HSV-2 Challenge Research Articles

Glycoprotein G of Herpes Simplex Virus 2 as a Novel Vaccine Antigen for Immunity to Genital and Neurological Disease

The envelope glycoproteins of herpes simplex virus 1 (HSV-1) and HSV-2, with the exception of glycoprotein G, elicit cross-reactive B- and T-cell responses. Human vaccine trials, using the cross-reactive glycoproteins B and D, have shown no protection against genital HSV-2 infection or disease. In this study, the mature form of glycoprotein G (mgG-2) of HSV-2 was used for immunization of mice, either alone or in combination with adjuvant CpG, followed by an intravaginal challenge with a lethal dose of a fully virulent HSV-2 strain. Mice immunized with mgG-2 plus CpG showed low disease scores and a significantly higher survival rate (73%) than mice immunized with mgG-2 alone (20%) or controls (0%). Accordingly, limited numbers of infectious HSV-2 particles were detected in the spinal cord of mice immunized with mgG-2 plus CpG. The observed protection was associated with a gamma interferon (IFN-γ) response by splenic CD4(+) T cells upon antigen restimulation in vitro and in vaginal washes 1 day postinfection. The majority of sera collected from mice immunized with mgG-2 plus CpG showed macrophage-mediated antibody-dependent cellular cytotoxicity and antibody-dependent complement-mediated cytolysis, while no neutralization activity was observed. In conclusion, we have shown that immunization with the type-specific mgG-2 protein in combination with CpG could elicit protective immunity against an otherwise lethal vaginal HSV-2 challenge. The mgG-2 protein may therefore constitute a promising HSV-2 vaccine antigen to be considered for future human trials.

IL-15 Can Signal via IL-15Rα, JNK, and NF-κB To Drive RANTES Production by Myeloid Cells

IL-15 plays many important roles within the immune system. IL-15 signals in lymphocytes via trans presentation, where accessory cells such as macrophages and dendritic cells present IL-15 bound to IL-15Rα in trans to NK cells and CD8(+) memory T cells expressing IL-15/IL-2Rβ and common γ chain (γ(c)). Previously, we showed that the prophylactic delivery of IL-15 to Rag2(-/-)γ(c)(-/-) mice (mature T, B, and NK cell negative) afforded protection against a lethal HSV-2 challenge and metastasis of B16/F10 melanoma cells. In this study, we demonstrated that in vivo delivery of an adenoviral construct optimized for the secretion of human IL-15 to Rag2(-/-)γ(c)(-/-) mice resulted in significant increases in spleen size and cell number, leading us to hypothesize that IL-15 signals differently in myeloid immune cells compared with lymphocytes, for which IL-15/IL-2Rβ and γ(c) expression are essential. Furthermore, treatment with IL-15 induced RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells, but the presence of γ(c) did not increase bone marrow cell sensitivity to IL-15. This IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) bone marrow cells occurred independently of the IL-15/IL-2Rβ and Jak/STAT pathways and instead required IL-15Rα signaling as well as activation of JNK and NF-κB. Importantly, we also showed that the trans presentation of IL-15 by IL-15Rα boosts IL-15-mediated IFN-γ production by NK cells but reduces IL-15-mediated RANTES production by Rag2(-/-)γ(c)(-/-) myeloid bone marrow cells. Our data clearly show that IL-15 signaling in NK cells is different from that of myeloid immune cells. Additional insights into IL-15 biology may lead to novel therapies aimed at bolstering targeted immune responses against cancer and infectious disease.

DNA vaccine delivery by densely-packed and short microprojection arrays to skin protects against vaginal HSV-2 challenge

There is an unmet medical need for a prophylactic vaccine against herpes simplex virus (HSV). DNA vaccines and cutaneous vaccination have been tried for many applications, but few reports combine this vaccine composition and administration route. We compared DNA administration using the Nanopatch™, a solid microprojection device coated with vaccine comprised of thousands of short (110 μm) densly-packed projections (70 μm spacing), to standard intramuscular DNA vaccination in a mouse model of vaginal HSV-2 infection. A dose-response relationship was established for immunogenicity and survival in both vaccination routes. Appropriate doses administered by Nanopatch™ were highly immunogenic and enabled mouse survival. Vaginal HSV-2 DNA copy number day 1 post challenge correlated with survival, indicating that vaccine-elicited acquired immune responses can act quickly and locally. Solid, short, densely-packed arrays of microprojections applied to the skin are thus a promising route of administration for DNA vaccines.

FSL-1, a bacterial-derived toll-like receptor 2/6 agonist, enhances resistance to experimental HSV-2 infection

BackgroundHerpes simplex virus type 2 (HSV-2) is a leading cause of genital ulceration that can predispose individuals to an increased risk of acquiring other sexually transmitted infections. There are no approved HSV-2 vaccines and current suppressive therapies require daily compound administration that does not prevent all recurrences. A promising experimental strategy is the use of toll-like receptor (TLR) agonists to induce an innate immune response that provides resistance to HSV-2 infection. Previous studies showed that anti-herpetic activity varied based on origin of the agonists and activation of different TLR indicating that activity likely occurs through elaboration of a specific innate immune response. To test the hypothesis, we evaluated the ability of a bacterial-derived TLR2/6 agonist (FSL-1) to increase resistance to experimental genital HSV-2 infection.MethodsVaginal application of FSL-1 at selected doses and times was evaluated to identify potential increased resistance to genital HSV-2 infection in the mouse model. The FSL-1 induced cytokine profile was quantified using kinetically collected vaginal lavages. Additionally, cytokine elaboration and organ weights were evaluated after single or multiple FSL-1 doses to establish a preliminary safety profile. Human vaginal EC cultures were used to confirm the mouse model outcomes.ResultsThe results showed that vaginally-applied FSL-1 created an environment resistant to a 25-fold higher HSV-2 challenge dose. Mechanistically, vaginal FSL-1 application led to transient elaboration of cytokines linked to anti-herpetic innate immune responses. No gross local or peripheral immunotoxicity was observed even after multiple dosing. FSL-1 also created an anti-herpetic environment in cultures of human vaginal epithelial cells (EC).ConclusionThe results showed, for the first time, that the bacterial-derived TLR2/6 agonist FSL-1 induced significant resistance to HSV-2 infection when applied in mice or human vaginal EC cultures. Cytokine evaluation illustrated that anti-herpetic activity correlated with induction of a specific profile. The identified anti-herpetic profile provides an invaluable resource for the future design of novel compounds to reduce genital HSV-2 transmission and improves understanding of the complex innate immune response to potential pathogens elicited by the vaginal mucosa.

The Mucosal Adjuvant Effect of α-Galactosylceramide for Induction of Protective Immunity to Sexually Transmitted Viral Infection

Development of mucosal adjuvants to generate immunity in the female genital tract may have important implications for the development of vaccines to counter sexually transmitted infections. alpha-Galactosylceramide (alpha-GalCer) is presented by CD1d molecule on APCs to invariant Valpha14(+) NKT (iNKT) cells, which upon activation rapidly produce large amounts of immunomodulatory cytokines, leading to activation of a variety of innate and adaptive immune cells. Here, we assessed whether alpha-GalCer could act as a mucosal adjuvant for induction of protective immunity against genital herpes. We found that intranasal immunization with HSV-2 glycoprotein D (gD) in combination with alpha-GalCer elicits strong systemic gD-specific IgG Ab response as well as lymphoproliferative response with a mixed Th1/Th2 cytokine profile in the spleen, mediastinal lymph nodes, and genital lymph nodes. Importantly, such an immunization scheme conferred complete protection against an otherwise lethal vaginal HSV-2 challenge. We could also show that intravaginal immunization with gD plus alpha-GalCer generates potent gD-specific lymphoproliferative and IFN-gamma responses in the genital lymph nodes and spleen. Furthermore, the vaginally immunized mice developed a strong systemic and mucosal IgG Ab response and protection against vaginal HSV-2 challenge. The mucosal adjuvant effect of alpha-GalCer was found to be mediated via CD1d molecule and appeared to be independent of the usage of the adaptor molecule MyD88. To our knowledge, this is the first report on the mucosal adjuvant effect of alpha-GalCer for induction of protective immunity against a sexually transmitted pathogen.

FimH Adhesin of Type 1 Fimbriae Is a Potent Inducer of Innate Antimicrobial Responses Which Requires TLR4 and Type 1 Interferon Signalling

Components of bacteria have been shown to induce innate antiviral immunity via Toll-like receptors (TLRs). We have recently shown that FimH, the adhesin portion of type 1 fimbria, can induce the innate immune system via TLR4. Here we report that FimH induces potent in vitro and in vivo innate antimicrobial responses. FimH induced an innate antiviral state in murine macrophage and primary MEFs which was correlated with IFN-β production. Moreover, FimH induced the innate antiviral responses in cells from wild type, but not from MyD88−/−, Trif−/−, IFN−α/βR−/− or IRF3−/− mice. Vaginal delivery of FimH, but not LPS, completely protected wild type, but not MyD88−/−, IFN-α/βR−/−, IRF3−/− or TLR4−/− mice from subsequent genital HSV-2 challenge. The FimH-induced innate antiviral immunity correlated with the production of IFN-β, but not IFN-α or IFN-γ. To examine whether FimH plays a role in innate immune induction in the context of a natural infection, the innate immune responses to wild type uropathogenic E. coli (UPEC) and a FimH null mutant were examined in the urinary tract of C57Bl/6 (B6) mice and TLR4-deficient mice. While UPEC expressing FimH induced a robust polymorphonuclear response in B6, but not TLR4−/− mice, mutant bacteria lacking FimH did not. In addition, the presence of TLR4 was essential for innate control of and protection against UPEC. Our results demonstrate that FimH is a potent inducer of innate antimicrobial responses and signals differently, from that of LPS, via TLR4 at mucosal surfaces. Our studies suggest that FimH can potentially be used as an innate microbicide against mucosal pathogens.

Virus-Encoded B7-2 Costimulation Molecules Enhance the Protective Capacity of a Replication-Defective Herpes Simplex Virus Type 2 Vaccine in Immunocompetent Mice

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-gamma-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.

Intranasal and subcutaneous immunization under the effect of estradiol leads to better protection against genital HSV-2 challenge compared to progesterone

This study examined the effect of hormonal environment on intranasal and subcutaneous routes of immunization in a genital herpes infection model. Ovariectomized mice were treated with estradiol (E2), progesterone (P4) or placebo hormone pellets and immunized intranasally (i.n.) or subcutaneously (s.c.) with attenuated HSV-2. Immunized mice were subsequently challenged, intravaginally, with wild-type HSV-2. Mice immunized under the influence of E2 showed higher survival rates, reduced pathology and significantly lower viral shedding compared with those immunized under the influence of P4 or placebo, by both i.n. and s.c. routes. Vaginal and serum anti-HSV-2 IgG, but not IgA, levels correlated with decreased pathology in E2-treated, i.n. immunized mice. We conclude that immunization under the influence of E2 afforded better protection compared to placebo and P4, by both routes of immunization. Female sex hormones can influence immune responses and outcome of viral challenge in the genital tract following systemic immunization.

Vaginal microbicides: the importance of effective distribution, retention and coating of the mucosa

In their response to our paper, das Neves et al.[1] argue that the recent failures of candidate microbicides could have been caused by poor drug delivery systems. They state ‘poor outcomes obtained in clinical trials may result mostly from poor drug delivery systems design, not from increased women's vulnerability to HIV due to microbicide local toxicity or differences in nonadherence between study arms as argued by van de Wijgert and Shattock’ [2]. We agree that the optimization of vaginal drug delivery needs more attention. We also agree that poor drug delivery may explain lack of efficacy in clinical trials. However, we believe that poor drug delivery alone is unlikely to explain increased HIV incidence in the microbicide arm of a clinical trial compared to the placebo arm, which was the focus of our paper. The presence of vaginal fluids dilutes vaginal gels by about 10–30%, with increased dilution over time [3–6]. A study [3,4] that simulated this dilution effect using simulant vaginal fluid found that the dilution not only resulted in better gel coverage of the cervical and vaginal mucosa, but also in more gel leakage and faster gel erosion. Dilution may also result in a lower concentration of the active ingredient in the gel. This is of concern for microbicide candidates containing antiretroviral drugs, but is less of a concern for the polyanions. Although these dilution effects may explain lack of efficacy, they are not likely to cause harm. It should be noted, however, that gel leakage often translates into poor acceptability, which in turn is likely to reduce adherence with gel use. As was discussed in our paper, differences in adherence between the microbicide and placebo arms of a clinical trial may indeed explain trial results in the direction of harm [2]. The effect of semen on microbicide efficacy is more complex. Semen not only introduces a much larger dilution effect than vaginal fluids, but also changes the vaginal pH and contains proteins that may competitively inhibit binding of the active ingredient in the microbicide to HIV or its receptors. A recent study [7] showed that seminal plasma (and particularly the proteins lactoferrin and fibronectin) significantly interfered with the in-vitro activity of the polyanions cellulose sulfate and PRO-2000, which translated into a reduction in protection from HSV2 challenge in the HSV2 mouse model. Again, this could explain lack of efficacy of these candidate microbicides, but would only lead to clinical trial results in the direction of harm if other biological mechanisms (such as changes in epithelial integrity or local immunity) are also present. The interaction between a microbicide and semen could result in such harmful biological mechanisms, and this needs to be carefully evaluated in preclinical and early clinical trials. Even more recently, it was shown that peptides in semen form amyloid fibrils that capture HIV and promote attachment to target cells [8]. The effects of different microbicides and placebos on this newly described mechanism of HIV transmission have not yet been studied. In summary, even though we believe that poor drug delivery alone is unlikely to explain harmful clinical trial results, we very much agree with das Neves et al.[1] that vaginal drug delivery needs more attention, and that antiviral activity and safety testing of candidate microbicides should be performed in the presence of genital secretions and semen.

Adaptive immune responses fail to provide protection against genital HSV‐2 infection in the absence of IL‐15

IL-15 plays a crucial role in innate defense against viral infections. The role of IL-15 in the generation and function of adaptive immunity, following mucosal immunization, against genital HSV-2 has not been studied. Here, we report that immunized IL-15(-/-) mice were able to generate antibody and T cell-mediated immune responses against HSV-2, comparable to those seen in immunized B6 mice. However, immunized IL-15(-/-) mice were not protected against subsequent HSV-2 challenge, compared to B6 immunized mice, even with a ten times lower challenge dose. We then examined if the adaptive immune responses generated in the absence of IL-15 could provide protection against HSV-2 in an IL-15-positive environment. Adoptive transfer of lymphocytes from immunized IL-15(-/-) to naive mice were able to provide protection against HSV-2 challenge similar to protection with immunized cells from control mice. This suggests that the adaptive immune responses raised in the absence of IL-15 are functional in vivo. Reconstitution of the innate components, particularly IL-15, NK cells and NK cell-derived IFN-gamma, in immunized IL-15(-/-) mice restored their protective adaptive immunity against subsequent genital HSV-2 challenge. Our results clearly suggest that innate antiviral activity of IL-15 is necessary for protective adaptive immunity against genital HSV-2 infection.

Antibody-Mediated Protection against Genital Herpes Simplex Virus type 2 Disease in Mice by Fc gamma Receptor -Dependent and -Independent Mechanisms (41.8)

Abstract The requirement for Ab -Fcγ receptor (FcγR) interactions or for virus neutralization in protection against genital HSV-2 challenge was examined. Serum IgG Ab isolated from HSV-immune mice protected normal mice against HSV-2 disease when administered prior to challenge. However, protection was significantly diminished in mice lacking the γ chain subunit utilized in FcγRI, FcγRIII, FcγRIV, and FcεRI and in normal mice depleted of FcγR+, Gr-1+ immune cells suggesting protection was largely mediated by an FcγR-dependent mechanism. To test if FcγR-independent antibody-mediated mechanisms might manifest protection differently, a highly neutralizing, HSV glycoprotein D -specific monoclonal antibody (mAb) was utilized. Administration of IgG1, IgG2a, or IgG2b switch variants of the mAb did not prevent infection of the genital tract but resulted in lower virus loads in the vaginal epithelium and provided significant protection against disease and acute infection of the sensory ganglia independently of host FcγR expression. Together, these data demonstrate two distinct antibody effector mechanisms capable of providing substantial protection against genital HSV-2 disease. The presence of either FcγR -dependent Ab or strongly neutralizing Ab did not completely prevent HSV-2 infection but limited initial infection of genital and neuronal tissues and diminished HSV-2 disease. NIH Grants AI42815 and AI054444.

Protective Immunity to Genital Herpes 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.

Protection against Genital Herpes Infection in Mice Immunized under Different Hormonal Conditions Correlates with Induction of Vagina-Associated Lymphoid Tissue

The present study was undertaken to examine the effect of the hormonal environment on immunization with an attenuated strain of herpes simplex virus type 2 (HSV-2 TK(-)) and subsequent protection against challenge. Ovariectomized mice were administered saline (S; control), estradiol (E(2)), progesterone (P(4)), or a combination of estradiol and progesterone (E+P) and immunized intravaginally (IVAG) with HSV-2 TK(-). Three weeks later, the immunized mice were challenged IVAG with wild-type HSV-2. Mice that were immunized following E treatment were not protected, whereas complete protection against the challenge was seen in mice from the S- and P(4)-treated groups. In the P(4)-treated group, 15% of mice developed chronic pathology following TK(-) immunization. Interestingly, about 40% of the E+P-treated mice were also protected. Upon examination of viral shedding in the vaginal secretions, it was clear that protection against challenge was dependent on the ability of the TK(-) virus to cause productive genital infection under different hormonal conditions. In the protected mice (the S and P groups and part of the E+P group), induced vagina-associated lymphoid tissues composed of CD11c(+) dendritic cells and CD3(+) and CD4(+) T cells were formed transiently in the vaginal lamina propria from day 2 to day 5 postchallenge. These aggregates were absent in the unprotected mice (the E group and part of the E+P group). Significant HSV-2-specific activation of lymphocytes was observed in the local draining lymph nodes of protected mice. This response was absent in the unprotected groups. High titers of gB-specific local immunoglobulin A (IgA) antibodies were present in the vaginal secretions of S- and P(4)-treated immunized mice following HSV-2 challenge. The S-treated group of mice also had high gB-specific IgG titers. These studies show that sex hormones modify the induction of protective immune responses following IVAG immunization.

Opposite Roles of B7.1 and CD28 Costimulatory Molecules for Protective Immunity against HSV-2 Challenge in a gD DNA Vaccine Model

Costimulation is a critical process in Ag-specific immune responses. Both B7.1 and CD28 molecules have been reported to stimulate T cell responses during antigen presentation. Therefore, we tested whether Ag-specific immune responses as well as protective immunity are influenced by coinjecting with B7.1 and CD28 cDNAs in a mouse HSV-2 challenge model system. Methods: ELISA was used to detect levels of antibodies, cytokines and chemokines while thymidine incorporation assay was used to evaluate T cell proliferation levels. Results: Ag-specific antibody responses were enhanced by CD28 coinjection but not by B7.1 coinjection. Furthermore, CD28 coinjection increased IgG1 production to a significant level, as compared to pgD+pcDNA3, suggesting that CD28 drives Th2 type responses. In contrast, B7.1 coinjection showed the opposite, suggesting a Th1 bias. B7.1 coinjection also enhanced Ag-specific Th cell proliferative responses as well as production of Th1 type cytokines and chemokines significantly higher than pgD+pcDNA3. However, CD28 coinjection decreased Ag-specific Th cell proliferative responses as well as production of Th1 types of cytokines and chemokine significantly lower than pgD+pcDNA3. Only MCP-1 production was enhanced by CD28. B7.1 coimmunized animals exhibited an enhanced survival rate as well as decreased herpetic lesion formation, as compared to pgD+pcDNA3. In contrast, CD28 vaccinated animals exhibited decreased survival from lethal challenge. Conclusion: This study shows that B7.1 enhances protective Th1 type cellular immunity against HSV-2 challenge while CD28 drives a more detrimental Th2 type immunity against HSV-2 challenge, supporting an opposite role of B7.1 and CD28 in Ag-specific immune responses to a Th1 vs Th2 type.

Toll‐Like Receptor (TLR)–3, but Not TLR4, Agonist Protects against Genital Herpes Infection in the Absence of Inflammation Seen with CpG DNA

We previously demonstrated that delivery of CpG oligodeoxynucleotide (ODN) to vaginal mucosa induced an innate mucosal antiviral state that protected against intravaginal challenge with herpes simplex virus (HSV)-2. We report that mucosal, but not systemic, delivery of ligands for Toll-like receptor (TLR)-3, but not TLR4, induced protection against genital HSV-2 challenge that was not accompanied by the local inflammation and splenomegaly seen after treatment with CpG ODN. Surprisingly, TLR4 messenger (m) RNA expression was shown to be higher than that of TLR3 or TLR9 in murine genital mucosa. Similarly, murine RAW264.7 cells were shown to express more mRNA for TLR4 than TLR3 or TLR9, yet treatment of these cells with double-stranded RNA provided greater protection than lipopolysaccharide or CpG ODN. These results indicate that TLR3 ligand induces a more potent antiviral response than TLR4 and TLR9 ligands and may be a safer means of protecting against sexually transmitted viral infections.

Poly(sodium 4-styrene sulfonate): evaluation of a topical microbicide gel against herpes simplex virus type 2 and Chlamydia trachomatis infections in mice

To investigate the in vivo activity of poly(sodium 4-styrene sulfonate) (T-PSS) gel formulations as topical microbicides. The ability of the gel formulations to reduce the incidence of infection when applied prior to pathogen challenge was examined in mouse models of vaginal herpes simplex type 2 (HSV-2) and Chlamydia trachomatis infection, and rectal HSV-2 infection. In the vaginal HSV-2 challenge studies, 10% T-PSS gel provided significant protection against infection, even when administered 60 min prior to virus challenge (P < 0.0001). Both 5% and 10% T-PSS gel formulations significantly reduced the incidence of upper genital tract C. trachomatis infection in animals treated up to 5 min before challenge (P < 0.001). However, no protection against C. trachomatis infection was seen in animals treated 30 min before challenge. In mice challenged rectally with HSV-2, both the 5% and 10% T-PSS gels significantly reduced infection at 20 s (P < 0.01 for both). However, only the 10% gel provided significant protection when administered 5 min before challenge (P < 0.01). T-PSS gel formulations have promising in vivo activity as topical microbicides.

Intranasal immunization with CpG oligodeoxynucleotides as an adjuvant dramatically increases IgA and protection against herpes simplex virus-2 in the genital tract.

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems

The immunogenicity of a type 2 herpes simplex virus (HSV-2) antigen preparation following its formulation into immunostimulating complexes (ISCOMs) or non-ionic surfactant vesicles (NISV) was investigated in a murine model. The immune responses induced by each formulation were characterised by antigen specific total and subclass serum responses, and by lymphocyte proliferation and cytokine (interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-γ)) production by in vitro restimulated spleen cells. The degree of protection afforded to mice by these various HSV-2 vaccine preparations against homologous (HSV-2) and heterologous (HSV-1) challenge infection was also determined. The findings suggest that formulation of the HSV-2 glycoprotein antigens with ISCOM or NISV delivery vehicles, and the methods used to prepare these formulations, influenced the immunogenicity of the final preparation. Higher IgG2a and neutralising antibody levels, IL-2 and IFN-γ levels and lymphoproliferative responses were noted in mice immunised with the HSV-2 ISCOM formulated vaccine preparation. Furthermore, although HSV-2 antigens formulated in dehydration–rehydration NISV, or entrapped in NISV by freeze–thawing at 30°C (HSV-2 NISV 30), also elicited relatively high antibody, IL-2 and IFN-γ levels and relatively high lymphoproliferative responses, formulation of HSV-2 antigens by freeze–thawing with NISV at 60°C (HSV-2 NISV 60) did not. There were no differences between any of the HSV-2 vaccine formulations in terms of IL-4 induction in in vitro stimulated spleen cell cultures. Almost complete protection against HSV-2 challenge was afforded by the HSV-2 ISCOM preparation, while partial protection against challenge infection was afforded by the HSV-2 NISV 30 vaccine formulation. The findings are discussed in relation to the nature of the immune mechanisms, particularly Th1- or Th2-like responses, that may be elicited by HSV-2 antigen preparations formulated into various delivery systems and the relevance of these immune responses to protection against HSV infection in the murine model.

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 ED 50≤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.

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 (gD 2) DNA vaccine formulated with bupivacaine was evaluated using murine and guinea pig models of genital herpes. Animals received three doses of 100 μg of gD 2 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 gD 2+bupivacaine was effective in reducing clinical disease and viral replication in both guinea pigs and mice.