Effect of Water-Based Microencapsulation on Protection against EDIM Rotavirus Challenge in Mice

We determined the capacity of microcapsules formed by the combination of sodium alginate, an aqueous anionic polymer, and spermine hydrochloride, an aqueous cationic amine, to enhance protection against rotavirus challenge in mice. Adult BALB/c mice were orally inoculated with either free or microencapsulated rotavirus (simian rotavirus strain RRV) and challenged 6 or 16 weeks later with murine rotavirus strain EDIM. Virus-specific humoral immune responses were determined at the time of challenge and 4 days after challenge by intestinal fragment culture. We found that spermine-alginate microcapsules enhanced protection against challenge 16 weeks after immunization but not 6 weeks after immunization. Quantities of virus-specific immunoglobulin A produced by small intestinal lamina propria lymphocytes were correlated with the degree of protection against challenge afforded by spermine-alginate microcapsules. Possible mechanisms by which microcapsules enhance protection against rotavirus challenge are discussed.     

Immunologic correlates of protection against rotavirus challenge after intramuscular immunization of mice.

The capacity of intramuscular (i.m.) inoculation of mice with homologous or heterologous host rotaviruses to induce protection from challenge was evaluated. i.m. inoculation with live, wild-type rotavirus (murine strain EDIM) induced complete protection from viral shedding after challenge for at least 6 weeks after inoculation; protection was correlated with production of virus-specific immunoglobulin A (IgA) by lamina propria (LP) lymphocytes. i.m. inoculation with inactivated EDIM, cell culture-adapted EDIM, or simian strain RRV was associated with partial protection, characterized by reduced viral shedding after challenge. Partial protection after challenge was not associated with production of virus-specific IgA by LP lymphocytes. The mechanisms by which i.m. inoculation induces virus-specific humoral immune responses in the small intestinal LP were examined.     

Induction of Mucosal B-Cell Memory by Intramuscular Inoculation of Mice with Rotavirus

We investigated the capacity of intramuscular (i.m.) immunization with heterologous-host rotavirus (simian strain RRV) to induce mucosal virus-specific memory B cells in mice. We found that prior i.m. immunization enhanced the magnitude of mucosal virus-specific immunoglobulin A (IgA) production but did not alter the site and timing of induction of virus-specific IgA responses after challenge.     

Migration of antigen-presenting B cells from peripheral to mucosal lymphoid tissues may induce intestinal antigen-specific IgA following parenteral immunization.

Parenterally administered immunizations have long been used to induce protection from mucosal pathogens such as Bordetella pertussis and influenza virus. We previously found that i.m. inoculation of mice with the intestinal pathogen, rotavirus, induced virus-specific Ab production by intestinal lymphocytes. We have now used adoptive transfer studies to identify the cell types responsible for the generation of virus-specific Ab production by gut-associated lymphoid tissue (GALT) after i.m. immunization. Three days after i.m. immunization with rotavirus, cells obtained from the draining peripheral lymph nodes of donor mice were transferred into naive recipient mice. We found that intestinal lymphocytes produced rotavirus-specific Igs (IgM, IgA, and IgG) 2 wk after transfer of either unfractionated cells, or unfractionated cells rendered incapable of cellular division by mitomycin C treatment. Additional studies demonstrated that rotavirus-specific IgA, but not IgG, was produced by intestinal lymphocytes after transfer of purified B cells. Ig allotype analysis revealed that rotavirus-specific IgA was produced by intestinal B cells of recipient origin, suggesting that migration of Ag-presenting B cells from peripheral lymphoid tissues to GALT may contribute to the generation of mucosal IgA responses after parenteral immunization. Strategies that promote Ag uptake and presentation by B cells may enhance mucosal IgA production following parenteral immunization.     

Antibody-Dependent and -Independent Protection following Intranasal Immunization of Mice with Rotavirus Particles

The ability to elicit protective immune responses after intranasal immunization with rotavirus particles, either with or without the attenuated Escherichia coli heat-labile enterotoxin LT(R192G) as an adjuvant, was examined in the adult mouse model. BALB/c mice were administered one or two inoculations of psoralen/UV-inactivated, triple-layered (tl) or double-layered (dl) purified rotavirus particles. Four weeks after immunization, mice were challenged with the murine rotavirus strain EDIM, and the shedding of rotavirus antigen was quantified. Rotaviruses used for immunization included EDIM and heterotypic simian (RRV), bovine (WC3), and human (89-12) strains. tl EDIM stimulated both systemic and intestinal rotavirus antibody responses and complete protection with as little as one 1-microgram dose. Inclusion of LT(R192G) (10 micrograms) significantly increased rotavirus antibody responses and reduced antigen concentrations needed for full protection. Both dl EDIM and heterotypic dl and tl particles stimulated protection, but they did so less than tl EDIM at comparable concentrations, either with or without LT(R192G). When B-cell-deficient microMt mice were immunized with tl EDIM particles, protection was reduced to levels similar to those induced with dl EDIM and heterotypic particles in BALB/c mice. However, dl EDIM particles induced similar levels of protection in both mouse strains. The protection stimulated by tl or dl EDIM particles was not diminished by CD8 cell depletion prior to immunization in either strain of mice. These results indicate that tl EDIM induced immunity at least partially through responses to its outer capsid proteins, presumably by stimulation of serotype-specific neutralizing antibody. In contrast, the other particles stimulated protection primarily by an antibody-independent mechanism. Finally, depletion of CD8 cells had no effect on protection by either mechanism.     

Systemic and mucosal immunity to respiratory syncytial virus induced by recombinant Streptococcus gordonii surface-displaying a domain of viral glycoprotein G

A conserved fragment comprising amino acid residues 130-230 of the G glycoprotein of human respiratory syncytial virus subtype A was expressed in the commensal bacterium Streptococcus gordonii. Recombinant streptococci displaying the G domain at the cell surface were used to immunize mice via both parenteral and mucosal routes. Subcutaneous immunization induced respiratory syncytial virus-specific serum immunoglobin G (IgG) capable of partially controlling virus replication in the lungs. Intranasal immunization with live bacteria stimulated the production of IgA against both the whole virus and the G domain in serum and bronchoalveolar fluid. Upon challenge, immunized animals had significantly lower virus titres in the lungs than the controls. Our results show for the first time that the G domain-expressing S. gordonii strain elicits both systemic and mucosal immunity that reduced respiratory syncytial virus replication in the lungs of mice.     

Mucosal adjuvant activity of oligomannose-coated liposomes for nasal immunization

In the present study, we investigated the effectiveness of liposomes coated with a neoglycolipid consisting of mannotriose and dipalmitoylphosphatidylcholine (Man3-DPPE) as an adjuvant for induction of mucosal immunity. Immunization of BALB/c mice with ovalbumin (OVA)-encapsulated Man3-DPPE-coated liposomes (oligomannose-coated liposomes; OMLs) by a nasal route produced high levels of OVA-specific IgG and IgA antibodies in serum of immunized mice 1 week after the last nasal immunization, whereas no significant serum antibody responses were observed in mice that received OVA in uncoated liposomes or OVA alone. Seven weeks after the last nasal immunization, nasal challenge with an excess amount of OVA in mice that had received OVA/OMLs led to an anamnestic response to the antigen that resulted in 5- to 10-fold increases of antigen-specific serum IgG and IgA antibodies. Only mice immunized nasally with OML/OVA secreted antigen-specific secretory IgA in nasal washes and produced interferon-gamma secreting cells in nasopharyngeal-associated lymphoreticular tissue. Taken together, these results show that nasal administration of OMLs induces mucosal and systemic immunity that are specific for the entrapped antigen in the liposomes. Thus, liposomes coated with synthetic neoglycolipids might be useful as adjuvants for induction of mucosal immunity.     

Association of gamma interferon and interleukin-17 production in intestinal CD4+ T cells with protection against rotavirus shedding in mice intranasally immunized with VP6 and the adjuvant LT(R192G)

Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the protein that comprises the intermediate capsid layer of the rotavirus particle, together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant, reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus challenge, and the only lymphocytes required for protection are CD4+ T cells. Because these cells produce cytokines with antiviral properties, the cytokines whose expression is upregulated in intestinal memory CD4+ T cells immediately after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or indirectly responsible for the rapid suppression of rotavirus shedding. This study was designed to identify which cytokines are significantly upregulated in intestinal effector sites and secondary lymphoid tissues of intranasally immunized BALB/c mice after challenge with murine rotavirus strain EDIM. Initially, this was done by using microarray analysis to quantify mRNAs for 96 murine common cytokines. With this procedure, the synthesis of mRNAs for gamma interferon (IFN-gamma) and interleukin-17 (IL-17) was found to be temporarily upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h after rotavirus challenge. These cytokines were also produced in CD4+ T cells obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in vitro exposure to VP6 as determined by intracellular cytokine staining and secretion of cytokines. Although genetically modified mice that lack receptors for either IFN-gamma or IL-17 remained protected after immunization, these results provide suggestive evidence that these cytokines may play direct or indirect roles in protection against rotavirus after mucosal immunization of mice with VP6/LT(R192G).     

Specificity of Acquired Resistance Produced by Immunization with Listeria monocytogenes and Listeria Fractions

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.     

Nasal Immunization of Mice with Human Papillomavirus Type 16 Virus-Like Particles Elicits Neutralizing Antibodies in Mucosal Secretions

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 μg of VLP given at weekly intervals to anesthetized mice induced high (>10⁴) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-μg VLP systemic priming followed by two 5-μg VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.     

Protection against rotavirus-induced gastroenteritis in a murine model by passively acquired gastrointestinal but not circulating antibodies.

Newborn mice suckled on dams immunized either orally or parenterally with primate rotavirus SA-11 were protected against diarrhea induced by SA-11 virus challenge. Experimental oral administration of milk from orally immunized dams protected suckling mice against challenge; protective activity was detected both in the anti-rotavirus immunoglobulin A (IgA) and IgG fractions, but IgA was more potent in vivo than IgG. Oral administration of milk from parentally immunized dams also protected suckling mice against challenge; in this case, protective activity was detected in the anti-rotavirus IgG fraction. In newborn mice foster-nursed by seronegative dams, circulating rotavirus-specific antibodies in high titer did not protect mice against oral SA-11 virus challenge. It appears that the most effective rotavirus vaccine will be that which induces an efficient production of antibodies active at the intestinal cell surface.     

CD4 T Cells Are the Only Lymphocytes Needed To Protect Mice against Rotavirus Shedding after Intranasal Immunization with a Chimeric VP6 Protein and the Adjuvant LT(R192G)

Intranasal immunization of mice with a chimeric VP6 protein and the mucosal adjuvant Escherichia coli heat labile toxin LT(R192G) induces nearly complete protection against murine rotavirus (strain EDIM [epizootic diarrhea of infant mice virus]) shedding for at least 1 year. The aim of this study was to identify the protective lymphocytes elicited by this new vaccine candidate. Immunization of mouse strains lacking one or more lymphocyte populations revealed that protection was dependent on alphabeta T cells but mice lacking gammadelta T cells and B cells remained fully protected. Furthermore, depletion of CD8 T cells in immunized B-cell-deficient mice before challenge resulted in no loss of protection, while depletion of CD4 T cells caused complete loss of protection. Therefore, alphabeta CD4 T cells appeared to be the only lymphocytes required for protection. As confirmation, purified splenic T cells from immunized mice were intraperitoneally injected into Rag-2 mice chronically infected with EDIM. Transfer of 2 x 10(6) CD8 T cells had no effect on shedding, while transfer of 2 x 10(5) CD4 T cells fully resolved shedding in 7 days. Interestingly, transfer of naive splenic CD4 T cells also resolved shedding but more time and cells were required. Together, these results establish CD4 T cells as effectors of protection against rotavirus after intranasal immunization of mice with VP6 and LT(R192G).     

重组SEF21脂质体口服疫苗对肠炎沙门菌感染的作用

目的探讨制备脂质体包裹重组SEF21疫苗,并评价其在预防肠炎沙门菌(S.enteritidis)感染中的作用。方法利用PCR获得SEF21基因,并连接至pET-28a(+)载体。将pET-28a(+)-SEF21在BL21(DE3)大肠埃希菌中表达,通过镍层析柱纯化高表达的rSEF21蛋白。制备脂质体包裹rSEF21疫苗,并对鸡进行2次免疫,然后利用S.enteritidis进行攻毒实验。ELISA检测血清以及肠内容物中的抗体效价。结果所有被免疫鸡的血清及肠黏液中产生了高效价的IgG和IgA抗体。脂质体包裹rSEF21所免疫的鸡的粪便样本中S.enteritidis数量明显下降。结论口服脂质体包裹的重组SEF21蛋白疫苗能有效保护鸡对抗S.enteritidis感染。     

A viral vaccine vector that expresses foreign genes in lymph nodes and protects against mucosal challenge.

A candidate live-virus vaccine strain of Venezuelan equine encephalitis virus (VEE) was configured as a replication-competent vector for in vivo expression of heterologous immunogens. Three features of VEE recommend it for use as a vaccine vector. (i) Most human and animal populations are not already immune to VEE, so preexisting immunity to the vector would not limit expression of the heterologous antigen. (ii) VEE replicates first in local lymphoid tissue, a site favoring the induction of an effective immune response. (iii) Parenteral immunization of rodents and humans with live, attenuated VEE vaccines protects against mucosal challenge, suggesting that VEE vaccine vectors might be used successfully to protect against mucosal pathogens. Upon subcutaneous (s.c.) inoculation into the footpad of mice, a VEE vector containing the complete influenza virus hemagglutinin (HA) gene expressed HA in the draining lymph node and induced anti-HA immunoglobulin G (IgG) and IgA serum antibodies, the levels of which could be increased by s.c. booster inoculation. When immunized mice were challenged intranasally with a virulent strain of influenza virus, replication of challenge virus in their lungs was restricted, and they were completely protected from signs of disease. Significant reduction of influenza virus replication in the nasal epithelia of HA vector-immunized mice suggested an effective immunity at the mucosal surface. VEE vaccine vectors represent an alternative vaccination strategy when killed or subunit vaccines are ineffective or when the use of a live attenuated vaccine might be unsafe.     

Evaluation of the Lon-deficient Salmonella strain as an oral vaccine candidate

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.     

Live attenuated influenza virus expressing human interleukin-2 reveals increased immunogenic potential in young and aged hosts

Despite the reported efficacy of commercially available influenza virus vaccines, a considerable proportion of the human population does not respond well to vaccination. In an attempt to improve the immunogenicity of live influenza vaccines, an attenuated, cold-adapted (ca) influenza A virus expressing human interleukin-2 (IL-2) from the NS gene was generated. Intranasal immunization of young adult and aged mice with the IL-2-expressing virus resulted in markedly enhanced mucosal and cellular immune responses compared to those of mice immunized with the nonrecombinant ca parent strain. Interestingly, the mucosal immunoglobulin A (IgA) and CD8(+) T-cell responses in the respiratory compartment could be restored in aged mice primed with the IL-2-expressing virus to magnitudes similar to those in young adult mice. The immunomodulating effect of locally expressed IL-2 also gave rise to a systemic CD8(+) T-cell and distant urogenital IgA response in young adult mice, but this effect was less distinct in aged mice. Importantly, only mice immunized with the recombinant IL-2 virus were completely protected from a pathogenic wild-type virus challenge and revealed a stronger onset of virus-specific CD8(+) T-cell recall response. Our findings emphasize the potential of reverse genetics to improve the efficacy of live influenza vaccines, thus rendering them more suitable for high-risk age groups.     

Recovery from chronic rotavirus infection in mice with severe combined immunodeficiency: virus clearance mediated by adoptive transfer of immune CD8+ T lymphocytes.

Severe combined immunodeficient (SCID) mice lack both functional T and B cells. These mice develop chronic rotavirus infection following an oral inoculation with the epizootic diarrhea of infant mice (EDIM) rotavirus. Reconstitution of rotavirus-infected SCID mice with T lymphocytes from immunocompetent mice allows an evaluation of a role of T-cell-mediated immunity in clearing chronic rotavirus infection. Complete rotavirus clearance was demonstrated in C.B-17/scid mice 7 to 9 days after the transfer of immune CD8+ splenic T lymphocytes from histocompatible BALB/c mice previously immunized intraperitoneally with the EDIM-w strain of murine rotavirus. The virus clearance mediated by T-cell transfer was restricted to H-2d-bearing T cells and occurred in the absence of rotavirus-specific antibody as determined by enzyme-linked immunosorbent assay, neutralization, immunohistochemistry, and radioimmunoprecipitation. Temporary clearance of rotavirus was observed after the transfer of immune CD8+ T cells isolated from the intestinal mucosa (intraepithelial lymphocytes [IELs]) or the spleens of BALB/c mice previously infected with EDIM by the oral route. Chronic virus shedding was transiently eliminated 7 to 11 days after spleen cell transfer and 11 to 12 days after IEL transfer. However, recurrence of rotavirus infection was detected 1 to 8 days later in all but one SCID recipient receiving cells from orally immunized donors. The viral clearance was mediated by IELs that were both Thy1+ and CD8+. These data demonstrated that the clearance of chronic rotavirus infection in SCID mice can be mediated by immune CD8+ T lymphocytes and that this clearance can occur in the absence of virus-specific antibodies.     

Protective Immunity Induced by Oral Immunization with a Rotavirus DNA Vaccine Encapsulated in Microparticles

DNA vaccines are usually given by intramuscular injection or by gene gun delivery of DNA-coated particles into the epidermis. Induction of mucosal immunity by targeting DNA vaccines to mucosal surfaces may offer advantages, and an oral vaccine could be effective for controlling infections of the gut mucosa. In a murine model, we obtained protective immune responses after oral immunization with a rotavirus VP6 DNA vaccine encapsulated in poly(lactide-coglycolide) (PLG) microparticles. One dose of vaccine given to BALB/c mice elicited both rotavirus-specific serum antibodies and intestinal immunoglobulin A (IgA). After challenge at 12 weeks postimmunization with homologous rotavirus, fecal rotavirus antigen was significantly reduced compared with controls. Earlier and higher fecal rotavirus-specific IgA responses were noted during the peak period of viral shedding, suggesting that protection was due to specific mucosal immune responses. The results that we obtained with PLG-encapsulated rotavirus VP6 DNA are the first to demonstrate protection against an infectious agent elicited after oral administration of a DNA vaccine.     

Temporal Analyses of Virus Replication, Immune Responses, and Efficacy in Rhesus Macaques Immunized with a Live, Attenuated Simian Immunodeficiency Virus Vaccine

Despite evidence that live, attenuated simian immunodeficiency virus (SIV) vaccines can elicit potent protection against pathogenic SIV infection, detailed information on the replication kinetics of attenuated SIV in vivo is lacking. In this study, we measured SIV RNA in the plasma of 16 adult rhesus macaques immunized with a live, attenuated strain of SIV (SIVmac239Δnef). To evaluate the relationship between replication of the vaccine virus and the onset of protection, four animals per group were challenged with pathogenic SIVmac251 at either 5, 10, 15, or 25 weeks after immunization. SIVmac239Δnef replicated efficiently in the immunized macaques in the first few weeks after inoculation. SIV RNA was detected in the plasma of all animals by day 7 after inoculation, and peak levels of viremia (10⁵ to 10⁷ RNA copies/ml) occurred by 7 to 12 days. Following challenge, SIVmac251 was detected in all of the four animals challenged at 5 weeks, in two of four challenged at 10 weeks, in none of four challenged at 15 weeks, and one of four challenged at 25 weeks. One animal immunized with SIVmac239Δnef and challenged at 10 weeks had evidence of disease progression in the absence of detectable SIVmac251. Although complete protection was not achieved at 5 weeks, a transient reduction in viremia (approximately 100-fold) occurred in the immunized macaques early after challenge compared to the nonimmunized controls. Two weeks after challenge, SIV RNA was also reduced in the lymph nodes of all immunized macaques compared with control animals. Taken together, these results indicate that host responses capable of reducing the viral load in plasma and lymph nodes were induced as early as 5 weeks after immunization with SIVmac239Δnef, while more potent protection developed between 10 and 15 weeks. In further experiments, we found that resistance to SIVmac251 infection did not correlate with the presence of antibodies to SIV gp130 and p27 antigens and was achieved in the absence of significant neutralizing activity against the primary SIVmac251 challenge stock.     

Protection of mice from rabies by intranasal immunization with inactivated rabies virus.

The mucosal immunization method is a needle-free alternative way of vaccination. This study evaluated the efficacy of mucosal immunization for rabies. Mice were intranasally administered five times with inactivated and concentrated rabies virus antigen (CRV) supplemented with or without cholera toxin (CT). The anti-rabies virus antibody titer of mice intranasally immunized with CRV plus CT (CRV/CT) was comparable to that of mice intraperitoneally immunized twice with the same amount of CRV. Virus neutralizing (VNA) titers of mice immunized intranasally with CRV/CT were slightly lower than those of intraperitoneally immunized mice. Both anti-rabies virus ELISA antibody and VNA titers of mice immunized with CRV without CT were significantly lower than those of mice immunized with CRV/CT. In mice intranasally immunized with CRV/CT, and intraperitoneally immunized mice, high levels of IgG(2a) antibody were detected, suggesting the activation of Th1-driven cellular immunity by the two ways of immunization. All immunized mice were challenged intracerebrally with a lethal dose of virulent rabies virus CVS strain. The survival rates of mice immunized with CRV/CT and CRV without CT were 67% and 17%, respectively, while the rate of intraperitoneally immunized mice was 100%. Antigen-specific whole IgG and IgG(2a), and VNA titers of survived mice were significantly higher than those of dead mice at the challenge day. These data suggest the possibility of intranasal immunization with inactivated antigen as a rabies vaccination strategy and the importance of a mucosal adjuvant such as CT.