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Miyata T.,University of Ryukyus | Harakuni T.,University of Ryukyus | Tsuboi T.,Ehime University | Sattabongkot J.,Armed Forces Research Institute of Medical science | And 6 more authors.
Infection and Immunity | Year: 2010

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. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source


Yamamoto S.,Japan BCG Laboratory
Kekkaku | Year: 2010

A potent immunostimulatory effect of DNA containing an unmethylated CpG motif was found in the course of research on water-soluble components from BCG possessing antitumor activity. Because such CpG motifs are relatively common in bacterial DNA, but rare in mammalian animal and plant DNA, they may be evolutionary adaptation augmenting innate immunity, most likely in response to pathogens that replicate within the host cells, such as viruses and intracellular bacteria. Microbial infection induces innate immunity by triggering pattern-recognition system. The infected cells produce proinflammatory cytokines that directly combat microbial invaders and express costimulating surface molecules, which develop adaptive immunity by inducing distinct T cell differentiation. Bacterial DNA with unmethylated CpG-DNA stimulates vertebrate immature immune cells to induce maturation and to produce Th1 cytokines as well as TNF-α. Therefore, CpG-DNA functions as an adjuvant for regulating the initiation of Th1 differentiation. DNA vaccine including genes encoding mycobacterial proteins either MPB64 or HSP65 was assessed the ability to prevent the growth of bacilli in the lungs and spleens of guinea pigs after pulmonary challenge of virulent Mycobacterium tuberculosis H37Rv. Immunization with two constructs such as MPB64 and HSP65 elicited protective responses compared to a vector control or saline control. The roles of immunostimulatory CpG motifs in DNA vaccine developments and therapeutic applications have been discussed. Source


Fujiwara N.,Osaka City University | Porcelli S.A.,Yeshiva University | Naka T.,Osaka City University | Naka T.,MBR Co. | And 9 more authors.
Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids | Year: 2013

Sphingobacterium spiritivorum has five unusual sphingophospholipids (SPLs). Our previous study determined the complete chemical structures of these SPLs. The compositions of the long-chain bases/fatty acids in the ceramide portion, isoheptadecasphingosine/isopentadecanoate or isoheptadecasphingosine/2-hydroxy isopentadecanoate, are characteristic. The immune response against bacterial lipid components is considered to play important roles in microbial infections. It is reported that several bacterial sphingolipids composed of ceramide are recognized by CD1-restricted T and NKT cells and that a non-peptide antigen is recognized by γδ T cells. In this study, we demonstrated that these bacterial SPLs activated murine bone marrow macrophages (BMMs) via Toll-like receptor (TLR) 4 but not TLR2, although they slightly activated CD1d-restricted NKT and γδT cells. Interestingly, this TLR 4-recognition pathway of bacterial SPLs involves the fatty acid composition of ceramide in addition to the sugar moiety. A non-hydroxy fatty acid composed of ceramide was necessary to activate murine BMMs. The bacterial survival was significantly higher in TLR4-KO mice than in TLR2-KO and wild-type mice. The results indicate that activation of the TLR4-dependent pathway of BMMs by SPLs induced an innate immune response and contributed to bacterial clearance. Source


Sato H.,Hokkaido University | Jing C.,Hokkaido University | Isshiki M.,Hokkaido University | Matsuo K.,Japan BCG Laboratory | And 5 more authors.
Vaccine | Year: 2013

We compared the effect of the very strong pSFJ1-10 and moderately strong p7.5 promoters on the immunogenicity and pathogenicity of the replication-competent vaccinia virus (VV) LC16m8δ (m8δ) vector harboring the SIV gag gene in a vaccination regimen consisting of a recombinant BCG-SIVGag (rBCG-SIVGag) prime followed by a recombinant vaccinia boost. m8δ/pSFJ/SIVGag synthesized more Gag protein than m8δ/p7.5/SIVGag but replicated less efficiently in vitro. In addition, m8δ/pSFJ/SIVGag was less pathogenic and elicited Gag-specific IFN-γ+, CD107a+, CD8+ cells more efficiently than m8δ/p7.5/SIVGag. Vaccination by this regimen elicited long-lasting Gag-specific CD8+ T cells, the majority of which showed a CCR7- phenotype at over 8 weeks post-boost. Tetramer staining analyses revealed maintenance of Gag specific tetramer+, CD62L-, CD8+ T cells for long time in vaccinated mice. However, Gag expression increased the neurotoxicity of the vaccinia vector, indicating the necessity of safety testing for each recombinant VV. We propose that this recombinant BCG prime-m8δ/pSFJ/HIVGag boost regimen would be a promising vaccination procedure for preventing HIV infection. © 2013 Elsevier Ltd. Source


Saitoh T.,Kitasato University | Yano I.,Japan BCG Laboratory | Kumazawa Y.,Iwaki Meisei University | Kumazawa Y.,Vino Science Japan Inc. | Takimoto H.,Kitasato University
Immunopharmacology and Immunotoxicology | Year: 2012

We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6′-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung. © 2012 Informa Healthcare USA, Inc. Source

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