Phosphoregulation of a Conserved Herpesvirus Tegument Protein by a Virally Encoded Protein Kinase in Viral Pathogenicity and Potential Linkage between Its Evolution and Viral Phylogeny.

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multifunctional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein, UL7, as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenetically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7s of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestor for HSV-1, HSV-2, and ChHV, and other members of the genus Simplexvirus that are phylogenetically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplex viruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1, in contrast to phosphorylation in HSV-2, had no effect on viral replication and pathogenicity in mice. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phosphoregulation of UL7 during their evolution because the phosphoregulation had an impact on viral fitness in vivo, whereas most other simplex viruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivoIMPORTANCE It has been hypothesized that the evolution of protein phosphoregulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phosphoregulation and the phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phosphoregulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo We also showed that the evolution of Us3 phosphoregulation of UL7 was linked to the phylogeny of viruses that are phylogenetically close to HSV-2 and to the phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have and have not evolved phosphoregulation, respectively. This study reports the first evidence showing that evolution of viral phosphoregulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phosphoregulation during viral evolution.

Immunofluorescence diagnosis of the herpesvirus stillborn infection.

Congenital herpes infection belongs to the category of actual problems of Perinatal Medicine. Pathological diagnosis of this disease is not effective in the routine method of autopsy studies without virological research. Objective. Determination of the value of the fluorescent antibody technique in the diagnosis of congenital herpes infection of the stillborn is a promising approach to medical diagnosis. subjects and methods. In 96 cases of stillbirth immunofluorescent identification of herpes simplex virus types 1 and 2 and cytomegalovirus in the placenta and internal organs (brain, heart, lungs, and liver) was implemented. The findings were compared with the results of a complete histological examination of the heart, including its rhythmogenic centers. Results. The herpes viruses were found in 51 observations (53.1%). Among them, HSV-1 were found in 16 observations (16.7%), HSV-2, in 19 (19.7%), CMV, in 16 (16.7%). In 34 stillbirths (35.8%) the pathological signs of herpetic atrial myocarditis were observed, which were regarded as the cause of death. Conclusion. The use of the fluorescent antibody technique in the autopsy practice is an effective way of diagnosis of intrauterine infection caused by the herpes simplex virus and cytomegalovirus.

Immunization with HSV-2 gB-CCL19 Fusion Constructs Protects Mice against Lethal Vaginal Challenge.

There is a lack of an HSV-2 vaccine, in part as the result of various factors that limit robust and long-term memory immune responses at the mucosal portals of viral entry. We previously demonstrated that chemokine CCL19 augmented mucosal and systemic immune responses to HIV-1 envelope glycoprotein. Whether such enhanced immunity can protect animals against virus infection remains to be addressed. We hypothesized that using CCL19 in a fusion form to direct an immunogen to responsive immunocytes might have an advantage over CCL19 being used in combination with an immunogen. We designed two fusion constructs, plasmid (p)gBIZCCL19 and pCCL19IZgB, by fusing CCL19 to the C- or N-terminal end of the extracellular HSV-2 glycoprotein B (gB) with a linker containing two (Gly4Ser)2 repeats and a GCN4-based isoleucine zipper motif for self-oligomerization. Following immunization in mice, pgBIZCCL19 and pCCL19IZgB induced strong gB-specific IgG and IgA in sera and vaginal fluids. The enhanced systemic and mucosal Abs showed increased neutralizing activity against HSV-2 in vitro. Measurement of gB-specific cytokines demonstrated that gB-CCL19 fusion constructs induced balanced Th1 and Th2 cellular immune responses. Moreover, mice vaccinated with fusion constructs were well protected from intravaginal lethal challenge with HSV-2. Compared with pgB and pCCL19 coimmunization, fusion constructs increased mucosal surface IgA(+) cells, as well as CCL19-responsive immunocytes in spleen and mesenteric lymph nodes. Our findings indicate that enhanced humoral and cellular immune responses can be achieved by immunization with an immunogen fused to a chemokine, providing information for the design of vaccines against mucosal infection by HSV-2 and other sexually transmitted viruses.

Immunogenicity, protective efficacy, and non-replicative status of the HSV-2 vaccine candidate HSV529 in mice and guinea pigs.

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine.

Role of Fas/FasL signaling in regulation of anti-viral response during HSV-2 vaginal infection in mice.

Fas receptor-Fas ligand (FasL) signaling is involved in apoptosis of virus-infected cells but increasing evidence accumulates on Fas receptor as a mediator of apoptosis-independent processes such as induction of activating and pro-inflammatory signals. In this study, we examined the role of Fas/FasL pathway in regulation of anti-viral response to genital HSV-2 infection using a murine model of HSV-2 infection applied to C57BL6/J, B6. MRL-Faslpr/J and B6Smn.C3-Faslgld/J mice. HSV-2 infection of Fas- and FasL-deficient mice led to decreased migration of IFN-γ expressing NK cells and CD4+ T cells, but not of γδ T cells, into the vaginal tissue. The vaginal tissues of HSV-2 infected Fas- and FasL-deficient mice showed increased production of IL-10, followed by low expression of the early CD69 activation marker on CD4+ and CD8+ T cells and increased numbers of regulatory T cells (Tregs). Experiments in co-cultures of CD4+ T cells and bone marrow derived dendritic cells showed that lack of bilateral Fas-FasL signaling led to expansion of Tregs and increased production of IL-10 and TGF-ß1. Our results demonstrate that Fas/FasL can regulate development of tolerogenic dendritic cells and expansion of Tregs early during HSV-2 infection, which further influences effective anti-viral response.

Vaginally delivered tenofovir disoproxil fumarate provides greater protection than tenofovir against genital herpes in a murine model of efficacy and safety.

Increased susceptibility to genital herpes in medroxyprogesterone-treated mice may provide a surrogate of increased HIV risk and a preclinical biomarker of topical preexposure prophylaxis safety. We evaluated tenofovir disoproxil fumarate (TDF) in this murine model because an intravaginal ring eluting this drug is being advanced into clinical trials. To avoid the complications of surgically inserting a ring, hydroxyethylcellulose (HEC)-stable formulations of TDF were prepared. One week of twice-daily 0.3% TDF gel was well tolerated and did not result in any increase in HSV-2 susceptibility but protected mice from herpes simplex virus 2 (HSV-2) disease compared to mice treated with the HEC placebo gel. No significant increase in inflammatory cytokines or chemokines in vaginal washes or change in cytokine, chemokine, or mitochondrial gene expression in RNA extracted from genital tract tissue was detected. To further evaluate efficacy, mice were treated with gel once daily beginning 12 h prior to high-dose HSV-2 challenge or 2 h before and after viral challenge (BAT24 dosing). The 0.3% TDF gel provided significant protection compared to the HEC gel following either daily (in 9/10 versus 1/10 mice, P < 0.01) or BAT24 (in 14/20 versus 4/20 mice, P < 0.01) dosing. In contrast, 1% tenofovir (TFV) gel protected only 4/10 mice treated with either regimen. Significant protection was also observed with daily 0.03% TDF compared to HEC. Protection was associated with greater murine cellular permeability of radiolabeled TDF than of TFV. Together, these findings suggest that TDF is safe, may provide substantially greater protection against HSV than TFV, and support the further clinical development of a TDF ring.

Live attenuated herpes simplex virus 2 glycoprotein E deletion mutant as a vaccine candidate defective in neuronal spread.

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.

Zinc acetate/carrageenan gels exhibit potent activity in vivo against high-dose herpes simplex virus 2 vaginal and rectal challenge.

Topical microbicides that block the sexual transmission of HIV and herpes simplex virus 2 (HSV-2) are desperately needed to reduce the incidence of HIV infections worldwide. Previously we completed phase 3 testing of the carrageenan-based gel Carraguard. Although the trial did not show that Carraguard is effective in preventing HIV transmission during vaginal sex, it did show that Carraguard is safe when used weekly for up to 2 years. Moreover, Carraguard has in vitro activity against human papillomavirus (HPV) and HSV-2 and favorable physical and rheological properties, which makes it a useful vehicle to deliver antiviral agents such as zinc acetate. To that end, we previously reported that a prototype zinc acetate carrageenan gel protects macaques against vaginal challenge with combined simian-human immunodeficiency virus reverse transcriptase (SHIV-RT). Herein, we report the safety and efficacy of a series of zinc acetate and/or carrageenan gels. The gels protected mice (75 to 85% survival; P < 0.001) against high-dose (10(6)-PFU) HSV-2 vaginal or rectal challenge. In contrast, zinc acetate formulated in HEC (hydroxyethylcellulose; or the Universal Placebo) failed to protect mice against the high-dose vaginal HSV-2 challenge (similar to aqueous zinc acetate solution and the placebo controls). The gels were found to be effective spreading gels, exhibited limited toxicity in vitro, caused minimal damage to the architecture of the cervicovaginal and rectal mucosae in vivo, and induced no increased susceptibility to HSV-2 infection in a mouse model. Our results provide a strong rationale to further optimize and evaluate the zinc acetate/carrageenan gels for their ability to block the sexual transmission of HIV and HSV-2.

Herpes simplex virus and pregnancy: a review of the management of antenatal and peripartum herpes infections.

Genital herpes is one of the most common sexually transmitted infections, affecting 1 in 6 people in the United States. Women are twice as likely to be infected as men and infections in women of reproductive age carry the additional risk of vertical transmission to the neonate at the time of delivery. Neonatal herpes infections can be devastating with up to 50% mortality for disseminated herpes simplex virus (HSV) infections in the newborn. Rates of transmission are affected by the viral type of HSV infection and whether the infection around delivery is primary or recurrent. Current management approaches decrease rates of active lesions at the time of delivery and thereby cesarean deliveries, but have not been shown to decrease the incidence of neonatal herpes infections. More research is needed to better elucidate the risk factors for transmission to the neonate and to improve our current management methodology to further decrease vertical transmission. In this review, we will discuss management of antenatal and peripartum herpes infections, considerations for mode of delivery, and the course of neonatal HSV infections.

Herpes simplex virus type 2-induced mortality following genital infection is blocked by anti-tumor necrosis factor alpha antibody in CXCL10-deficient mice.

The role of tumor necrosis factor alpha (TNF-alpha) was evaluated for CXCL10-deficient (CXCL10(-/-)) mice which succumbed to genital herpes simplex virus type 2 (HSV-2) infection and possessed elevated levels of virus and TNF-alpha but not other cytokines in the central nervous system (CNS) and vaginal tissue within the first 7 days following virus exposure. Anti-TNF-alpha but not control antibody treatment offsets the elevated mortality rate of CXCL10(-/-) mice, despite increased CNS viral titers. In addition, TNF-alpha neutralization suppressed recruitment of leukocyte subpopulations into the CNS, which is associated with reduced CCL2 and CXCL9 expression. Collectively, the results implicate TNF-alpha as the principal mediator of mortality in response to genital HSV-2 infection.

A comparison of one year of episodic or suppressive treatment of recurrent genital herpes with valacyclovir.

OBJECTIVES: We conducted this study to compare episodic and suppressive therapy for genital herpes about disease characteristics, disease burden, and psychologic impact. STUDY DESIGN: The authors conducted a randomized, open-label, 1-year treatment trial. RESULTS: Forty subjects were randomized to episodic therapy with valacyclovir (500 mg twice daily for 5 days) and 40 to suppressive therapy (500 mg daily). Subjects in the episodic arm experienced more outbreaks (7.87 +/- 4.65) than those in the suppressive arm (1.59 +/- 1.93) (P <0.001). Subjects in the episodic arm also had significantly more days with pain and lesions (6.5 +/- 7.3) than those in the suppressive arm (1.1 +/- 3.3) (P < 0.001), and other quantitative measures of disease burden also favored the suppressive arm. Both treatment groups had significant improvements in their recurrent genital herpes quality-of-life scores from baseline that persisted throughout the study; however, there was no significant difference between the treatment arms in these scores. CONCLUSIONS: Suppressive therapy of recurrent genital herpes with valacyclovir has a greater impact on measures of disease frequency and burden than episodic therapy. However, both treatment strategies lead to significant improvements in recurrent genital herpes quality-of-life scores. Both treatment strategies benefit patients with recurrent genital herpes.

Neonatal herpes simplex virus infection presenting as acute liver failure: prevalent role of herpes simplex virus type I.

BACKGROUND: Acute liver failure (ALF) in neonates is rare but carries a high mortality without liver transplantation. Herpes simplex virus (HSV) is one of the microbes that more commonly causes ALF and is potentially treatable; hence, early diagnosis and treatment are important to avoid progression to liver failure. PATIENTS AND RESULTS: We have analysed retrospectively the case notes of 11 patients with HSV-induced ALF. A history of possible herpes infection was elicited in 5 parents, but HSV had not been suspected clinically. All patients were asymptomatic when discharged from postnatal units and were presented with nonspecific symptoms of poor feeding and lethargy within 2 weeks from birth. Seven of the 11 patients had HSV-1 infection, 4 HSV-2. Only 2 patients who received early treatment with intravenous acyclovir survived. CONCLUSIONS: HSV-related ALF in the neonatal period carries high morbidity and mortality and needs a high index of suspicion so that life-saving treatment can be started promptly. Both HSV-1 and HSV-2 can cause severe neonatal infection. It is important to recognise HSV infection in women of childbearing age and their sexual partners.

Type-specific screening for asymptomatic herpes infection in pregnancy: a decision analysis.

OBJECTIVE: To evaluate the merits of serum screening for herpes simple virus (HSV) in pregnant women with no history of prior HSV infection. DESIGN: Clinical decision analysis. POPULATION: Hypothetical cohort of pregnant women in first trimester with no clinical history of HSV infection. METHODS: We used decision analysis techniques to compare three strategies for antepartum screening for HSV in women with no history of infection: (1) universal screening; (2) targeted screening in women estimated to be at high risk for infection; and (3) current care (no screening). For the screening strategies, we considered screening at 35 weeks of gestation, with prophylactic antiviral therapy for seropositive women. For all women, we assumed caesarean delivery in the setting of symptomatic infection at delivery. We performed a literature review of English-language publications to derive probability estimates for the rate of HSV seropositivity in asymptomatic pregnant women, and the risks of symptomatic HSV infection and asymptomatic shedding at the time of delivery. We determined the modification of rates of viral shedding, symptomatic lesions and caesarean section with the use of prophylactic suppression therapy for seropositive women based on available data. We chose neonatal herpes with severe sequelae, neonatal death, as well as caesarean delivery as clinically relevant outcomes. MAIN OUTCOME MEASURES: Number of cases of neonatal death, neonatal HSV with severe sequelae, neonatal HSV with moderate sequelae, patients screened, patients treated and caesarean section with each strategy. RESULTS: Universal maternal screening reduced the total number of deaths and severe sequelae secondary to neonatal HSV. Universal screening required treatment of 3849 women to prevent one case of neonatal death or disease with severe sequelae from HSV. Targeted screening of high risk women treatment of 2277 women to prevent one death or case of severe disease. Universal screening reduced the rate of neonatal HSV attributable to recurrent HSV by 79.3%. Caesarean delivery was reduced with both screening strategies. We used one-way sensitivity analyses to evaluate the robustness of our model. CONCLUSIONS: Maternal screening reduced the number of cases of neonatal HSV. Screening also reduced the rate of caesarean delivery. However, employing universal screening will likely result in a significant expenditure of medical resources because the number needed to treat to avert a single case of neonatal herpes is high.

Protective immunity to genital herpes simplex [correction of simpex] virus type 2 infection is mediated by T-bet.

We show, for the first time, that the transcription factor T-bet, which is implicated in IFN-gamma production, is required for the induction of vaccine-induced antiviral immune protection. T-bet was found to be important in both the innate and acquired immune protection against genital HSV-2 infection. T-bet(-/-) and T-bet(+/+) mice were infected vaginally with HSV-2 and examined daily for disease and mortality. T-bet(-/-) mice had significantly higher virus titers than T-bet(+/+) mice following a primary HSV-2 infection, and succumbed significantly earlier to the infection. This result was associated with an impaired NK cell cytotoxic capacity and NK cell-mediated IFN-gamma production in the T-bet(-/-) mice. To assess the induction of acquired antiviral immune protection, mice were vaccinated with an attenuated virus before infection. Vaccinated T-bet(-/-) mice could not control viral replication following an HSV-2 challenge and had significantly higher virus titers and mortality rates than vaccinated T-bet(+/+) mice that remained healthy. The impaired acquired immune protection in T-bet(-/-) mice was associated with a significantly decreased HSV-2-specific delayed-type hypersensitivity response and a significantly reduced HSV-2-specific IFN-gamma production from CD4(+) T cells. However, T-bet deficiency did not impair either the IFN-gamma production or the cytotoxic capacity of HSV-2-specific CD8(+) T cells. We conclude that T-bet plays a crucial role in both the innate defense and the generation of vaccine-induced immunity against genital HSV-2 infection in mice.

Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice.

In the present study, the protective effect of 1T1, a lambda-carrageenan extracted from the red seaweed Gigartina skottsbergii was evaluated in a murine model of herpes simplex virus type 2 (HSV-2) genital infection. Six to eight-week-old female BALB/c mice were intravaginally inoculated with a lethal dose of HSV-2 (MS strain) and pre- or post-infection treated with different doses of a 10mg/ml solution of 1T1. A single topical administration of 1T1 shortly before infection of BALB/c mice with HSV-2 protected 9 out of 10 mice from HSV-2-induced lesions and mortality, compared with only 10% survival in control mice. In addition, 1T1 produced a total blockade in virus shedding in the vaginal secretions. When 1T1 pre-treatment was reinforced with a second dose 2h after infection, total protection was observed even when the prophylactic administration had taken place at 60min before infection. The irreversible virucidal action of 1T1 against herpes virus seems to be responsible of its protective effect against virus replication and mortality following vaginal HSV-2 infection.

A locus on mouse chromosome 6 that determines resistance to herpes simplex virus also influences reactivation, while an unlinked locus augments resistance of female mice.

During studies to determine a role for tumor necrosis factor (TNF) in herpes simplex virus type 1 (HSV-1) infection using TNF receptor null mutant mice, we discovered a genetic locus, closely linked to the TNF p55 receptor (Tnfrsf1a) gene on mouse chromosome 6 (c6), that determines resistance or susceptibility to HSV-1. We named this locus the herpes resistance locus, Hrl, and showed that it also mediates resistance to HSV-2. Hrl has at least two alleles, Hrl(r), expressed by resistant strains like C57BL/6 (B6), and Hrl(s), expressed by susceptible strains like 129S6 (129) and BALB/c. Although Hrl is inherited as an autosomal dominant gene, resistance to HSV-1 is strongly sex biased such that female mice are significantly more resistant than male mice. Analysis of backcrosses between resistant B6 and susceptible 129 mice revealed that a second locus, tentatively named the sex modifier locus, Sml, functions to augment resistance of female mice. Besides determining resistance, Hrl is one of several genes involved in the control of HSV-1 replication in the eye and ganglion. Remarkably, Hrl also affects reactivation of HSV-1, possibly by interaction with some unknown gene(s). We showed that Hrl is distinct from Cmv1, the gene that determines resistance to murine cytomegalovirus, which is encoded in the major NK cell complex just distal of p55 on c6. Hrl has been mapped to a roughly 5-centimorgan interval on c6, and current efforts are focused on obtaining a high-resolution map for Hrl.

Calcium phosphate nanoparticles induce mucosal immunity and protection against herpes simplex virus type 2.

Previously we reported that calcium phosphate nanoparticles (CAP) represented a superior alternative to alum adjuvants in mice immunized with viral protein. Additionally, we showed that CAP was safe and elicited no detectable immunoglobulin E (IgE) response. In this study, we demonstrated that following mucosal delivery of herpes simplex virus type 2 (HSV-2) antigen with CAP, CAP adjuvant enhanced protective systemic and mucosal immunity versus live virus. Mice were immunized intravaginally and intranasally with HSV-2 protein plus CAP adjuvant (HSV-2+CAP), CAP alone, phosphate-buffered saline, or HSV-2 alone. HSV-2+CAP induced HSV-specific mucosal IgA and IgG and concurrently enhanced systemic IgG responses. Our results demonstrate the potency of CAP as a mucosal adjuvant. Furthermore, we show that systemic immunity could be induced via the mucosal route following inoculation with CAP-based vaccine. Moreover, neutralizing antibodies were found in the sera of mice immunized intranasally or intravaginally with HSV-2+CAP. Also, the results of our in vivo experiments indicated that mice vaccinated with HSV-2+CAP were protected against live HSV-2 infection. In conclusion, these preclinical data support the hypothesis that CAP may be an effective mucosal adjuvant that protects against viral infection.

B7 costimulation plays an important role in protection from herpes simplex virus type 2-mediated pathology.

We have used mice lacking both B7-1 and B7-2 costimulation molecules (B7KO) to investigate the effects of B7 costimulation on herpes simplex virus type 2 (HSV-2) pathogenesis. B7KO mice infected intravaginally with virulent HSV-2 showed more severe genital and neurologic disease and higher mortality rates than their wild-type counterparts. These results suggest that B7 costimulation molecules play an important role in the development of primary immune responses protective against HSV-2.

Modulation of cellular responses by plasmid CD40L: CD40L plasmid vectors enhance antigen-specific helper T cell type 1 CD4+ T cell-mediated protective immunity against herpes simplex virus type 2 in vivo.

Engineering gene therapy vectors to modulate the immune response is an important goal. In this regard, costimulation of T cells is a critical determinant in immune activation. The costimulatory molecule CD40, expressed on antigen-presenting cells, is thought to interact with CD40 ligand (CD40L) expressed on activated CD4(+) or CD8(+) T cells to further drive interleukin-2 receptor (IL-2R) expression and antigen-specific T cell expansion necessary for both class II and class I responses. To compare the specific roles of these two costimulatory molecules in immune induction in a herpes simplex virus (HSV) model, we constructed plasmid DNAs expressing CD40 and CD40L, coimmunized these molecules with a gD plasmid vaccine, and then analyzed immune modulatory effects as well as protection against lethal HSV-2 challenge. We observed that gD-specific IgG production was unaffected by these molecules. However, a higher production of IgG2a isotype was induced by CD40L coinjection, suggesting that CD40L drives immune responses towards a helper T cell type 1 (Th1) phenotype. CD40L also enhanced Th cell proliferative responses and production of Th1-type cytokines (IL-2 and IFN-gamma) and beta-chemokines (RANTES and MIP-1alpha) from splenocytes. In contrast, CD40 showed slightly increasing effects on T cell proliferation responses and cytokine and chemokine production. When animals were challenged with a lethal dose of HSV-2, CD40L-coimmunized animals exhibited a significantly enhanced survival rate, as compared with CD40 coinjection or gD DNA vaccine alone. This enhanced protection appears to be mediated by Th1-type CD4(+) T cells, as determined by in vitro and in vivo T cell subset deletion. CD40L also promoted migration of CD4(+) T cells into the muscle sites. These studies demonstrate that CD40L can play an important role in protective antigen-specific immunity in a gene-based model system through increased expansion of the CD4(+) Th1 T cell subset in vivo.