murine and guinea pig models of genital herpes. Mice were administered a 20% solution of UA in polyethylene glycol (PEG) vehicle, vehicle alone or phosphate buffered saline (PBS) intravaginally immediately prior to vaginal challenge with HSV-2. Pre-treatment with UA decreased the number of mice that became infected (P < 0.001 vs. PBS or vehicle control), developed symptoms (P <0.001) or died (P <0.001). However, when treatment was extended to either 5 min prior to or after viral inoculation, protection was lost. Similar findings were found using the guinea pig model, where UA treatment completely prevented HSV-2 vaginal infection when given immediately prior to HSV-2 inoculation (P<0.001 vs. PBS or vehicle control). Thus, UA, an approved OTC medication, provided significant protection against HSV disease and infection only when applied immediately before viral inoculation, indicating that better formulations were needed to extend the duration of protection.

Effects of cytokines and R-837, a cytokine inducer, on UV-irradiation augmented recurrent genital herpes in guinea pigs.

We have recently reported that latently HSV-2-infected guinea pigs exhibit a three- to four-fold increase in recurrent lesions after exposure to ultraviolet radiation (UV), allowing rapid evaluation of antiviral drugs in treating recurrent HSV disease. In this report we examine the effect of alpha interferon (IFN-alpha), interleukin-2 (IL-2), and a cytokine inducer (R-837) on UV-induced recurrent genital herpes. We have previously shown that topical R-837 is a biologic response modifier with no in vitro anti-HSV activity, but with potent anti-HSV activity in vivo due to cytokine induction and enhancement of cell-mediated immune responses. Three-day regimens of intravaginal R-837, or five-day intraperitoneal (i.p.) administration of IFN-alpha or of IL-2 each significantly reduced recurrent genital HSV-2 disease that occurred within 7 days of UV exposure, suggesting that cytokines or cytokine inducers may be useful in the treatment of recurrent HSV disease. This model using ultraviolet radiation to induce recurrent herpes simplex virus infection proved useful in the evaluation of immunoactive agents as putative antiviral drugs.

Evaluation of HPMPC therapy for primary and recurrent genital herpes in mice and guinea pigs.

The nucleoside analogue (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC) inhibited the replication of herpes simplex virus (HSV) types 1 and 2 in tissue culture cells at about 1.0 micrograms/ml, whereas Acyclovir (ACV) had an EC50 of about 0.10-0.50 micrograms/ml. The purpose of these studies was to evaluate the efficacy of topically applied HPMPC in animal models of primary and recurrent genital HSV-2 infections. Mice treated with 5%, 1% or 0.5% HPMPC three times daily, beginning 6 or 24 h after virus inoculation had reduced vaginal viral replication regardless of time of initiation of therapy. ACV at 5% also reduced vaginal viral replication, but not as effectively as HPMPC. In primary infection of guinea pigs, therapy with 5% or 1% HPMPC beginning at 24 h but not 72 h significantly altered lesion development. However, 5% HPMPC was highly toxic to guinea pigs. Vaginal viral replication was reduced significantly with either 1% or 0.3% HPMPC initiated at 24 h. In these studies, HPMPC was also more efficacious than 5% ACV. Topical treatment with 1% HPMPC did not reduce the incidence or severity of spontaneous or UV-induced recurrent genital lesions. These results indicate that topical therapy with 1%, 0.5% or 0.3% HPMPC was more effective than 5% ACV in the treatment of primary genital HSV-2 infections of guinea pigs and mice and suggest that HPMPC should be considered for topical use in humans.

Herpes simplex virus glycoprotein treatment of recurrent genital herpes reduces cervicovaginal virus shedding in guinea pigs.

Treatment of previously infected guinea pigs with herpes simplex virus (HSV) glycoproteins reduces the frequency and severity of subsequent genital recurrences. An effective vaccine should also reduce episodes of viral shedding. In this study, HSV glycoproteins B and D treatment of animals experiencing recurrent genital herpes reduced the frequency of both clinical recurrences and cervicovaginal viral shedding. When virus was shed, however, the peak viral titer and duration of shedding was unaltered.

Herpes simplex virus glycoprotein immunotherapy of recurrent genital herpes: factors influencing efficacy.

Recombinant herpes simplex virus type 1 (HSV-1) glycoproteins B (gB) and D (gD) were used as immunotherapeutic agents for the treatment of recurrent genital herpes in guinea pigs. Administration of a gBgD vaccine eight to 21 days after intravaginal HSV-2 inoculation significantly increased the titer of anti-HSV antibodies (P less than 0.005) while significantly reducing the frequency of subsequent herpetic recurrences (P less than 0.05). The effectiveness of gBgD immunotherapy was influenced by both the co-administration of adjuvant, the type of adjuvant, and by the timing and route of administration. These data demonstrate that recurrent HSV disease in animals with established latent infection may be favorably altered by the administration of immunogenic viral proteins.

Recurrent genital herpes in the guinea pig augmented by ultraviolet irradiation: effects of treatment with acyclovir.

The guinea pig model of genital herpes simplex virus infection has proven useful in the evaluation of antiviral drugs. We have recently demonstrated that recurrent herpetic infections can be induced in latently infected guinea pigs by ultraviolet irradiation. In this report we have examined the effect of acyclovir on ultraviolet radiation-induced recurrent genital herpes. Prophylactic topical acyclovir decreased the severity but not the incidence of ultraviolet radiation-induced recurrences while intraperitoneal acyclovir initiated before ultraviolet irradiation reduced both the incidence and severity of induced recurrences. When treatment was begun after ultraviolet exposure, neither topical nor intraperitoneal acyclovir were effective in reducing the incidence or severity of induced recurrent disease. The effectiveness of acyclovir in the control of induced recurrent genital infections in the guinea pig is similar to what has been observed in human trials. This model of ultraviolet radiation-induced recurrent herpes simplex virus infection should prove useful in the evaluation on new putative antiviral drugs.

Introduction: Is viral shedding a surrogate marker for transmission of genital herpes?

Genital herpes, caused by either herpes simplex virus type 1 or 2 (HSV-1 and HSV-2), is a significant public health problem worldwide. It increases the risk of infection with HIV, upregulates HIV after infection and can be associated with serious morbidity and mortality. It is now known that clinical and subclinical viral reactivation with resultant shedding from anogenital mucosa occurs frequently, resulting in transmission during sexual contact. Sexual transmission of HSV infection is common, even between monogamous individuals. Antiviral therapy reduces the frequency and degree of viral shedding and lowers the transmission rate in discordant monogamous couples, although transmission can still occur in people prescribed antiviral therapy. These encouraging data raise important questions for the management of genital HSV infection, particularly with regard to the prevention of transmission. Although the quantity of virus present is clearly important in transmission of some viruses, it is not clear whether this is the case for HSV transmission. Ideally, a surrogate marker needs to be able to identify individuals with detectable amounts of virus, and differentiate them from individuals with detectable amounts of virus that are transmissible. The aim of this supplement is to explore the issues surrounding the validation of surrogate markers of transmission of HSV, using examples from other human viral diseases, and to review the available evidence. In the future, exploration of these issues may shed light on management and prevention strategies. In particular, the results may clarify what evidence is required to warrant prescribing a drug for reducing HSV transmission, and for which patient populations this strategy is appropriate.

Lessons from HIV and hepatitis viruses.

Surrogate markers are an important component in the process of investigating management and prevention strategies, and for increasing understanding of viral diseases. The importance of surrogate markers and applied statistical models is particularly true for HIV. For HIV infection, the development of such methods provides new approaches for evaluation of HIV therapies and vaccines, and for the study of HIV transmission and its pathogenesis. The complex natural history of hepatitis B infection demonstrates that viral load is not the only predictor of transmission of this virus; for hepatitis C infection, viral load per se is not a prognostic factor for disease progression, but cumulative viral load may affect the outcome, and therapy is aimed at eliminating active viral replication.