Anaplasma marginale : análise da variabilidade do gene msp1 a e avaliação imunogênica da vacina de DNA contendo genes para MSP1a, MSP1b e MSP5 em camundongos BALB/c
Anaplasma marginale is an important tick-transmitted rickettsial pathogen of cattle that invades and multiplies within erythrocytes, causing severe hemolytic anemia during acute infection. Immunization with purified outer membrane proteins (OMP) induces protection against acute A. marginale disease. Of 21 OMP described six major surface proteins (MSPs 1a, 1b, 2-5) have been well-characterized. The complex MSP1 (MSP1a and 1b) is an adhesin for bovine erythrocyte, and MSP1a vary in size and sequence due to the number of tandem 28-29-amino acid repeats. DNA vaccine against anaplasmosis has been investigated using the msp1a and msp1? genes, and the results related cellular and humoral response in mice and cattle. Immunization with DNA plasmids encoding antigens of interest represents a novel and promising method in vaccine research and development. Multiepitope DNA vaccine is a new experience to increase immunogenicity and protection for vaccinated animal as compared in single epitope. The objectives of this study were to analyze the variability of the msp1a gene of A. marginale strains from Parana State, evaluate the capacity of the recombinant plasmids to express MSP1a, MSP1b, and MSP5 in eukaryotic cells, and evaluate the immunogenicity of BALB/c mice immunized with these DNA vaccines encoding MSPs of A. marginale PR1 strain individually or in association. The analysis of the msp1a gene identified the presence of six, five and three tandem repeats in PR1, PR2 PR3, respectively; however, the region of MSP1a responsible for immunogenicity was conserved. The plasmids pcDNA-msp1?, pcDNA-msp1? and pcDNA-msp5, which encode the MSPs genes under the control of cytomegalovirus enhancer/promoter and intron A, were constructed, multiplied in TOP10 E. coli and purified. Expression of MSP1a, MSP1b, and MSP5 in vitro was performed into Vero cells using lipofectamine 2000, following Indirect Immunofluorescen Assay (IFA) using monoclonal antibodies. Seven experimental groups of mice were immunized to evaluate the production of whole IgG and to determinate IgG1 and IgG2a isotype: G1-100 ml PBS; G2-100 mg empty vector; G3-100 mg A. marginale initial bodies + Freund´s adjuvant; G4-100 mg pcDNA-msp1a; G5-100 mg pcDNA-msp1b; G6-100 mg pcDNA-msp5; and G7-pool of recombinant plasmids (33 mg for each). Three weeks after the last immunization, mice were sacrified to evaluate spleen cells proliferation. Vero cells transfected with recombinants plasmids reacted with specific monoclonal antibodies, demonstrating the expression of msp genes. Specific IgG against MSP1a and MSP5 were detected in either by ELISA and Western blot. The groups that received pcDNA-msp1a and pcDNA-msp5 exhibited predominance for IgG2a production and splenocytes proliferation, suggesting that these recombinant plasmids are good candidates for elicited T helper 1 immune response. The association of three recombinant plasmids (pcDNA-msp1a, pcDNA-msp1b and pcDNA-msp5) used in the immunization of mice induced high antibodies response by ELISA and reacted with all recombinant proteins (rMSP1a, rMSP1b, and rMSP5) of A. marginale by Western blot. Also, the combination of plasmids provide strong lymphoproliferation (SI = 12,2), whereas the genes to MSP1a provide significant splenocytes (SI = 2,6) and the genes to MSP1b and MSP5 did not provide significant proliferation (SI<2). The results showed no suppression when the recombinant plasmids were taken in association, and demonstrated that they can generate significant T-cell lymphocyte. Thus, the immunization in association of recombinant plasmids encoding MSPs can be an effective strategy for immunoprofilaxy of anaplasmosis.
Advisor:Odilon Vidotto; Marilda Carlos Vidotto [Orientador].; Amauri A. Alfieri; João Luis Garcia
Source Type:Master's Thesis
Cattle - Diseases
Date of Publication:04/27/2007