Vaginal immunity in the HSV-2 mouse model

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against such infections. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.     

VAGINAL IMMUNITY IN THE HSV-2 MOUSE MODEL

Herpes simplex virus type 2 (HSV-2) is a sexually transmitted pathogen that infects the genital tract. Efforts to develop vaccines to protect women against this and other sexually transmitted pathogens would be facilitated by a better understanding of the immune mechanisms that protect the female reproductive tract against such infections. Such information would be invaluable in developing vaccine strategies to promote the type and magnitude of immune responses in the genital tract that would effectively protect against infection. This review focuses on recent studies using a progestin-treated adult mouse model to explore mucosal immunity to HSV-2 in the vagina. Evidence indicating a major role for both humoral and T cell immunity is presented.     

Recent progress in herpes simplex virus immunobiology and vaccine research

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) cause prevalent, chronic infections that have serious outcomes in some individuals. Neonatal herpes may occur when the infant traverses the cervix during maternal genital herpes. Genital herpes is a major risk factor for human immunodeficiency virus type 1 transmission. Considerable efforts have been made to design and test vaccines for HSV, focusing on genital infection with HSV-2. Several protein subunit vaccines based on HSV-2 envelope glycoproteins have reached advanced-phase clinical trials. These antigens were chosen because they are the targets of neutralizing-antibody responses and because they elicit cellular immunity. Encouraging results have been reported in studies of treatment of HSV-seronegative women with a vaccine consisting of truncated glycoprotein D of HSV-2 and a novel adjuvant. Because most sexual HSV transmission occurs during asymptomatic shedding, it is important to evaluate the impact of vaccination on HSV-2 infection, clinically apparent genital herpes, and HSV shedding among vaccine recipients who acquire infection. There are several other attractive formats, including subunit vaccines that target cellular immune responses, live attenuated virus strains, and mutant strains that undergo incomplete lytic replication. HSV vaccines have also been evaluated for the immunotherapy of established HSV infection.     

Vaccination strategies to prevent genital herpes and neonatal herpes simplex virus (HSV) disease

Vaccination strategies to prevent genital and neonatal herpes simplex virus (HSV) disease have a history of apparent efficacy in animal studies followed by failure in clinical trials. Further study of the immune response induced by natural HSV infections in both adults and neonates will provide insight into the requirements for vaccination against acute disease and recurrences. Lessons can also be learnt from the recent partial success of a HSV-2 glycoprotein D vaccine coupled with monophosphoryl lipid A adjuvant, which induced protection from clinical disease in HSV-1 and HSV-2 seronegative women. Its efficacy has been attributed to enhancement of Th-1 immunity by the adjuvant. Newer vaccine vectors including DNA vaccines, recombinant viral vaccines and specific HSV mutants are being developed but better animal models are required for more rapid progress. This review examines the history of HSV vaccine development, describes recent progress towards an effective prophylactic vaccine against HSV and outlines further improvements required to make current vaccines immunogenic to a wide population.     

Comparison of different forms of herpes simplex replication-defective mutant viruses as vaccines in a mouse model of HSV-2 genital infection

Some subunit vaccines composed of herpes simplex virus (HSV) glycoproteins have been shown to protect guinea pigs against primary and recurrent genital infection by HSV-2. However, these vaccines were ineffective or only marginally effective in clinical trials. To attempt to define an animal model that would better discriminate the protective capacity of different vaccine formulations, we have examined the requirements for vaccine-induced protection against HSV-2 infection and disease in a mouse genital model. Unlike the guinea pig model where inactivated viral vaccines can protect nearly as well as live viral vaccines, inactivated viral vaccine afforded little protection in this mouse model. Using replication-defective mutant viruses as a form of live viral vaccine, we found that the extent of protection conferred by live vaccine was proportional to the amount of replication-defective mutant virus inoculated, over doses from 10(4) to 10(6) PFU. Furthermore, the mouse genital model showed quantitative differences in the degree of protection induced by various viral vaccine constructs. An HSV-2 replication-defective mutant virus protected better than an HSV-1 replication-defective mutant that expressed HSV-2 glycoprotein D, which in turn protected better than an HSV-2 replication-defective mutant virus. We conclude that this mouse genital model can rank different vaccine constructs for their capacity to induce protective immunity. Thus, genital infection of the mouse with HSV-2 may provide a stringent animal model that can predict the relative capacity of viral vaccines to stimulate protective immunity against HSV-2.     

Immunization with a replication-defective herpes simplex virus 2 mutant reduces herpes simplex virus 1 infection and prevents ocular disease

Ocular infections with herpes simplex virus 1 can lead to corneal scarring and blindness, with herpes keratitis being the major infectious cause of blindness. There is currently no clinically approved vaccine and nearly all developmental vaccines are targeted against HSV-2 and genital herpes. We tested the ability of an HSV-2 replication-defective virus, a genital herpes vaccine candidate, to protect against HSV-1 corneal infection. Immunization with HSV-2 dl5-29 reduced viral replication in the cornea, prevented ocular disease and reduced latent infection by the HSV-1 strain. Therefore, this HSV-2 replication-defective mutant strain may have applications for prevention of herpes keratitis and genital herpes due to HSV-1 infection.     

Induction of innate immune responses in the female genital tract: friend or foe of HIV-1 infection?

Heterosexual transmission of HIV-1 and HSV-2 across the genital tract epithelial tissue is one of the primary routes for dissemination of these viral infections. Mucosal innate immunity is the first line of defense against invading pathogens. A vast majority of mucosal HIV-1 exposures do not result in productive infections which may indicate that the innate mucosal immune system is highly protective. It has been shown that Toll-like receptors (TLR)-induced innate antiviral immunity in the genital mucosa lead to induction of type I and III interferon and prevention of HSV-2 infection. The innate antiviral function of type I and III interferons and other innate factors at genital mucosa against HIV-1 is not well defined. In this review, we summarize our current understanding and advances of the innate mucosal response to genital viral infections, including HIV-1 and HSV-2, focusing on those factors that may prevent or accelerate initial infection. Understanding how each of these components contributes to mucosal innate antiviral immunity may lead to the development of novel and effective strategies to use microbicides or antiviral agents to control HIV-1 acquisition and/or transmission.     

The role of toll-like receptor ligands/agonists in protection against genital HSV-2 infection

Control of virus replication initially depends on rapid activation of the innate immune responses. Toll-like receptor (TLR) ligands are potent inducers of innate immunity against viral infections, including herpes simplex virus (HSV). HSV-2 is currently one of the most common sexually transmitted infections in developed nations and is becoming more prevalent in adolescents. HSV-2 infects the genital mucosa and is associated with an increased risk of obtaining other sexually transmitted infections such as HIV. There is currently no vaccine available against HSV-2. In the last several years, there has been an interest in utilizing Toll-like receptor (TLR) ligands to initiate innate immune responses in order to provide an early line of defence against viral replication. This review highlights recent studies investigating the effect of various TLR ligands on genital HSV-2 infection. A considerable body of information has been published on the effect of local delivery of TLR ligands on HSV-2 replication in genital mucosa. We have outlined ligands that have a potential to provide protection against HSV-2 infection. In addition, we have presented possible mechanisms by which the local delivery of TLR ligands provides innate protection against genital HSV-2.     

Subclinical chlamydial infection of the female mouse genital tract generates a potent protective immune response: implications for development of live attenuated chlamydial vaccine strains

Chlamydia trachomatis is a major cause of sexually transmitted disease (STD) for which a vaccine is needed. CD4(+) T-helper type 1 (Th1) cell-mediated immunity is an important component of protective immunity against murine chlamydial genital infection. Conventional vaccine approaches have not proven effective in eliciting chlamydial-specific CD4 Th1 immunity at the genital mucosa. Thus, it is possible that the development of a highly efficacious vaccine against genital infection will depend on the generation of a live attenuated C. trachomatis vaccine. Attenuated strains of C. trachomatis do not exist, so their potential utility as vaccines cannot be tested in animal models of infection. We have developed a surrogate model to study the effect of chlamydial attenuation on infection and immunity of the female genital tract by treating mice with a subchlamydiacidal concentration of oxytetracycline following vaginal infection. Compared to untreated control mice, antibiotic-treated mice shed significantly fewer infectious organisms (3 log(10)) from the cervico-vagina, produced a minimal inflammatory response in urogenital tissue, and did not experience infection-related sequelae. Antibiotic-treated mice generated levels of chlamydia-specific antibody and cell-mediated immunity equivalent to those of control mice. Importantly, antibiotic-treated mice were found to be as immune as control untreated mice when rechallenged vaginally. These findings demonstrate that subclinical chlamydial infection of the murine female genital tract is sufficient to stimulate a potent protective immune response. They also present indirect evidence supporting the possible use of live attenuated chlamydial organisms in the development of vaccines against chlamydial STDs.     

Glycoprotein D adjuvant herpes simplex virus vaccine

Herpes simplex virus (HSV) Type-1 and -2 are common infections that can cause primary and recurrent herpes labialis and genitalis, as well as gingivostomatitis, keratoconjunctivitis, encephalitis, disseminated infections in immunocompromised persons and neonatal infections. Despite several decades of HSV vaccine development, no effective vaccine has been developed until recently. The following review of the genital HSV-2 glycoprotein D (gD2t, t is for truncated) subunit vaccine formulated with a new adjuvant (AS04) containing alum and 3-O deacylated monophosphoryl lipid A (MPL) provides a background in which to evaluate the vaccine as well as a brief review of other approaches to herpes vaccines. The gD2t-AS04 vaccine has been demonstrated to be safe in several large clinical trials. In two trials, the vaccine reduced genital herpes disease by 73 and 74%, but only in females with no previous HSV infection. A large ongoing trial in HSV seronegative females will provide additional data on protection from HSV disease and infection.     

Protective immunity to genital herpes simplex virus type 1 and type 2 provided by self-adjuvanting lipopeptides that drive dendritic cell maturation and elicit a polarized Th1 immune response

Genital herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) infections are a significant health problem worldwide. While it is believed that CD4+ Th1 cells are among the effectors to herpes immunity, developing an epitope-based clinical vaccine capable of inducing an effective anti-herpes CD4+ Th1-mediated protection is still under investigation. Few molecules achieve this target without the aid of external immuno-adjuvant. The present study was undertaken to examine the immunogenicity in mice of five CD4+ T cell epitope peptides (gD1-29, gD49-82, gD146-179, gD228-257, and gD332-358), recently identified from the HSV-1 glycoprotein D (gD), covalently linked to a palmitic acid moiety (lipopeptides) using the high-yielding chemoselective ligation method and delivered subcutaneously in free-adjuvant saline. Their protective efficacy was evaluated in a progestin-induced susceptibility mouse model of genital herpes following intravaginal challenge with either HSV-1 or HSV-2. Four out of five gD lipopeptides effectively induced virus-specific CD4+ Th1 responses associated with a reduction of virus replication in the genital tract and protection from overt signs of genital disease. A cocktail of three highly immunogenic lipopeptides provoked maturation of dendritic cells, induced interferon gamma (IFN-gamma)-producing CD4+ T cells, and protected against both HSV- 1 and HSV-2 infections. Depletion of specific T cell subsets from lipopeptideimmunized mice before intravaginal HSV challenges demonstrated that CD4+ T cells were primarily responsible for this protection. The strength of induced T cell immunity, together with the ease of construction and safety of these totally synthetic self-adjuvanting lipopeptides, provide a molecularly defined formulation that could combat genital herpes and other human viral infections for which induction of Th1 immunity is crucial.     

Delayed but effective induction of mucosal memory immune responses against genital HSV-2 in the absence of secondary lymphoid organs

To examine whether local immunization in the absence of secondary lymphoid organs (SLOs) could establish effective antiviral memory responses in the female genital tract, we examined immunity in the vaginal tracts of LTα−/− mice, LTα−/− SPL (splenectomized), and control C57BL/6 (WT) mice. All three groups of mice were immunized intravaginally (IVAG) with attenuated thymidine kinase-negative (TK⁻) Herpes simplex virus type 2 (HSV-2) and challenged 4–6 weeks later with wild-type (WT) HSV-2. Both groups of LTα−/− mice exhibited delayed viral clearance and prolonged genital pathology after immunization. Following IVAG WT HSV-2 challenge, LTα−/− and LTα−/− SPL mice had significantly lower levels of HSV-2-specific IgG and IgA in the vaginal secretions. Although the frequency of B and T cells in the vaginal mucosa was comparable or higher in both groups of LTα−/− mice, lower frequency of HSV-2-specific interferon-γ (IFNγ)-producing CD3+ T cells was seen after immunization and after challenge, compared with WT group. Despite this, immunized mice in all three groups showed complete sterile protection against IVAG WT HSV-2 challenge. These results show that even in the absence of SLOs, IVAG immunization generates effector memory immune responses at genital mucosa that can provide antiviral protection against subsequent viral exposures. This will inform new strategies to design mucosal vaccines against sexually transmitted infections.     

PmpG303-311, a protective vaccine epitope that elicits persistent cellular immune responses in Chlamydia muridarum-immune mice

Urogenital Chlamydia serovars replicating in reproductive epithelium pose a unique challenge to host immunity and vaccine development. Previous studies have shown that CD4 T cells are necessary and sufficient to clear primary Chlamydia muridarum genital tract infections in the mouse model, making a protective CD4 T cell response a logical endpoint for vaccine development. Our previous proteomics studies identified 13 candidate Chlamydia proteins for subunit vaccines. Of those, PmpG-1 is the most promising vaccine candidate. To further that work, we derived a PmpG(303-311)-specific multifunctional Th1 T cell clone, designated PmpG1.1, from an immune C57BL/6 mouse and used it to investigate the presentation of the PmpG(303-311) epitope by infected epithelial cells. Epithelial presentation of the PmpG(303-311) epitope required bacterial replication, occurred 15 to 18 h postinfection, and was unaffected by gamma interferon (IFN-γ) pretreatment. Unlike epitopes recognized by other Chlamydia-specific CD4 T cell clones, the PmpG(303-311) epitope persisted on splenic antigen-presenting cells (APC) of mice that cleared primary genital tract infections. PmpG1.1 was activated by unmanipulated irradiated splenocytes from immune mice without addition of exogenous Chlamydia antigen, and remarkably, activation of PmpG1.1 by unmanipulated immune splenocytes was stronger 6 months postinfection than it was 3 weeks postinfection. Enhanced presentation of PmpG(303-311) epitope on splenic APC 6 months postinfection reflects some type of "consolidation" of a protective immune response. Understanding the antigen-presenting cell populations responsible for presenting PmpG(303-311) early (3 weeks) and late (6 months) postinfection will likely provide important insights into stable protective immunity against Chlamydia infections of the genital tract.     

A genital tract peptide epitope vaccine targeting TLR-2 efficiently induces local and systemic CD8+ T cells and protects against herpes simplex virus type 2 challenge

The next generation of needle-free mucosal vaccines is being rationally designed according to rules that govern the way in which the epitopes are recognized by and stimulate the genital mucosal immune system. We hypothesized that synthetic peptide epitopes extended with an agonist of Toll-like receptor 2 (TLR-2), that are abundantly expressed by dendritic and epithelial cells of the vaginal mucosa, would lead to induction of protective immunity against genital herpes. To test this hypothesis, we intravaginally (IVAG) immunized wild-type B6, TLR-2 (TLR2^−/−) or myeloid differentiation factor 88 deficient (MyD88^−/−) mice with a herpes simplex virus type 2 (HSV-2) CD8⁺ T-cell peptide epitope extended by a palmitic acid moiety (a TLR-2 agonist). IVAG delivery of the lipopeptide generated HSV-2-specific memory CD8⁺ cytotoxic T cells both locally in the genital tract draining lymph nodes and systemically in the spleen. Moreover, lipopeptide-immunized TLR2^−/− and MyD88^−/− mice developed significantly less HSV-specific CD8⁺ T-cell response, earlier death, faster disease progression, and higher vaginal HSV-2 titers compared to lipopeptide-immunized wild-type B6 mice. IVAG immunization with self-adjuvanting lipid-tailed peptides appears to be a novel mucosal vaccine approach, which has attractive practical and immunological features.     

Upper respiratory tract immunity

Most viral infections occur via mucosal surfaces like the respiratory, gastrointestinal, or genital epithelium. The mucosal immune system is an important component of the body's defense against such infections and consequently induction of mucosal, in addition to systemic immunity, might improve vaccine efficacy. Several orally administered vaccines, for example, against poliovirus and gastrointestinal bacterial infections, have been developed and are widely used. In contrast, to date most vaccines against respiratory pathogens are applied parenterally and thus do not induce significant mucosal immunity. For the development of effective mucosal vaccines a more profound understanding of the immune mechanisms operative at mucosal surfaces and of the interplay between different mucosal compartments is needed. Moreover, factors like the dose, form of application, and type of mucosal adjuvants are critical to the induction of effective mucosal immunity. This brief review will focus mainly on the nasal route and will summarize some recent findings concerning the function of the mucosal immune system of the upper respiratory tract. Furthermore, routes of cross-immunization between distinct mucosal compartments and how they might be relevant to vaccine development will be addressed. Finally, I will outline critical factors for the rational design of nasal vaccines and in this context highlight some recent preclinical and clinical developments in the field.     

Sperm-mediated host-derived DNA transfer as a new mechanism for immune system evasion of sexually transmitted genital tract pathogens

Over one century of extensive efforts directed towards investigating the immune response and the immuno-protection associated with sexually transmitted infections have failed to produce any effective vaccines against most of the major pathogens, among them Neisseria gonorrhea, herpes simplex virus type 2, and Chlamydia trachomatis. Attempts to design and develop protective vaccines against them have also yielded disappointing results. It has long been felt that there might be another yet undiscovered complicating factor, in addition to the recognized difficulties, which might be impeding the development of successful vaccines. Unlike the other body organs and systems, the genital tract and the elements found within it (e.g., spermatozoa) are endowed with unique features, some of which are associated with inherent DNA transferability skills as physiologically required from such an environment. We hypothesize that there is a novel evasion mechanism that involves an unusual sperm-mediated host-derived DNA transfer by which sexually transmitted genital tract microorganisms can express brand new chimeric antigens and epitopes and, by doing so, thus evade the surveillance of the immune system. This hypothesis may describe what would be the long-awaited breakthrough in the search for a vaccine against sexually transmitted infections. It may also assist in developing better-designed vaccines in general, and may have implications on other microorganism-related challenges (e.g., antibiotic resistance).     

女性下生殖道HPV感染和HPV相关的宫颈肿瘤

子宫颈病变是女性最常见的疾患之一.在女性癌瘤中,宫颈癌的发病率仅次于乳腺癌.人乳头状瘤病毒(HPV)感染在宫颈肿瘤的发病机制中起着重要的作用,许多学者关注HPV疫苗的预防和治疗.在女性生殖道HPV传播及继发性感染是局部性的,因此,针对这种局部性传播疫苗的有效性最好能用局部免疫的参数来评价.     

Herpes simplex virus type 2 vaccines: new ground for optimism?

The development of effective prophylactic and therapeutic vaccines against genital herpes has proven problematic. Difficulties are associated with the complexity of the virus life cycle (latency) and our relatively poor understanding of the mechanism of immune control of primary and recurrent disease. The types of effector cells and the mechanisms responsible for their activation and regulation are particularly important. Studies from my and other laboratories have shown that recurrent disease is prevented by virus-specific T helper 1 (Th1) cytokines (viz., gamma interferon) and activated innate immunity. Th2 cytokines (viz., interleukin-10 [IL-10]) and regulatory (suppressor) T cells downregulate this immune profile, thereby allowing unimpeded replication of reactivated virus and recurrent disease. Accordingly, an effective therapeutic vaccine must induce Th1 immunity and be defective in Th2 cytokine production, at least IL-10. These concepts are consistent with the findings of the most recent clinical trials, which indicate that (i) a herpes simplex virus type 2 (HSV-2) glycoprotein D (gD-2) vaccine formulated with a Th1-inducing adjuvant has prophylactic activity in HSV-2- and HSV-1-seronegative females, an activity attributed to the adjuvant function, and (ii) a growth-defective HSV-2 mutant (ICP10DeltaPK), which is deleted in the Th2-polarizing gene ICP10PK, induces Th1 immunity and has therapeutic activity in both genders. The ICP10DeltaPK vaccine prevents recurrent disease in 44% of treated subjects and reduces the frequency and severity of recurrences in the subjects that are not fully protected. Additional studies to evaluate these vaccines are warranted.     

Vaccination to protect against infection of the female reproductive tract

Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.     

Vaccination to protect against infection of the female reproductive tract

Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.