Deliverables
The Sm14 antigen of Schistosoma mansoni was cloned and expressed in Mycobacterium bovis BCG as a fusion with the Mycobacterium fortuitum beta-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.
We have obtained a strain expressing Sm14 from Schistosoma mansoni using a codon-optimized sm14 gene. This construct increased expression levels 4-fold. This strain induced a cellular Th1-type immune response specific for Sm14. We have also obtained expression of Sm14 in rBCG by a complemented LeuD- BCG auxotroph.
Mycobacterium bovis BCG has been studied as a live vaccine vector with the aim of developing an efficient multivalent vaccine. Most mycobacterial cloning vectors rely on antibiotic resistance genes as selectable markers. We report the use of auxotrophic complementation as a selectable marker. BCG auxotrophic for the amino acid leucine was constructed by knocking out the leuD gene by unmarked homologous recombination. Expression of leuD in a plasmid not only allowed complementation, but also acted as a selectable marker. Removal of the kanamycin resistance gene, still necessary for plasmid manipulation in E. coli, was accomplished by two different methods: restriction enzyme digestion followed by re-ligation before BCG transformation, or by Cre-loxP in vitro recombination mediated by the bacteriophage P1 Cre recombinase. Both methods worked well, however digestion and re-ligation was more efficient resulting in higher transformation efficiency. Stability of the plasmid was evaluated during in vitro and in vivo growth of the recombinant BCG in comparison to selection by antibiotic resistance. The new system was highly stable even during in vivo growth, as the selective pressure is maintained, whereas the conventional vector was unstable in the absence of selective pressure.
We demonstrated the capability of BCG expressing Rap-1 to stimulate an specific immune response against the B.bovis antigen in mice. The specific anti RAP-1 cellular immune response was analyzed measuring gIFN produced by antigen-stimulated splenocytes and the humoral immune response was followed up by Western blot. The groups of mice that received a prime/booster schema showed better specific immnue response. Moreover and in accordance to stability data the highest gIFN mesurements were observed in animals vaccinated with the most stable rBCG(pMip12:rap1) and boostered with rRap-1. The results are in accordance to previous publications showing a specific T limphocyte priming by BCG and its usefulnes as part of a combined vaccine strategy in order to induce long lasting humoral an cellular inmune response.
As a second step we perfomed a bovine inoculation assay of rBCG to test for protection against Babesia bovis in cattle as there is no experimental laboratory model for bovine babesiosis. Male Holstein calves of 200kg were used for evaluation of the rBCG. Five groups of 5 calves were employed for the vaccination trial.
Two groups were injected with rBCG-Rap, the three other were the control groups injected with BCG, Babesia bovis R1A (B. bovis attenuated strain) and non vaccinated animals respectively. A biopsy of the subscapular lymphonode of eight animals was performed the day after inoculation for identification of BCG by PCR and in vitro culture of the sample. Although all of the samples rendered negative results for the in vitro culture, 40% were PCR positive using specific primers for Mycobacteria .
Antibody response against rRap-1 in sera of immunized cattle were quentified by enzyme- lynked immunosorbent assays (ELISA) using rRap-1. No anti rRap-1 antibodies were detected in any of the groups until 22 days after challenge. At day 36 after challenge a slight incresase of antibody titer was detected for the BCG innoculated group and even higher in the rBCG- pMIP12-RAP-1 innoculated group.
Whether or not it is an specifici response will be confirmed by Immunoflurescence assay and Western blotting. At day 45 after innoculation production of bovine g-IFN in complete blood sample was detected using the commercial available kit. No specific stimulation was detected at that time point in any of the tested groups. Although no protection was obtained a slight improvement in clinical parameters post challenge in the 3 innoculated groups were observed in comparison with the control group. However most likely reflects a nonspecific effect associated with BCG.
This study represents the first application of rBCG technology to the development of candidate vaccines for the control of a veterinary protozoan pathogen.
The results obtained so far provide encouragement for continued research into the development of rBCG as a convenient and practical means through which to control bovine infections with B. bovis.
Current immunodiagnostic tests for tuberculosis (TB) do not allow to differentiate between vaccination with Mycobacterium bovis BCG or infection with M. bovis or M. tuberculosis. This is a major drawback for the use of BCG as a vector to develop new candidate vaccines both against TB and other pathogens. Thus the development of differential diagnostics is a major issue. As a proof of concept, we have deleted the genes encoding two well-characterized immunodominant antigens, MPB70 and MPB83 from M. bovis BCG. Since we feared that these antigens might be of high importance for BCG persistence, we decided to construct our mutant strain in a naturally low-producing strain i.e. the BCG Pasteur strain 1173P2. The double knock-out strain did not show impaired persistence in mice after intravenous inoculation as compared to wildtype BCG. Moreover, both strains induced similar protection against an aerosol challenge with virulent M. tuberculosis H37Rv in mice. These data indicate that MPB70 and 83 antigens are not fully required for inducing protection by BCG vaccination in preclinical models. Thus, these two antigens might become good candidates to dicriminate BCG vaccination from infection with bacteria from the M. tuberculosis complex.
We have generated recombinant BCG strains that express the Schistosoma mansoni antigen, Sm14, in fusion with the beta-lactamase protein and signal sequence. Expression was obtained using a plasmid containing the upregulated beta-lactamase promoter, pBlaF. We have expressed Sm14 in BCG from the native gene. These rBCG-Sm14 strains induced a cellular Th1-type immune response, demonstrated by IFN-g production of cultured splenocytes upon stimulation with rSm14.
Genes encoding the antigens mpb63 and mpb83 were eliminated from BCG Pasteur by allelic exchange to generate BCG-delta strains.
These BCG-delta strains may be useful as alternative BCG vaccine strains.
Vaccination with these strains will generate immunity against tuberculosis but complementary diagnostic tests that target the mpb63 and mpb83 antigens may be used to distinguish BCG vaccination from natural infection with M. bovis or M. tuberculosis.
We have generated two recombinant Mycobacterium bovis BCG expressing the MSP1a antigen of Anaplasma marginale. The msp1a gene was amplified by PCR and cloned into the mycobacterial expression vectors pUS2000 and pMIP12. Stability of the vector was demosntrated in vitro and inside bovine cells. Expression of the recombinant antigen was confirmed by Western blotting using a monoclonal antibody against the Msp1 angigen.
Two recombinant BCG (rBCG) expressing the antigen RAP-1 from Babesia bovis. The rBCG contains expression vectors named pUS2001 and pMIP12 were a fragment comprising the rap-1 gene devoid of signal sequence was inserted. Both constructs have different promoters, ribosome binding sites and heterologous signal sequences. Western blot using specific anti RAP-1 monoclonal antibodies demonstrated the expression of rRAP-1 in BCG. Subcellular localization experiments indicated that rRAP-1. The construction in pMIP12 demonstrated to be stable in vitro and in vivo.
Humoral and cellular immune responses of mice inoculated with recombinant Mycobacterium bovis BCG expressing the MSP1a antigen of Anaplasma marginale were evaluated. The msp1a gene was amplified by PCR and cloned into the mycobacterial expression vectors pUS2000 and pMIP12. Immunization of isogenic BALB/c mice with the rBCG/pUS2000-msp1a construct induced significant seroconversion to MSP1a (p<0,001), which was 26 times above pre-immunization levels at day 63 post initial immunization and which remained stable for the duration of the experiment (6 months). In contrast, rBCG/pMIP12-msp1a induced seroconversion at a level of 6 times above pre-immunization values, which peaked at day 63. Western blot analysis showed that sera derived from mice vaccinated with either rBCG construct recognized both native and recombinant forms of A. marginale MSP1a. In contrast to the humoral response data, immunization with rBCG/pMIP12-msp1a was found to induce a markedly stronger cellular response than that recorded for BCG/pUS2000-msp1a. These results clearly demonstrated the immunogenicity of rBCG expressing the MSP1a antigen and suggested that the immune responses were influenced by the level of antigen expression. Recombinant BCG expressing MSP1a constitutes a potential strategy of immunization against bovine anaplasmosis.
Prior to testing candidate vaccines in cattle it is necessary to characterise the rBCG strains in terms of their ability to survive in vivo, express recombinant antigen and stimulate an immune response. In the context of the current project it was considered essential to develop a model system with which to rapidly, reproducibly and meaningfully assess the behaviour of candidate rBCG constructs in the bovine intracellular environment. A method was established for the use of cultured bovine PBMCs so that rBCG vaccine candidate constructs most likely to function in cattle could be selected.
I recombinant BCG strain was generated, rBCG-leuD, in which the leuD gene was deleted. The strain was therefore an auxotroph for the amino acid leucine. This allowed the use of plasmid vectors that provide the leuD gene in trans that complement the leuD mutation. This complementation allows the stable expression of foreign genes in BCG in the absence of antibiotic resistance markers.
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