Geraldo R. G. Armôa

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

ABSTRACT 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.

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

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.

Kinetoplastid membrane protein-11 is present in promastigotes and amastigotes of Leishmania amazonensis and its surface expression increases during metacyclogenesis

Kinetoplastid membrane protein-11 (KMP-11), a protein present in all kinetoplastid protozoa, is considered a potential candidate for a leishmaniasis vaccine. A suitable leishmaniasis vaccine candidate molecule must be expressed in amastigotes, the infective stage for mammals. However, the expression of KMP-11 in Leishmania amastigotes has been a subject of controversy. We evaluated the expression of this molecule in logarithmic and stationary growth phase promastigotes, as well as in amastigotes, of Leishmania amazonensis by immunoblotting, flow cytometry and immunocytochemistry, using a monoclonal antibody against KMP-11. We found that KMP-11 is present in promastigotes and amastigotes. In both stages, the protein was found in association with membrane structures (at the cell surface, flagellar pocket and intracellular vesicles). More importantly, its surface expression is higher in amastigotes than in promastigotes and increases during metacyclogenesis. The increased expression of KMP-11 in metacyclic promastigotes, and especially in amastigotes, indicates a role for this molecule in the parasite relationship with the mammalian host. The presence of this molecule in amastigotes is consistent with the previously demonstrated immunoprotective capacity of vaccine prototypes based on the KMP-11-coding gene and the presence of humoral and cellular immune responses to KMP-11 in Leishmania-infected humans and animals.

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

UNLABELLED MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic 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 are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. AVAILABILITY MamMiBase is available at http://www.mammibase.lncc.br

A comparison of the T cell delayed-type hypersensitivity epitopes of the 19-kD antigens from Mycobacterium tuberculosis and Myco. intracellulare using overlapping synthetic peptides.

Mycobacterial disease remains a serious international public health concern. Improved methods to rapidly and specifically detect mycobacterial infections would greatly enhance clinical management of these diseases. To define species‐specific T cell epitopes that may be useful for the immunodiagnosis of mycobacterial infections, polymerized synthetic peptides from the 19‐kD Mycobacterium tuberculosis and Myco. intracellulare protein homologues were tested in guinea pig DTH assays. Five Myco. tuberculosis and eight Myco. intracellulare peptides evoked skin test responses. Although all of the active Myco. tuberculosis and seven of the Myco. intracellulare peptides elicited non‐specific DTH reactions, the peptide IN13 induced a Myco intracellulare‐specific skin test reaction, and thus represents a specific Myco intracellulare T cell DTH epitope. This result suggests that the development of monospecific peptide‐based immunodiagnostic reagents may be feasible for future clinical use.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15 cells using the expression vector pQE-30. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

A highly immunogenic putative Mycobacterium kansasii lipoprotein

The resurgence of tuberculosis, the emergence of multiple drug resistant tuberculosis, and the increasing prevalence of mycobacterial disease in AIDS patients have increased the importance of defining new mycobacterial antigens that can be utilized in the development of improved diagnostic reagents and more effective vaccines. In this report, a highly immunogenic Mycobacterium kansasii protein (MK35) and the gene encoding this antigen were characterized. MK35 gene probes reacted with genomic DNA from M. avium, M. bovis BCG, M. intracellulare and M. tuberculosis but not with DNA isolated from nine other mycobacterial species. Nucleotide sequence analysis showed that the MK35 gene encodes a 26 kDa protein which contains a consensus bacterial lipoprotein processing sequence. In addition, detergent-phase separation studies strongly suggested that MK35 is a lipoprotein. Skin test assays demonstrated that MK35 elicited a strong response in guinea pigs sensitized with M. kansasii but did not react in M. tuberculosis-sensitized guinea pigs. These results further suggest that mycobacterial lipoproteins are immunogenic antigens that should be considered in the development of new mycobacterial vaccines and diagnostic reagents.