Endocytic trafficking factor VPS45 is essential for spatial regulation of lens fiber differentiation in zebrafish.

In vertebrate lens, lens epithelial cells cover the anterior half of the lens fiber core. Lens epithelial cells proliferate, move posteriorly and start to differentiate into lens fiber cells at the lens equator. Although FGF signaling promotes this equatorial commencement of lens fiber differentiation, the underlying mechanism is not fully understood. Here, we show that lens epithelial cells abnormally enter lens fiber differentiation without passing through the equator in zebrafish vps45 mutants. VPS45 belongs to the Sec1/Munc18-like protein family and promotes endosome trafficking, which differentially modulates signal transduction. Ectopic lens fiber differentiation in vps45 mutants does not depend on FGF, but is mediated through activation of TGFß signaling and inhibition of canonical Wnt signaling. Thus, VPS45 normally suppresses lens fiber differentiation in the anterior region of lens epithelium by modulating TGFß and canonical Wnt signaling pathways. These data indicate a novel role of endosome trafficking to ensure equator-dependent commencement of lens fiber differentiation.

Enhanced effect of BCG vaccine against pulmonary Mycobacterium tuberculosis infection in mice with lung Th17 response to mycobacterial heparin-binding hemagglutinin adhesin antigen.

Although the BCG vaccine can prevent tuberculosis (TB) in infants, its ability to prevent adult pulmonary TB is reportedly limited. Therefore, development of a novel effective vaccine against pulmonary TB has become an international research priority. We have previously reported that intranasal vaccination of mice with a mycobacterial heparin-binding hemagglutinin adhesin (HBHA) plus mucosal adjuvant cholera toxin (CT) enhances production of IFN-γ and anti-HBHA antibody and suppresses extrapulmonary bacterial dissemination after intranasal infection with BCG. In the present study, the effects of intranasal HBHA + CT vaccine on murine pulmonary Mycobacterium tuberculosis (Mtb) infection were examined. Intranasal HBHA + CT vaccination alone failed to reduce the bacterial burden in the infected lung. However, a combination vaccine consisting of s.c. BCG priming and an intranasal HBHA + CT booster significantly enhanced protective immunity against pulmonary Mtb infection on day 14 compared with BCG vaccine alone. Further, it was found that intranasal HBHA + CT vaccine enhanced not only IFN-γ but also IL-17A production by HBHA-specific T cells in the lung after pulmonary Mtb infection. Therefore, this combination vaccine may be a good candidate for a new vaccine strategy against pulmonary TB.

Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule.

Vaccines against Shiga toxin (Stx)-producing Escherichia coli (STEC) have not yet been developed. Two immunologically distinct serotypes of Stx (Stx1 and Stx2) are the main virulence factors of STEC. Thus, blocking their B subunits (StxB) from binding to the cell surface receptor globotriaosylceramide (Gb3) efficiently prevents the action of these toxins. We expressed Stx1B and Stx2B in E. coli inclusion bodies and reassembled them into pentamers by a stepwise dialysis. Stx1B pentamer fully protected mice against Stx1 challenge, but Stx2B pentamer failed to protect mice against Stx2 challenge. To explain those observations, we proposed that the pentamer of Stx2B readily dissociates into its constituent monomers, especially under in vivo conditions, thus being unable to induce pentamer-specific immunity. To increase pentamer stability, we fused the B subunit to a pentameric coiled-coil domain of the cartilage oligomeric matrix protein (COMP). This "five-to-five" fusion hybrid molecule (Stx2B-COMP) was shown to be protective against Stx2 challenge, demonstrating that the Stx2B subunit when leashed and bundled by a rigid pentameric coiled-coil domain mount a pentamer-specific immune response and efficiently neutralize the toxin both in vitro and in vivo. Our data strongly suggest that the Stx2B subunit moiety fluctuates between a pentameric and monomeric state within the fusion protein, which may increase the likelihood of the immune system recognizing the pentameric conformation for toxin neutralization.

A bio-nanocapsule containing envelope protein domain III of Japanese encephalitis virus protects mice against lethal Japanese encephalitis virus infection.

An engineered bio-nanocapsule (BNC) comprising modified hepatitis B surface antigen L protein was used as a physical scaffold for envelope protein domain III (D3) of Japanese encephalitis virus (JEV). At the N terminus, the BNC contained a two-tandem repeat of the Z domain (ZZ) derived from Staphylococcus aureus protein A (ZZ-BNC). The Lys-rich ZZ moiety exposed on the surface of ZZ-BNC was used for chemical conjugation with the JEV D3 antigen, which had been expressed and purified from Escherichia coli. Immunization of mice with D3 loaded on the surface of ZZ-BNC (ZZ-BNC:D3) augmented serum IgG response against JEV and increased protection against lethal JEV infection. The present study suggests that innocuous recombinant antigens, when loaded on the surface of ZZ-BNC, can be transformed to immunogenic antigens.

Expression and secretion of cholera toxin B subunit in lactobacilli.

Lactic acid bacteria (LAB) are used in various fields, including in food and medical supplies. There has been a great deal of research into vaccine development using LAB as carriers due to their "generally recognized as safe" status. Cholera is an infectious disease that causes diarrhea due to cholera toxin (CT) produced by Vibrio cholerae. The pentameric cholera toxin B (CTB) subunit has no toxicity, and is used as an antigen in cholera vaccines and as a delivery molecule in vaccines to various diseases. In this study, we generated recombinant LAB expressing and secreting CTB. Here, we first report that CTB expressed and secreted from LAB bound to GM1 ganglioside. The secreted CTB was purified, and its immunogenicity was determined by intranasal administration into mice. The results of the present study suggested that it may be useful as the basis of a new oral cholera vaccine combining LAB and CTB.

Tricomponent immunopotentiating system as a novel molecular design strategy for malaria vaccine development.

The creation of subunit vaccines to prevent malaria infection has been hampered by the intrinsically weak immunogenicity of the recombinant antigens. We have developed a novel strategy to increase immune responses by creating genetic fusion proteins to target specific antigen-presenting cells (APCs). The fusion complex was composed of three physically linked molecular entities: (i) a vaccine antigen, (ii) a multimeric α-helical coiled-coil core, and (iii) an APC-targeting ligand linked to the core via a flexible linker. The vaccine efficacy of the tricomponent complex was evaluated using an ookinete surface protein of Plasmodium vivax, Pvs25, and merozoite surface protein-1 of Plasmodium yoelii. Immunization of mice with the tricomponent complex induced a robust antibody response and conferred substantial levels of P. vivax transmission blockade as evaluated by a membrane feed assay, as well as protection from lethal P. yoelii infection. The observed effect was strongly dependent on the presence of all three components physically integrated as a fusion complex. This system, designated the tricomponent immunopotentiating system (TIPS), onto which any recombinant protein antigens or nonproteinaceous substances could be loaded, may be a promising strategy for devising subunit vaccines or adjuvants against various infectious diseases, including malaria.

Plasmodium vivax ookinete surface protein Pvs25 linked to cholera toxin B subunit induces potent transmission-blocking immunity by intranasal as well as subcutaneous immunization.

The nontoxic cholera toxin B subunit (CTB) was evaluated as a potential delivery molecule for the Plasmodium vivax ookinete surface protein, Pvs25. Recombinant Pvs25 was expressed as a secreted protein in the yeast Pichia pastoris, as a mixture of isoforms including multimers and the A and B monomers. The A isoform with the presumed native protein fold was the most abundant, accounting for more than 40% of all expressed protein. The molecularly uniform A isoform was chemically conjugated to CTB via its primary amines, and the fusion protein, retaining GM1-ganglioside affinity, was administered to BALB/c mice by the subcutaneous (s.c.) or intranasal (i.n.) route. Immunization of mice with conjugated Pvs25 without supplemental adjuvant induced antisera that specifically recognized P. vivax ookinetes in vitro. Furthermore, the antisera, when mixed with parasitized blood isolated from P. vivax patients from Thailand, was found to reduce parasite transmission to mosquitoes, conferring a 93 to 98% (s.c.) or a 73 to 88% (i.n.) decrease in oocyst number. Unconjugated Pvs25 alone conferred only a 23 to 60% (s.c.) or a 0 to 6% (i.n.) decrease in oocyst number. Coadministration of extraneous adjuvants, however, further enhanced the vaccine efficacy up to complete blockade. Taken together, we conclude that a weakly immunogenic Pvs25 by itself, when linked to CTB, transforms into a potent transmission-blocking antigen in both i.n. and s.c. routes. In addition, the present study is, to the best of our knowledge, the first demonstration of the immune potentiating function of CTB for a vaccine antigen delivered by the s.c. route.

Malaria ookinete surface protein-based vaccination via the intranasal route completely blocks parasite transmission in both passive and active vaccination regimens in a rodent model of malaria infection.

Malaria vaccines based on ookinete surface proteins (OSPs) of the malaria parasites block oocyst development in feeding mosquitoes and hence disrupt the parasite life cycle and prevent the disease from being transmitted to other individuals. To investigate whether a noninvasive mucosal vaccination regimen effectively blocks parasite transmission in vivo, Plasmodium yoelii Pys25, a homolog of the Pfs25 and Pvs25 OSPs of Plasmodium falciparum and Plasmodium vivax, respectively, was intranasally (i.n.) administered using a complement-deficient DBA/2 mouse malaria infection model, in which a highly elevated level of oocysts develops in feeding mosquitoes. Vaccinated mice developed a robust antibody response when the vaccine antigen was given together with cholera toxin adjuvant. The induced immune serum was passively transferred to DBA/2 mice 3 days after infection with P. yoelii 17XL, and Anopheles stephensi mosquitoes were allowed to feed on the infected mice before or after serum transfusion. This passive immunization completely blocked oocyst development; however, immune serum induced by the antigen or adjuvant alone did not have such a profound antiparasite effect. Further, when i.n. vaccinated mice were infected with the parasite and then mosquitoes were allowed to directly feed on the infected mice, complete blockage of transmission was again observed. To our knowledge, this is the first time that mucosal vaccination has been demonstrated to be efficacious for directly preventing parasite transmission from vaccinated animals to mosquitoes, and the results may provide important insight into rational design of nonparenteral vaccines for use against human malaria.

Mucosal vaccination approach against mosquito-borne Japanese encephalitis virus.

To investigate the potential applicability of mucosal vaccines against mucosa-unrelated pathogens, a non-parenteral vaccination approach was taken as a prophylactic strategy against mosquito-borne Japanese encephalitis virus (JEV). Intranasal (i.n.) immunization with a mouse brain-derived formalin-inactivated JE vaccine induced a robust virus-neutralizing antibody in mice, and this induction was augmented by co-administration with cholera toxin (CT) and pertussis toxin, but not with killed Bordetella pertussis. The antibody response induced by the i.n. administration of the JE vaccine with bacterial toxins was comparable in intensity to that induced by a parenteral immunization regime, and the former was considerably more effective in terms of delayed-type hypersensitivity and local antibody response. In addition, the adjuvant effects of bacterial toxins were much more prominent for the mucosal than the parenteral route. Two other non-invasive routes, oral and transcutaneous administration, were examined, but the i.n. route was by far the most effective. Finally, the vaccine efficacy of a chimeric fusion protein between the B subunit of CT and the JEV envelope protein showed some promise for the development of non-invasive JE vaccine. Our results suggest that the mucosal vaccination approach is feasible for a non-mucosal pathogen such as JEV, but that the adjuvant, carrier molecule, and administration route must be optimized for construction of an effective vaccine platform.

Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination.

The cholera toxin B subunit (CTB) is secreted in its pentameric form from Escherichia coli if its leader peptide is replaced with one of E. coli origin. However, the secretion of the pentamer is generally severely impaired when the molecule is mutated or fused to a foreign peptide. Therefore, we attempted to regenerate pentameric CTB from the inclusion bodies (IBs) of E. coli. Stepwise dialysis of the IBs solubilized in guanidine hydrochloride predominantly generated soluble high-molecular-mass (HMM) aggregates and only a small fraction of pentamer. Three methods to reassemble homogeneous pentameric molecules were evaluated: (i) using a pentameric coiled-coil fusion partner, expecting it to function as an assembly core; (ii) optimizing the protein concentration during refolding; and (iii) eliminating contaminants before refolding. Coiled-coil fusion had some effect, but substantial amounts of HMM aggregates were still generated. Varying the protein concentration from 0.05 mg/mL to 5mg/mL had almost no effect. In contrast, eliminating the contaminants before refolding had a robust effect, and only the pentamer was regenerated, with no detectable HMM aggregates. Surprisingly, the protein concentration at refolding was up to 5mg/mL when the contaminants were removed, with no adverse effects on refolding. The regenerated pentamer was indistinguishable in its biochemical and immunological characteristics from CTB secreted from E. coli or choleragenoid from Vibrio cholerae. This study provides a simple but very efficient strategy for pentamerizing CTB with a highly homogeneous molecular conformation, with which it may be feasible to engineer CTB derivatives and CTB fusion antigens.

Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subunit vaccine against egg-drop syndrome.

Egg-drop syndrome (EDS) virus is an avian adenovirus that causes a sudden drop in egg production and in the quality of the eggs when it infects chickens, leading to substantial economic losses in the poultry industry. Inactivated EDS vaccines produced in embryonated duck eggs or cell culture systems are available for the prophylaxis of EDS. However, recombinant subunit vaccines that are efficacious and inexpensive are a desirable alternative. In this study, we engineered chimeric fusion proteins in which the trimeric fiber knob domain lacking the triple ß-spiral motif in the fiber shaft region was genetically fused to trimeric coiled coils, such as those of the engineered form of the GCN4 leucine zipper peptide or chicken cartilage matrix protein (CMP). The fusion proteins were expressed predominantly as soluble trimeric proteins in Escherichia coli at levels of 15-80mg/L of bacterial culture. The single immunization of chickens with the purified fusion proteins, at a dose equivalent to 10µg of the knob moiety, elicited serum antibodies with high hemagglutination inhibition (HI) activities, similar to those induced by an inactivated EDS vaccine. A dose-response analysis indicated that a single immunization with as little as 1µg of the knob moiety of the CMP-knob fusion protein was as effective as the inactivated vaccine in inducing antibodies with HI activity. The immunization of laying hens had no apparent adverse effects on egg production and effectively prevented clinical symptoms of EDS when the chickens were challenged with pathogenic EDS virus. This study demonstrates that the knob domain lacking the shaft sequence but fused to a trimeric coiled coil is a promising candidate subunit vaccine for the prophylaxis of EDS in chickens.

Cholera toxin B subunit-five-stranded α-helical coiled-coil fusion protein: "five-to-five" molecular chimera displays robust physicochemical stability.

To create a physicochemically stable cholera toxin (CT) B subunit (CTB), it was fused to the five-stranded α-helical coiled-coil domain of cartilage oligomeric matrix protein (COMP). The chimeric fusion protein (CTB-COMP) was expressed in Pichia pastoris, predominantly as a pentamer, and retained its affinity for the monosialoganglioside GM1, a natural receptor of CT. The fusion protein displayed thermostability, tolerating the boiling temperature of water for 10min, whereas unfused CTB readily dissociated to its monomers and lost its affinity for GM1. The fusion protein also displayed resistance to strong acid at pHs as low as 0.1, and to the protein denaturant sodium dodecyl sulfate at concentrations up to 10%. Intranasal administration of the fusion protein to mice induced anti-B subunit serum IgG, even after the protein was boiled, whereas unfused CTB showed no thermostable mucosal immunogenicity. This study demonstrates that CTB fused to a pentameric α-helical coiled coil has a novel physicochemical phenotype, which may provide important insight into the molecular design of enterotoxin-B-subunit-based vaccines and vaccine delivery molecules.

Tricomponent fusion complex comprising a viral antigen, a pentameric α-helical coiled-coil, and an immunoglobulin-binding domain as an effective antiviral vaccine.

The pentameric coiled-coil domain of cartilage oligomeric matrix protein (COMP) genetically fused to the Z domain of Staphylococcus aureus protein A, an immunoglobulin-binding domain (IBD), was evaluated as a viral antigen carrier complex. In a proof-of-concept study, recombinant Japanese encephalitis virus (JEV) E protein domain III (D3) was loaded onto the COMP-Z fusion protein by chemical conjugation, and the tricomponent complex generated, COMP-Z/D3, was evaluated for its vaccine efficacy in a mouse JEV infection model. Immunization with the complex conferred substantially greater protection against lethal JEV infection than the unloaded antigen. Next, a tricomponent complex was engineered in which the three molecular entities (the D3 antigen, COMP coiled-coil domain, and Z domain) were genetically connected in tandem to create the D3-COMP-Z tricomponent complex, or its reversal oriented construct, Z-COMP-D3. The fusion complexes were produced as inclusion bodies in Escherichia coli, but could be refolded to biologically active pentamers that retained the E protein antigenicity and the IBD function. Immunization with the refolded complexes conferred a high level of protection against lethal JEV infection, similar in efficacy to that of the tricomponent complex generated by chemical conjugation. These results demonstrate that the tricomponent complex, whether generated by chemical or genetic fusion, is a promising molecular design for the creation of effective subunit vaccines against viral infections.

Merozoite surface protein-1 of Plasmodium yoelii fused via an oligosaccharide moiety of cholera toxin B subunit glycoprotein expressed in yeast induced protective immunity against lethal malaria infection in mice.

Methylotrophic yeast (Pichia pastoris) secreted cholera toxin B subunit (CTB) predominantly as a biologically active pentamer (PpCTB) with identical ganglioside binding affinity profiles to that of choleragenoid. Unlike choleragenoid, however, the PpCTB did not induce a footpad edema response in mice. Of the two potential glycosylation sites (NIT(4-6) and NKT(90-92)) for this protein, a N-linked oligosaccharide was identified at Asn4. The oligosaccharide, presumed to extend from the lateral circumference of the CTB pentamer ring structure, was exploited as a site-specific anchoring scaffold for the C-terminal 19-kDa merozoite surface protein-1 (MSP1-19) of the rodent malaria parasite, Plasmodium yoelii. Conjugation of MSP1-19 to PpCTB via its oligosaccharide moiety induced higher protective efficacy against lethal parasite infection than conjugation directly to the PpCTB protein body in both intranasal and subcutaneous immunization regimes. Such increased protection was potentially due to the higher antigen loading capacity of CTB achieved when the antigen was linked to the extended branches of the oligosaccharide. This might have allowed the antigen to reside in more spacious molecular environment with less steric hindrance between the constituent molecules of the fusion complex.

Physicochemically stable cholera toxin B subunit pentamer created by peripheral molecular constraints imposed by de novo-introduced intersubunit disulfide crosslinks.

We attempted to generate a physicochemically stable cholera toxin B subunit (CTB) by de novo-introduction of intersubunit disulfide bonds between adjacent subunits. Genes encoding double mutant CTB (dmCTB) encompassing a pair of amino acids to be replaced with cysteine residues either at the N-terminal (T1C/T92C, Q3C/T47C), C-terminal (F25C/N103C, Y76C/N103C), or at the internal α-helix region (L77C/T78C), were engineered. One mutant with the N-terminal constraint [dmCTB(T1C/T92C)], expressed as pentamer retained monosialoganglioside G(M1) (GM1) binding affinity, and exhibited robust thermostability. However, when the mutant CTB was heat-treated in the presence of a reducing agent, the thermostable phenotype was abolished, indicating the observed phenotype is due to the introduction of intersubunit disulfide bonds. The mutant CTB also exhibited a strong acid stability at a pH as low as 1.2, as well as stability against incubation with sodium dodecyl sulfate at concentrations as high as 10%. Furthermore, intranasal administration of the mutant CTB to mice induced CTB-specific serum IgG even after heat treatment, while the wildtype CTB failed to show such heat-resistant mucosal immunogenicity. This study demonstrated that an enterotoxin B subunit could be transformed into a physicochemically stable pentamer by the de novo-introduction of peripherally arranged intersubunit disulfide crosslinks, which may prove to be a useful strategy for the development of molecularly stable enterotoxin B subunit-based vaccines and delivery molecules.

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice.

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1-3), domains I and II (D1-2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1-3, D1-2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1-3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.

Adenovirus-vectored Plasmodium vivax ookinete surface protein, Pvs25, as a potential transmission-blocking vaccine.

Adjuvants or delivery vehicles are essential components to expedite malaria vaccine development. In this study, replication-defective human adenovirus serotype 5 (rAd) was genetically engineered to express the Plasmodium vivax ookinete surface protein (OSP), Pvs25 (AdPvs25). BALB/c mice immunized with the AdPvs25 through various routes including intramuscular, subcutaneous and intranasal routes were analyzed for induction of antigen-specific transmission-blocking immunity. Parenteral but not mucosal immunization induced high serum immunoglobulin G (IgG) responses specific to P. vivax ookinetes isolated from P. vivax volunteer patients from Thailand. The membrane feeding assay revealed that antisera conferred a transmission blockade of up to 99% reduction in the average oocyst numbers per mosquito, while immunization with a rAd expressing Pfs25 from Plasmodium falciparum, a homolog of Pvs25, conferred only a background level of blockade, suggesting that a species-specific transmission-blocking immunity was induced. Vaccine efficacy of AdPvs25 was slightly higher than to a recombinant Pvs25 protein mixed with aluminum hydroxide, but less efficacious than the protein emulsified with incomplete Freund's adjuvant. This study, the first preclinical evaluation of adenovirus-vectored malaria OSPs, implicates a potential inclusion of malaria transmission-blocking vaccine antigens in viral vector systems.

Intranasal administration of Schistosoma japonicum paramyosin induced robust long-lasting systemic and local antibody as well as delayed-type hypersensitivity responses, but failed to confer protection in a mouse infection model.

To investigate intranasal (i.n.) immunization efficacy of Schistosoma japonicum 97-kDa myofibrillar protein paramyosin (PM), a vaccine candidate for Asian schistosomiasis, BALB/c mice were i.n. immunized with Escherichia coli-expressed recombinant PM (rPM). I.n. immunization using rPM mixed with cholera toxin (CT) was more potent than subcutaneous (s.c.) immunization with rPM emulsified in incomplete Freund's adjuvant for induction of serum (IgG, IgE, and IgA) and mucosal (IgA in nose, lung, and intestine) antibody and delayed-type hypersensitivity (DTH) responses. The second i.n. immunization was sufficient to induce maximal serum IgG and DTH responses, which were almost completely maintained for more than 6 months. Next, to evaluate protective efficacy of the rPM against S. japonicum infection, immunized mice were infected with S. japonicum cercariae at 2 weeks after the second immunization. At 7 weeks after infection, we observed no reduction in worm burden or fecundity in both i.n. and s.c. immunized groups. Results showed that i.n. immunization with rPM/CT failed to provide protection against parasite infection, albeit the antigen was a very potent mucosal immunogen. These results may emphasize the need to innovate new mucosal adjuvants or delivery molecules to overcome such hurdles in the construction of a mucosal antiparasite vaccine platform.

Mucosal immunization with recombinant heparin-binding haemagglutinin adhesin suppresses extrapulmonary dissemination of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in infected mice.

It is generally accepted that cellular immunity plays a critical role in the protection against Mycobacterium tuberculosis, an intracellular pathogen. Recently, however, an increasing number of reports indicate the important contribution of humoral immunity against mycobacterial infection. Since M. tuberculosis establishes its primary lesion in the lung, induction of humoral immunity in the airway tract by mucosal immunization regime could provide protective immunity against tuberculosis. In this study, mycobacterial heparin-binding haemagglutinin adhesin (HBHA) was used as an immunization antigen because HBHA is an essential virulence factor required for the infection of lung epithelial cells and extrapulmonary dissemination of mycobacteria. The effects of intranasal immunization with a yeast-expressed recombinant (r) HBHA co-administered with a mucosal adjuvant cholera toxin (CT) on the induction of humoral and cellular immunity were examined, and its protective efficacy against pulmonary challenge infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG) was evaluated. HBHA-specific antibodies were induced in serum and airway tract of immunized mice, which specifically recognized native HBHA expressed on M. bovis BCG. Th1-type immunity against mycobacterial antigens was also enhanced in the lung of immunized mice after pulmonary BCG infection. Furthermore, the immunization suppressed bacterial load in the spleen after pulmonary BCG infection. These results indicate that systemic and local humoral immunity induced by the HBHA-based mucosal vaccine impairs extrapulmonary dissemination, thus providing immune protection against mycobacterial infection.

Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems.

Noninvasive mucosal vaccines are attractive alternatives to parenteral vaccines. Although the conjugation of vaccine antigens with the B subunit of cholera toxin (CTB) is one of the most promising strategies for vaccine delivery to mucosal immune systems, the molecule cannot tolerate large-protein fusion, as it severely impairs pentamerization and loses affinity for GM1-ganglioside. Here we report a new strategy, in which steric hindrance between CTB-antigen fusion subunits is significantly reduced through the integration of unfused CTB "molecular buffers" into the pentamer unit, making them more efficiently self-assemble into biologically active pentamers. In addition, the chimeric protein took a compact configuration, becoming small enough to be secreted, and one-step affinity-purified proteins, when administered through a mucosal route, induced specific immune responses in mice. Since our results are not dependent on the use of a particular expression system or vaccine antigen, this strategy could be broadly applicable to bacterial enterotoxin-based vaccine design.