Mycobacterium bovis BCG-mediated suppression of Th17 response in mouse experimental autoimmune encephalomyelitis.

INTRODUCTION: Multiple sclerosis (MS) is an autoimmune disease mediated by a pro-inflammatory immune response. Experimental autoimmune encephalomyelitis (EAE) induced by immunization of mice with a myelin oligodendrocyte glycoprotein (MOG) peptide emulsified in killed Mycobacterium tuberculosis-containing complete Freund's adjuvant (CFA-EAE) is used as a model of MS. Mycobacterium bovis BCG has been reported to ameliorate clinical symptoms of CFA-EAE, although the precise mechanism has not yet been documented. Since CFA-EAE uses adjuvant with mycobacterial antigens, mycobacterial antigen-specific T cells induced by CFA may cross-react with BCG and modulate EAE. METHODS: To exclude the influence of cross-reactivity, a modified murine EAE model (cell wall skeleton (CWS)-EAE) that does not induce mycobacterial antigen-specific T cells was established and used to reevaluate the therapeutic effects of BCG on EAE. RESULTS: Inoculation with BCG 6 d after CWS-EAE induction successfully ameliorated EAE symptoms, suggesting that the therapeutic effects of BCG are independent of the mycobacterial antigen-specific T cells induced by the CFA-EAE protocol. BCG inoculation into the CWS-EAE mice resulted in reduced levels of MOG-specific Th17 in the central nervous system (CNS) with reduced demyelinated lesions of the spinal cord. In the draining lymph nodes of the MOG-immunized sites, BCG inoculation resulted in an increase in MOG-specific Th17 and Th1 cells at an early stage of immune response. CONCLUSION: The results suggest that BCG inoculation suppresses the Th17 response in the CNS of EAE mice via a mechanism that may involve the suppression of egress of encephalitogenic T cells from lymphoid organs.

Interleukin-17A is involved in enhancement of tumor progression in murine intestine.

Interleukin (IL)-17A is a cytokine involved in neutrophilic inflammation but the role of IL-17A in anti-tumor immunity is controversial because both pro- and anti-tumor activities of IL-17A have been reported. We hypothesized that constitutive expression of IL-17A in intestinal environment modifies tumor growth. To address the issue, mice were inoculated into subserosa of cecum (i.c.) with murine EL4 lymphoma expressing a model tumor antigen, and tumor growth was monitored. IL-17A-producing cells were detected both in tumor mass and in normal intestinal tissue of i.c. tumor-bearing wild type mice. Tumor size in the wild-type mice was significantly higher than that in the cecum of IL-17A gene-knockout mice. Furthermore, anti-IL-17A monoclonal antibody treatment of wild-type mice resulted in decreased tumor size in the cecum. Model tumor-antigen-specific interferon-γ production was not modified in draining mesenteric lymph node cells in the absence or after neutralization of IL-17A. All the results suggest that constitutive expression of IL-17A in intestine enhances tumor growth, and anti-IL-17A antibody treatment is a candidate of a new anti-tumor immunotherapy against intestinal tumors.

The therapeutic effects of R8-liposome-BCG-CWS on BBN-induced rat urinary bladder carcinoma.

BACKGROUND: The present gold standard for bladder cancer is Mycobacterium bovis bacillus Calmette-Guérin (BCG) immunotherapy, but serious side-effects are common. We previously reported that C3H/HeN mice vaccinated with a mixture of MBT-2 cells and artificial BCG, octaarginine-modified liposomes incorporating the cell wall of BCG (R8-liposome-BCG-CW), significantly inhibited growth of R8-liposome-BCG-CW pretreated MBT-2 cells. Our aim was to determine if a non-live bacterial agent could be as efficacious as live BCG in a model of bladder cancer. We investigated the suppressive effect of liposome-incorporating cell wall skeleton (BCG-CWS) on N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced urinary bladder carcinogenesis in rats. MATERIALS AND METHODS: F344 rats were fed with BBN and sodium ascorbate for 8 weeks, after which all rats were confirmed to have excreted atypical epithelial cells in the urine. Rats were administered BCG-CW(1.0 mg/rat) or R8-liposome-BCG-CWS (0.1 or 1.0 mg/rat) intravesically once/week for 8 weeks from week 28 to 35 of the experimental protocol. RESULTS: Rats receiving R8-liposome-BCG-CWS intravesically showed significantly inhibited numbers of tumors, especially those of simple hyperplasia, in comparison with the control rats. CONCLUSION: R8-liposome-BCG-CWS administration had inhibitory effects on rat bladder carcinogenesis. These results may indicate a novel adoptive immunotherapy against bladder cancers.

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.

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.

Suppressed induction of mycobacterial antigen-specific Th1-type CD4+ T cells in the lung after pulmonary mycobacterial infection.

Although the importance of T(h)1-type immune response in protection against mycobacterial infection is well recognized, its regulatory mechanism in the Mycobacterium tuberculosis (Mtb)-infected lung is not well characterized. To address this issue, we analyzed kinetics of induction of mycobacterial antigen-specific CD4(+) T(h)1 T cells after mycobacterial infection in P25 TCR-transgenic (Tg) mice which express TCR alpha and beta chains from a mycobacterial Ag85B-specific MHC class II A(b)-restricted CD4(+) T-cell clone. To supply normal regulatory T-cell repertoire, we transferred normal spleen T cells into the P25 TCR-Tg mice before infection. High dose subcutaneous infection with Mtb or Mycobacterium bovis bacillus Calmette-Guérin (BCG) induced P25 TCR-Tg CD4(+) T(h)1 cells within a week. In contrast, high-dose Mtb or BCG infection into the lung failed to induce P25 TCR-Tg CD4(+) T(h)1 cells at the early stage of the infection. Furthermore, low-dose Mtb infection into the lung induced P25 TCR-Tg CD4(+) T(h)1 cells on day 21 in the mediastinal lymph node but not in the lung. IL-10 was partially involved in the suppression of T(h)1 induction in the lung because pretreatment of mice with anti-IL-10 antibody resulted in increase of P25 TCR-Tg CD4(+) T(h)1 cells in the Mtb-infected lung on day 21 of the infection, whereas neutralization of transforming growth factor-beta, another important suppressive cytokine in the lung, showed no effects on the T(h)1 induction. Our data suggest that induction of anti-mycobacterial CD4(+) T(h)1 cells is suppressed in the mycobacteria-infected lung partially by IL-10.

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.

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.