Protective effect of nasal immunization of influenza virus hemagglutinin with recombinant cholera toxin B subunit as a mucosal adjuvant in mice.

To develop an efficient nasal influenza vaccine, influenza A and B virus HA with rCTB as a mucosal adjuvant were administered to mice intranasally. Serum anti-HA IgG and IgA antibody responses for both HA vaccines were significantly increased in the presence of rCTB. Higher HI and neutralizing antibody titers and higher mucosal IgA antibody responses in the respiratory tract were detected when rCTB was added than without rCTB. When mice were immunized with HA vaccine with or without rCTB and challenged by intranasal administration of mouse-adapted pathogenic influenza A virus, all mice immunized with HA plus rCTB survived for seven days without any inflammatory changes in the lungs, while not all the mice immunized with HA without rCTB survived, and all of them had lung consolidations. These results demonstrate that intranasal co-administration of rCTB as a mucosal adjuvant with influenza virus HA is necessary not only for the induction of systemic and mucosal HA antibodies, but also for the protection of mice from morbidity and mortality resulting from virus infection.

Expression of Escherichia coli heat-labile enterotoxin B subunit (LTB) in Saccharomyces cerevisiae.

Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately 1.9% of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

Recombinant cholera toxin B subunit activates dendritic cells and enhances antitumor immunity.

Activation of dendritic cells (DC) is crucial for priming of cytotoxic T lymphocytes (CTL), which have a critical role in tumor immunity, and it is considered that adjuvants are necessary for activation of DC and for enhancement of cellular immunity. In this study, we examined an adjuvant capacity of recombinant cholera toxin B subunit (rCTB), which is non-toxic subunit of cholera toxin, on maturation of murine splenic DC. After the in vitro incubation of DC with rCTB, the expression of MHC class II and B7-2 on DC was upregulated and the secretion of IL-12 from DC was enhanced. In addition, larger DC with longer dendrites were observed. These data suggest that rCTB induced DC maturation. Subsequently, we examined the induction of tumor immunity by rCTB-treated DC by employing Meth A tumor cells in mice. Pretreatment with subcutaneous injection of rCTB-treated DC pulsed with Meth A tumor lysate inhibited the growth of the tumor cells depending on the number of DC. Moreover, intratumoral injection of rCTB-treated DC pulsed with tumor lysate had therapeutic effect against established Meth A tumor. Immunization with DC activated by rCTB and the tumor lysate increased number of CTL precursor recognizing Meth A tumor. The antitumor immune response was significantly inhibited in CD8+ T cell-depleted mice, although substantial antitumor effect was observed in CD4+ T cell-depleted mice. These results indicated that rCTB acts as an adjuvant to enhance antitumor immunity through DC maturation and that CD8+ T cells play a dominant role in the tumor immunity. Being considered to be safe, rCTB may be useful as an effective adjuvant to raise immunity for a tumor in clinical application.

Recombinant cholera toxin B subunit (rCTB) as a mucosal adjuvant enhances induction of diphtheria and tetanus antitoxin antibodies in mice by intranasal administration with diphtheria-pertussis-tetanus (DPT) combination vaccine.

Recombinant cholera toxin B subunit (rCTB) which is produced by Bacillus brevis carrying pNU212-CTB acts as a mucosal adjuvant capable of enhancing host immune responses specific to unrelated, mucosally co-administered vaccine antigens. When mice were administered intranasally with diphtheria-pertussis-tetanus (DPT) combination vaccine consisting of diphtheria toxoid (DTd), tetanus toxoid (TTd), pertussis toxoid (PTd), and formalin-treated filamentous hemagglutinin (fFHA), the presence of rCTB elevated constantly high values of DTd- and TTd-specific serum ELISA IgG antibody titres, and protective levels of diphtheria and tetanus toxin-neutralizing antibodies but the absence of rCTB did not. Moreover, the addition of rCTB protected all mice against tetanic symptoms and deaths. DPT combination vaccine raised high levels of serum anti-PT IgG antibody titres regardless of rCTB and protected mice from Bordetella pertussis challenge. These results suggest that co-administration of rCTB as an adjuvant is necessary for induction of diphtheria and tetanus antitoxin antibodies on the occasion of intranasal administration of DPT combination vaccine.

Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB.

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.

Frequent nasal administrations of recombinant cholera toxin B subunit (rCTB)-containing tetanus and diphtheria toxoid vaccines induced antigen-specific serum and mucosal immune responses in the presence of anti-rCTB antibodies.

Vaccination via a mucosal route is a very attractive means for immunization, because both local and systemic immune responses are inducible and vaccines can be administered easily and safely from infants to elderly persons. For developing widely applicable mucosal vaccines using recombinant cholera toxin B subunit (rCTB) as a safe adjuvant, we examined whether frequent nasal administrations of rCTB-containing same and different vaccines could induce antigen-specific immune responses without induction of systemic tolerance and suppression by pre-existing anti-rCTB immunity. Ten repetitive nasal administrations to mice of tetanus toxoid (TT) + rCTB or diphtheria toxoid (DT) + rCTB raised and maintained high levels of antigen- and rCTB-specific serum IgG including high levels of tetanus/diphtheria antitoxin titres and raised nasal, salivary, lung, vaginal and fecal secreted IgA, suggesting that the regimen did not induce systemic tolerance to TT/DT and rCTB. Mice successively received repetitive five doses of TT as the first antigen and subsequent five doses of DT as the second antigen, and vice versa, raised serum IgG to the second antigen at various levels including low but sufficient protective levels of antitoxin titres and induced mucosal IgA in the lungs, the vaginas and feces, but hardly in the nasal secretions and salivas. After an interval of 22 weeks between the dosage of the first and second antigens, mice induced serum IgG to the second antigen at high levels and mucosal IgA in all sites. In conclusion, anti-TT and -DT serum and mucosal antibody responses induced by repeated intranasal immunization using rCTB adjuvant lasted for a long period, and for improving the effectivity of vaccination, different rCTB-containing vaccines should be administered at appropriate intervals.

Mucosal and systemic antibody responses against an acellular pertussis vaccine in mice after intranasal co-administration with recombinant cholera toxin B subunit as an adjuvant.

To investigate the possibility of intranasal immunization with an acellular pertussis vaccine, groups of mice were administered intranasally with aluminium-non-adsorbed pertussis toxoid (PTd; 0.5 or 5 microg) and formalin-treated filamentous hemagglutinin (fFHA; 5 microg) with and without recombinant cholera toxin B subunit (rCTB; 10 microg) as a mucosal adjuvant. At a low concentration of PTd, the following things became clear: (1) earlier and higher elevation of serum anti-PTd and anti-FHA IgG antibody titres in the presence of rCTB than in its absence, (2) higher serum anti-PTd and anti-FHA IgG antibody titres than 200 and 100 ELISA units ml(-1) (EU ml(-1)) in all mice, respectively, in the presence of rCTB, which were obtained by calibration against a reference anti-pertussis mouse serum, and (3) in an intranasal challenge experiment with Bordetella pertussis, slightly more rapid elimination of the bacteria from the lungs of mice intranasally immunized in the presence of rCTB, suggesting the effectiveness of rCTB as a mucosal adjuvant. However, irrespective of rCTB and dose of PTd, mice which were immunized four times and sacrificed on day 35 developed high levels of anti-PTd serum IgG antibodies, high or moderate levels of anti-FHA serum IgG antibodies and mucosal anti-PTd IgA antibodies in the lungs; only a slight or no increase of anti-FHA mucosal IgA antibodies was observed in the lung. These facts suggested the immunogenicity and mucosal adjuvanticity of PTd, and therefore, the mucosal adjuvanticity of rCTB seemed to be inconspicuous. Moreover, the addition of rCTB induced higher anti-PTd serum IgE antibody responses than no addition of it depending on dose of PTd. These results show that dose of PTd included in an acellular pertussis vaccine had better be low as possible and the addition of rCTB may not be always necessary in case of this nasal vaccine alone unlike tetanus and diphtheria toxoids and hepatitis B virus vaccine reported before.

Effects of recombinant cholera toxin B subunit on IL-1beta production by macrophages in vitro.

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. As a clue to the mechanism of the adjuvant effect of rCTB, the profile of cytokines secreted in vitro by the mouse peritoneal macrophage (Mphi) treated with rCTB was examined. IL-1beta secretion, intracellular production, and expression of its mRNA of LPS-stimulated Mphi was greatly enhanced by treatment with rCTB. IL-1beta production in response to other microbial stimulators, such as Pansorbin, Sansorbin, insoluble peptidoglycan, and Taxol, was also potentiated by rCTB. Mphi pretreated with rCTB before 24 hr could maintain the ability to produce a high level of IL-1beta, suggesting that this ability may be involved in the adjuvant activity of rCTB on Mphi stimulation. The possibility of close association between rCTB and signal transduction of a Toll-like receptor family in Mphi is discussed.

Immune responses in mice to intranasal and intracutaneous administration of a DNA vaccine encoding Helicobacter pylori-catalase.

We previously reported that the intracutaneous injection of DNA vaccines encoding Helicobacter pylori heat shock proteins elicited specific immune responses, and led to reduced infection in mice. In this study, we constructed DNA vaccine encoding H. pylori-catalase (pcDNA3.1-kat) and investigated the immune responses to intranasal and intracutaneous administration of pcDNA3.1-kat. C57/BL6 mice were immunized intracutaneously with 10 microg of pcDNA3.1-kat or intranasally with 50 microg of pcDNA3.1-kat. Catalase-specific IgG antibody was detected in the sera of intranasal and intracutaneous immunized mice. Both intranasal and intracutaneous immunized mice were significantly protected from colonization by H. pylori and had significantly reduced degrees of gastritis. These results demonstrate that DNA vaccine encoding H. pylori-catalase can induce an immune response against H. pylori, and that intranasal immunization works as well as intracutaneous immunization.