Dissociation between serum neutralizing and glycoprotein antibody responses of infants and children who received inactivated respiratory syncytial virus vaccine.

The serum antibody response of infants and children immunized with Formalin-inactivated respiratory syncytial virus (RSV) vaccine 20 years ago was determined by using an enzyme-linked immunosorbent assay specific for the RSV fusion (F) and large (G) glycoproteins and a neutralization assay. Twenty-one young infants (2 to 6 months of age) developed a high titer of antibodies to the F glycoprotein but had a poor response to the G glycoprotein. Fifteen older individuals (7 to 40 months of age) developed titers of F and G antibodies comparable to those in children who were infected with RSV. However, both immunized infants and children developed a lower level of neutralizing antibodies than did individuals of comparable age with natural RSV infections. Thus, the treatment of RSV with Formalin appears to have altered the epitopes of the F or G glycoproteins or both that stimulate neutralizing antibodies, with the result that the immune response consisted largely of "nonfunctional" (i.e., nonneutralizing) antibodies. Subsequent natural infection of the vaccinees with wild-type RSV resulted in enhanced pulmonary disease. Despite this potentiation of illness, the infected vaccinees developed relatively poor G, F, and neutralizing antibody responses. Any or all of three factors may have contributed to the enhancement of disease in the RSV-infected vaccinees. First, nonfunctional antibodies induced by the inactivated RSV vaccine may have participated in a pulmonary Arthus reaction during RSV infection. Second, the poor antibody response of infants to the G glycoprotein present in the Formalin-inactivated vaccine may have been inadequate to provide effective resistance to subsequent wild-type virus infection. Third, the relatively reduced neutralizing antibody response of the infant vaccinees to wild-type RSV infection may have contributed to their enhanced disease by delaying the clearance of virus from their lungs.     

Understanding respiratory syncytial virus (RSV) vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and children worldwide. In addition, RSV causes serious disease in elderly and immune compromised individuals. RSV infection of children previously immunized with a formalin-inactivated (FI)-RSV vaccine is associated with enhanced disease and pulmonary eosinophilia that is believed to be due to an exaggerated memory Th2 response. As a consequence, there is currently no licensed RSV vaccine and detailed studies directed towards prevention of vaccine-associated disease are a critical first step in the development of a safe and effective vaccine. The BALB/c mouse model of RSV infection faithfully mimics the human respiratory disease. Mice previously immunized with either FI-RSV or a recombinant vaccinia virus (vv) that expresses the attachment (G) glycoprotein exhibit extensive lung inflammation and injury, pulmonary eosinophilia, and enhanced disease following challenge RSV infection. CD4 T cells secreting Th2 cytokines are necessary for this response because their depletion eliminates eosinophilia. Intriguing recent studies have demonstrated that RSV-specific CD8 T cells can inhibit Th2-mediated pulmonary eosinophilia in vvG-primed mice by as yet unknown mechanisms. Information gained from the animal models will provide important information and novel approaches for the rational design of a safe and efficacious RSV vaccine.     

Involvement of antibody, complement and cellular immunity in the pathogenesis of enhanced respiratory syncytial virus disease

In 1966, infants and children in the USA were immunized with a formalin-inactivated vaccine against respiratory syncytial virus. The vaccine was immunogenic but elicited mainly nonprotective antibody. Upon exposure to respiratory syncytial virus in the community, immunized children developed severe pulmonary disease characterized by bronchoconstriction and pneumonia. Two immunized infants died as toddlers after respiratory syncytial virus infection. Exploration of the mechanisms of disease has dominated the literature for decades. In this review, the pathogenesis of enhanced respiratory disease is discussed and the characteristics of protective and pathogenic respiratory syncytial virus vaccines are examined.     

A recombinant influenza virus vaccine expressing the F protein of respiratory syncytial virus

Infections with influenza and respiratory syncytial virus (RSV) rank high among the most common human respiratory diseases worldwide. Previously, we developed a replication-incompetent influenza virus by replacing the coding sequence of the PB2 gene, which encodes one of the viral RNA polymerase subunits, with that of a reporter gene. Here, we generated a PB2-knockout recombinant influenza virus expressing the F protein of RSV (PB2-RSVF virus) and tested its potential as a bivalent vaccine. In mice intranasally immunized with the PB2-RSVF virus, we detected high levels of antibodies against influenza virus, but not RSV. PB2-RSVF virus-immunized mice were protected from a lethal challenge with influenza virus but experienced severe body weight loss when challenged with RSV, indicating that PB2-RSVF vaccination enhanced RSV-associated disease. These results highlight one of the difficulties of developing an effective bivalent vaccine against influenza virus and RSV infections.     

Detection of respiratory syncytial virus (RSV) infected cells by in situ hybridization in the lungs of cotton rats immunized with formalin-inactivated virus or purified RSV F and G glycoprotein subunit vaccine and challenged with RSV

The replication of RSV in unimmunized cotton rats was evaluated by quantitating the amount of infectious virus in the lung and the number of RSV infected cells in a histopathological section of lung by in situ hybridization. RSV infected cells were detected only in alveoli and bronchioles and constituted only a small minority of the cell population. The temporal patterns of rise to the peak number of infected cells (day 4) and the peak titer of infectious virus (day 3) were similar. The clearance of both infected cells and infectious virus was nearly complete by day 7. In animals previously immunized with purified RSV glycoproteins or formalin-inactivated RSV there also was a good correlation between the number of infected cells detected by in situ hybridization and the amount of infectious virus recovered. It was previously demonstrated that cotton rats immunized with formalin-inactivated vaccine developed enhanced pulmonary histopathology following challenge with RSV. In such animals, there was approximately a 90% reduction in the number of infected cells compared to control unimmunized, RSV-challenged animals. Formalin-inactivated RSV vaccine-enhanced lung histopathology developed despite the effective elimination of virus and virus-infected cells suggesting that the enhanced pathology is the result of an exaggeration of normal immune mechanisms involved in clearance of virus infection, an aberrant immune response during infection, or both.     

How Innate Immune Mechanisms Contribute to Antibody-Enhanced Viral Infections

Preexisting antibodies may enhance viral infections. In dengue, nonneutralizing antibodies raised by natural infection with one of four dengue viruses (DENVs) may enhance infection with a different virus by a process we term “intrinsic antibody-dependent enhancement” (iADE). In addition, nonprotective antibodies raised by formalin-inactivated respiratory syncytial virus (RSV) and measles virus vaccines have led to enhanced disease during breakthrough infections. Infections under iADE conditions not only facilitate the process of viral entry into monocytes and macrophages but also modify innate and adaptive intracellular antiviral mechanisms, suppressing type 1 interferon (IFN) production and resulting in enhanced DENV replication. The suppression observed in vitro has been documented in patients with severe (dengue hemorrhagic fever [DHF]) but not in patient with mild (dengue fever [DF]) secondary dengue virus infections. Important veterinary viral infections also may exhibit iADE. It is thought that use of formalin deconforms viral epitopes of RSV, resulting in poor Toll-like receptor (TLR) stimulation; suboptimal maturation of dendritic cells with reduced production of activation factors CD40, CD80, and CD86; decreased germinal center formation in lymph nodes; and the production of nonprotective antibodies. These antibodies fail to neutralize RSV, allowing replication with secondary stimulation of RSV-primed Th2 cells producing more low-avidity antibody, resulting in immune complexes deposited into affected tissue. However, when formalin-inactivated RSV was administered with a TLR agonist to mice, they were protected against wild-type virus challenge. Safe and effective vaccines against RSV/measles virus and dengue virus may benefit from a better understanding of how innate immune responses can promote production of protective antibodies.Over the past 4 decades different lines of scientific inquiry have contributed to improved understanding of how antibody-mediated mechanisms control the severity of diseases that accompany heterotypic viral infections or that follow incomplete immunization. In the case of heterotypic infection, independent studies on the cellular and host responses to acute and chronic human and animal viral diseases provide evidence that linking of immune complexes with Fcγ receptors enhance infection severity by a mechanism we term “intrinsic antibody-dependent enhancement” (iADE) (8). Parallel studies on immunization with respiratory syncytial virus (RSV) antigens demonstrate how use of formalin-inactivated viral immunogens yields deficient Toll-like receptor (TLR) activation of B cells, defective affinity maturation, and nonprotective antibodies (14, 39). The severe wild-type viral diseases occurring in the presence of these antibodies are characterized by eosinophilia, complement fixation, and Arthus-like phenomena (7, 11, 20, 40). The research histories of these two innate immune response-triggered antibody-mediated viral immunopathologies are reviewed.     

Role of dendritic cells in enhancement of herpes simplex virus type 1 latency and reactivation in vaccinated mice

Ocular infection with herpes simplex virus type 1 (HSV-1) frequently leads to recurrent infection, which is a major cause of corneal scarring. Thus, the prevention of the establishment of latency should be a primary goal of vaccination against HSV-1. To this end, we have examined the contribution of dendritic cells (DCs) to the efficacy of a vaccine against ocular HSV-1 infection. Transgenic mice (expressing a CD11c-diphtheria toxin receptor-green fluorescent protein construct) with a BALB/c background were immunized with a vaccine consisting of DNA that encodes five HSV-1 glycoproteins or were immunized with vector control DNA. The vaccinated mice were then depleted of their DCs through the injection of diphtheria toxin before and after ocular challenge with HSV-1. Analyses of HSV-1 replication in the eye, blepharitis, corneal scarring, and the survival of the infected mice upon primary infection indicated that DC depletion neither promoted nor compromised the efficacy of the vaccine. In contrast, DC depletion was associated with an approximately fivefold reduction in the level of latent virus in the trigeminal ganglia (TGs) of latently infected mice, as well as a significant reduction in the reactivation rate of latent virus. The possibility that DCs enhance the latency of HSV-1 in the TGs of ocularly infected mice suggests for the first time that DCs, rather than acting as “immune saviors,” can exacerbate disease and compromise vaccine efficacy by enhancing viral latency and reactivation.     

Formalin-inactivated respiratory syncytial virus vaccine induces antibodies to the fusion glycoprotein that are deficient in fusion-inhibiting activity.

The fusion (F) glycoprotein of respiratory syncytial virus (RSV) induces neutralizing antibodies and antibodies that inhibit fusion of infected cells (FI antibody). It was previously shown that infants and children immunized with Formalin-inactivated RSV 20 years ago developed antibodies that bound to the F glycoprotein but were deficient in neutralizing activity. A reexamination of these sera indicated that they were also deficient in FI activity. Thus, Formalin-inactivated RSV vaccine stimulated an unbalanced immune response in which an unusually large proportion of the induced antibodies were directed against nonprotective epitopes rather than against the epitopes that induce functional antibodies, i.e., neutralizing and FI antibodies. This deficiency in stimulation of functional antibodies probably decreased the protective efficacy of the vaccine and could have contributed to potentiation of disease in the vaccines during subsequent RSV infection.     

Biological challenges and technological opportunities for respiratory syncytial virus vaccine development

Respiratory syncytial virus (RSV) is an important cause of respiratory disease causing high rates of hospitalizations in infants, significant morbidity in children and adults, and excess mortality in the elderly. Major barriers to vaccine development include early age of RSV infection, capacity of RSV to evade innate immunity, failure of RSV-induced adaptive immunity to prevent reinfection, history of RSV vaccine-enhanced disease, and lack of an animal model fully permissive to human RSV infection. These biological challenges, safety concerns, and practical issues have significantly prolonged the RSV vaccine development process. One great advantage compared to other difficult viral vaccine targets is that passively administered neutralizing monoclonal antibody is known to protect infants from severe RSV disease. Therefore, the immunological goals for vaccine development are to induce effective neutralizing antibody to prevent infection and to avoid inducing T-cell response patterns associated with enhanced disease. Live-attenuated RSV and replication-competent chimeric viruses are in advanced clinical trials. Gene-based strategies, which can control the specificity and phenotypic properties of RSV-specific T-cell responses utilizing replication-defective vectors and which may improve on immunity from natural infection, are progressing through preclinical testing. Atomic level structural information on RSV envelope glycoproteins in complex with neutralizing antibodies is guiding design of new vaccine antigens that may be able to elicit RSV-specific antibody responses without induction of RSV-specific T-cell responses. These new technologies may allow development of vaccines that can protect against RSV-mediated disease in infants and establish a new immunological paradigm in the host to achieve more durable protection against reinfection.     

The live attenuated subgroup B respiratory syncytial virus vaccine candidate RSV 2B33F is attenuated and immunogenic in chimpanzees, but exhibits partial loss of the ts phenotype following replication in vivo

The cold-adapted (ca), temperature-sensitive (ts) respiratory syncytial virus (RSV) subgroup B vaccine candidate, designated RSV 2B33F, was found previously to be restricted in replication, immunogenic, and protective against wild-type (wt) virus challenge in rodents and African green monkeys. We sought to investigate the level of attenuation, immunogenicity and genetic stability of this vaccine candidate in seronegative chimpanzees. The 2B33F vaccine candidate was attenuated in chimpanzees and manifested a ten- and 1000-fold restriction in replication in the upper and lower respiratory tracts respectively, compared with its wt RSV 2B parent virus. Despite this attenuation, chimpanzees immunized with RSV 2B33F were completely resistant to respiratory tract disease and virus replication upon challenge with wt virus. The ts phenotype of the RSV 2B33F mutant exhibited some alteration during replication in vivo in three of four chimpanzees tested. Virus present in nasopharyngeal swab or tracheal lavage secretions of these three chimpanzees was biologically cloned by plaque passage in Vero cells at permissive temperature. The plaque progeny retained the ts phenotype, but uniformly produced plaques at 39 and 40 degrees C to a level intermediate between that of the 2B33F input virus and the 2B wt parent virus, indicating that partial loss of the level of temperature sensitivity occurred following replication in vivo. The implications of these findings for RSV vaccine development are discussed.     

Respiratory syncytial virus-induced immunoprotection and immunopathology

Respiratory syncytial virus (RSV), a member of the Paramyxoviridae family, is a major clinical problem causing yearly epidemics of severe lower airway disease in both infants and the elderly. Attempts at vaccination have been frustrated by both the poor immunogenicity of this virus, and the severe immunopathology observed in early vaccine trials. Primary infection generally occurs in infancy, with approximately 5% of infected infants requiring hospitalization. Equally problematic is the apparent link between severe RSV disease and the later development of allergy and asthma. While there is no evidence that natural infection promotes Th2 predominance, development of enhanced eosinophilic disease in children receiving inactivated virus administered with a commonly used adjuvant demonstrated how easily the balance between immune-mediated protection and immune-mediated pathology can be perturbed. In this review we have focused on studies carried out in the mouse model aimed at determining the correlates of RSV protection and explaining the mechanism of vaccine enhanced immunopathology.     

Antibodies to the central conserved region of respiratory syncytial virus (RSV) G protein block RSV G protein CX3C-CX3CR1 binding and cross-neutralize RSV A and B strains

Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein-CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein-CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.     

Isolation and characterization of a chimpanzee monoclonal antibody to the G glycoprotein of human respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory tract disease in infants and young children. In this study a hybridoma line secreting a chimpanzee monoclonal antibody that neutralizes RSV was isolated. Two chimpanzees were immunized with recombinant vaccinia viruses that express the RSV F or G surface glycoprotein and 1 month later were infected intranasally with the wild-type RSV strain A2. Peripheral blood lymphocytes obtained from the animals were transformed with Epstein-Barr virus, and lymphoblastoid cell lines that secreted anti-RSV antibodies were identified by an RSV antigen-binding enzyme-linked immunosorbent assay. Supernatants from RSV antibody-secreting lymphoblastoid cell lines were tested for in vitro virus neutralization before being fused to the heteromyeloma cell GLI-H7. A chimpanzee antibody [immunoglobulin G3(lambda) subclass] produced from a hybridoma line designated E1.4/2 was shown to bind to the RSV G glycoprotein and neutralize a panel of subgroup A viruses, but not subgroup B viruses, at low (nanomolar) concentrations. Mice passively immunized with this antibody were partially resistant to RSV strain A2 challenge. The usefulness of such antibodies in immunoprophylaxis and immunotherapy of RSV infection is discussed.     

A gammaherpesvirus licenses CD8 T cells to protect the host from pneumovirus-induced immunopathologies

Human respiratory syncytial virus (RSV) is a pneumovirus that causes severe infections in infants worldwide. Despite intensive research, safe and effective vaccines against RSV have remained elusive. The main reason is that RSV infection of children previously immunized with formalin-inactivated-RSV vaccines has been associated with exacerbated pathology, a phenomenon called RSV vaccine-enhanced respiratory disease. In parallel, despite the high RSV prevalence, only a minor proportion of children develop severe diseases. Interestingly, variation in the immune responses against RSV or following RSV vaccination could be linked with differences of exposure to microbes during childhood. Gammaherpesviruses (γHVs), such as the Epstein–Barr virus, are persistent viruses that deeply influence the immune system of their host and could therefore affect the development of pneumovirus-induced immunopathologies for the long term. Here, we showed that a previous ɣHV infection protects against both pneumovirus vaccine-enhanced disease and pneumovirus primary infection and that CD8 T cells are essential for this protection. These observations shed a new light on the understanding of pneumovirus-induced diseases and open new perspectives for the development of vaccine strategies.     

Respiratory syncytial virus vaccine development

The importance of RSV as a respiratory pathogen in young children made it a priority for vaccine development shortly after it was discovered. Unfortunately, after over 50 years of vaccine development no vaccine has yet been licensed and it is not certain which if any vaccines being developed will be successful. The first candidate vaccine, a formalin inactivated RSV vaccine (FI-RSV), was tested in children in the 1960s and predisposed young recipients to more serious disease with later natural infection. The ongoing challenges in developing RSV vaccines are balanced by advances in our understanding of the virus, the host immune response to vaccines and infection, and pathogenesis of disease. It seems likely that with efficient and appropriately focused effort a safe and effective vaccine is within reach. There are at least 4 different target populations for an RSV vaccine, i.e. the RSV naïve young infant, the RSV naïve infant >4–6 months of age, pregnant women, and elderly adults. Each target population has different issues related to vaccine development. Numerous vaccines from live attenuated RSV to virus like particle vaccines have been developed and evaluated in animals. Very few vaccines have been studied in humans and studies in humans are needed to determine which vaccines are worth moving toward licensure. Some changes in the approach may improve the efficiency of evaluating candidate vaccines. The complexity of the challenges for developing RSV vaccines suggests that collaboration among academic, government, and funding institutions and industry is needed to most efficiently achieve an RSV vaccine.     

Lymphocyte transformation response of calves to respiratory syncytial virus

Virus-specific cell-mediated immunity, as determined by in vitro lymphocyte transformation (LT), was demonstrated in calves following infection and vaccination with respiratory syncytial virus (RSV). After experimental infection, 4 of 6 gnotobiotic calves and 6 of 21 conventional calves developed a significant LT response to RSV. By means of a whole blood assay, the LT responses of calves were examined after vaccination with an inactivated vaccine, which consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with a bovine strain of RSV (GC), a live modified bovine strain of RSV (MV), or a live temperature-sensitive mutant of a human strain of RSV (ts-l). Three weeks after vaccination, a virus-specific LT response was detected in 6 of 6 calves given the GC vaccine, 0 of 4 calves given the MV vaccine, and 2 of 4 calves given the ts-l vaccine. The magnitude of the response was greatest in those animals given the GC vaccine. There was no significant correlation between the magnitude of the LT response and levels of serum neutralising antibody. However, the LT response did correlate with serum antibody measured by the single radial haemolysis test 3 weeks after the first vaccination. LT activity to RSV was associated with T and not B lymphocytes. The development of a virus-specific LT response in calves given an inactivated RSV vaccine was not associated with an increase in respiratory disease following challenge with live virus, but rather was related to increased resistance to RSV infection.     

Plasmid DNA encoding the respiratory syncytial virus G protein is a promising vaccine candidate

Respiratory syncytial virus (RSV) remains a major cause of severe respiratory diseases in infants, young children, and the elderly. However, development of a RSV vaccine has been hampered by the outcome of the infant trials in the 1960s with a formalin-inactivated RSV preparation. Enhanced lung disease was induced by the vaccination post-RSV exposure. Previous studies in mice primed with RSV G protein either formulated in adjuvants or delivered by recombinant vaccinia viruses have indicated that enhanced lung pathology resulted from a Th2-type host immune response against the viral G protein. However, in the present report, we have demonstrated that vaccination with plasmid vectors encoding either a full-length or a secreted G protein (DNA-G) clearly elicited balanced systemic and pulmonary Th1/Th2 cytokine responses in mice and did not induce an atypical pulmonary inflammatory reaction post-RSV challenge in cotton rats. DNA-G immunization also induced marked virus neutralizing antibody responses and protection against RSV infection of the lower respiratory tract of both mice and cotton rats. So far, only genetic immunization has been able to induce a balanced Th1/Th2 response with the RSV G protein, reminiscent of that induced by live RSV. Therefore, DNA-G is a promising immunogen for inclusion in a nucleic acid RSV vaccine.     

Role of histo-blood group antigens in primate enteric calicivirus infections

Human noroviruses (NoV) are associated with large proportion of non-bacterial diarrhea outbreaks together with > 50% of food-associated diarrheas. The function of histo-blood group antigens (HBGAs) in pathogenesis of virus infection was implicated. Until recently however, due to lack of a robust animal and in vitro models of human NoV infection, only the partial knowledge concerning the virus pathogenesis (receptor, co-receptor and target cell) and absence of viable vaccine candidates were the frequently referenced attributes of this acute diarrheal illness. Recently, a novel group of enteric caliciviruses (CV) of rhesus macaque host origin was discovered and described. The new genus within the family Caliciviridae was identified: Rhesus Enteric CV, i.e., “Recovirus” (ReCV). ReCVs are genetically and biologically close relatives of human NoVs, exhibit similar genetic and biological features and are capable of being propagated in cell culture. ReCVs cause symptomatic disease (diarrhea and fever) in experimentally inoculated macaques. Formulation and evaluation of efficient NoV vaccine might take several years. As suggested by recent studies, inhibition of HBGAs or HBGA-based antivirals could meanwhile be exploited as vaccine alternatives. The purpose of this minireview is to provide the guidance in respect to newly available primate model of enteric CV infection and its similarities with human NoV in utilizing the HBGAs as potential virus co-receptors to indirectly address the unresolved questions of NoV pathogenesis and immunity.     

Measles virus and inactivated canine distemper virus induce incomplete immunity to canine distemper

Summary Pairs of specific pathogen free dogs were immunized with two injections of heat inactivated canine distemper virus (CDV) or one injection of a live CDV or live measles virus (MV) vaccine. Three unimmunized dogs were used as controls. All 9 dogs were challenged with virulent CDV (Snyder Hill strain). The three unimmunized dogs developed severe signs of disease with a lethal infection in one. The two dogs immunized with live CDV vaccine developed a strong humoral as well as cellular immune response after immunization and were protected against virus replication. Animals immunized with either inactivated CDV or modified live MV failed to develop a measurable cellular immune response after immunization and had a comparatively weak humoral immune response to distemper antigens. They showed mild signs of infection after challenge and responded with strong anamnestic cellular and humoral immunity. The measles vaccine immunized dogs had a moderate serum titer of measles hemolysin-inhibiting antibodies which, after exposure to distemper virus, was boosted to high levels. It is proposed that this response plays a role in the mitigation of the virulent distemper infection in these animals.With 1 Figure     

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.