Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

Submitted by Sandra Infurna ([email protected]) on 2018-02-22T12:01:16Z No. of bitstreams: 1 geraldo_armoa_etal_IOC_2017.pdf: 715759 bytes, checksum: 233c3857f976dd336cedb7de9789c154 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2018-02-22T12:12:49Z (GMT) No. of bitstreams: 1 geraldo_armoa_etal_IOC_2017.pdf: 715759 bytes, checksum: 233c3857f976dd336cedb7de9789c154 (MD5)Made available in DSpace on 2018-02-22T12:12:49Z (GMT). No. of bitstreams: 1 geraldo_armoa_etal_IOC_2017.pdf: 715759 bytes, checksum: 233c3857f976dd336cedb7de9789c154 (MD5) Previous issue date: 2017Universidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilUniversidade Federal de Pelotas. Campus Universitário. Núcleo de Biotecnologia. Pelotas, RS, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ. Brasil.Universidade Federal Rural do Rio de Janeiro. Instituto de Veterinária. Seropédica, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Rio de Janeiro, RJ, Brasil.Universidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilUniversidade do Estado do Rio de Janeiro. Faculdade de Ciências Médicas. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilDiphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti- diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtb PW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay

Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics

Artigo disponível em acesso aberto no link da Editora.Submitted by Repositório Arca ([email protected]) on 2019-03-07T16:42:11Z No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5)Approved for entry into archive by monique santos ([email protected]) on 2019-03-08T15:39:22Z (GMT) No. of bitstreams: 2 F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5)Made available in DSpace on 2019-03-08T15:39:22Z (GMT). No. of bitstreams: 2 F83-1481999a.pdf: 399135 bytes, checksum: c78054a1227902e3a657c5fde27c4759 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Universidade Federal de Pelotas. Centro de Biotecnologia. Pelotas, RS, Brasil.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Departamento de Bioquímica e Biologia Molecular. Rio de Janeiro, RJ, Brasil.Universidade de Surrey. Escola de Biológica Ciências. Guildford, Surrey, UK.Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil.Mycobacterium vaccae represents an alternative mycobacterial cloning host that has been largely overlooked to date. The main reason for this may be the reported non-transformability of this species, specifically the so-called Stanford strain (NCTC 11659), with expression vectors that use kanamycin resistance as a selection method. However, this strain can be transformed using hygromycin resistance as an alternative selectable phenotype. The present study has shown that in contrast to previous reports, M. vaccae (ATCC 15483) is capable of being transformed with a range of vectors encoding kanamycin resistance as the selectable marker. Thereafter, the expression of the lacZ reporter gene in M. vaccae, Mycobacterium bovis BCG and Mycobacterium smegmatis mc(2)155 was evaluated using a range of characterized mycobacterial promoter sequences (hsp60, hsp70, PAN, 18kDa and 16S rRNA) cloned in the same promoter probe vector. In general, the promoters showed similar levels of activity in the three species, demonstrating that existing expression systems can readily be employed with M. vaccae (ATCC 15483). This was further confirmed by the observation that M. vaccae was capable of stable, in vitro expression of recombinant S1 subunit of pertussis toxin at levels equivalent to those obtained with BCG and M. smegmatis. Analysis of structural and functional stability of a range of vectors demonstrated that the incidence of instability noted for M. vaccae was lower than that recorded for M. smegmatis. Taken together, the results indicate that M. vaccae is an additional cloning host which may prove useful for specific aspects of mycobacterial biology and provide increased flexibility to the field of recombinant protein technology for mycobacteria

Recombinant Mycobacterium bovis BCG Expressing the Sm14 Antigen of Schistosoma mansoni Protects Mice from Cercarial Challenge

The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum β-lactamase protein under the control of its promoter, pBlaF*; the protein was localized in the bacterial cell wall. The rBCG-Sm14 strain was shown to be relatively stable in cultured murine and bovine monocytes in terms of infectivity, bacterial persistence, and plasmid stability. The immunization of mice with rBCG-Sm14 showed no induction of anti-Sm14 antibodies; however, splenocytes of immunized mice released increased levels of gamma interferon upon stimulation with recombinant Sm14 (rSm14), indicating an induction of a Th1-predominant cellular response against Sm14. Mice immunized with one or two doses of rBCG-Sm14 and challenged with live S. mansoni cercaria showed a 48% reduction in worm burden, which was comparable to that obtained by immunization with three doses of rSm14 purified from Escherichia coli. The data presented here further enhance the status of Sm14 as a promising candidate antigen for the control of schistosomiasis and indicate that a one-dose regimen of rBCG-Sm14 could be considered a convenient means to overcome many of the practical problems associated with the successful implementation of a multiple-dose vaccine schedule in developing countries

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

Summary: MamMibase, the Mammalian Mitochondrial Genome Database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phyloge-netic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that is useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, that are also given by Mammibase. A simple phylogenetic tree (neighborjoining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. Availability: MamMiBase is available at www.mammibase.lncc.br Contact