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Master's Dissertation
DOI
10.11606/D.60.2008.tde-31072009-083709
Document
Author
Full name
Carolina Nunes Costa Corgozinho
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2008
Supervisor
Committee
Bentley, Maria Vitoria Lopes Badra (President)
Castelo, Arlete Aparecida Martins Coelho
Freitas, Luis Alexandre Pedro de
Title in Portuguese
Desenvolvimento de vacina baseada em sistema de liberação sustentada contendo proteína recombinante
Keywords in Portuguese
Bm86
Microesfera
Proteina recombinante
Vacina
Abstract in Portuguese
No Brasil, e em outros paises de clima tropical, os carrapatos se tornaram um enorme problema economico de^$de que a industria do gado se desenvolveu. O carrapato Boophilus microplus, um dos artropodes mais importantes na veterinaria, causa efeitos direto, como suc,cao de sangue, e indireto, como a transmissao de uma grande variedade de patogenos que normalmente resulta em infec,c~es letais. As vacinas genicas contendo o antigeno Bm86, uma proteina ligada a membrana do intestino do carrapato B. microplus, representam uma alternativa atrativa aos acaricidas para controlar as infesta~oes por carrapatos em contrapartida aos inconvenientes produtos quimicos. Devido sua administra,cao ser feita em 4 doses no primeiro ano, seguida de refor,cos a cada seis meses, estas formula,coes vacinais nao s3c adequadas para paises com cria,cao extensiva de gado, como no Brasil. Visando uma libera~ao sustentada do antigeno Bm86, neste trabalho desenvolveuse uma vacina de dose unica baseada em microesferas polimericas. Para obter o padrao de liberac,ao desejavel, diferentes formula,coes e parametros de processo foram variados, como a composi,cao do polimero, a taxa entre os monomeros ^Uacido latico:acido glicolico" e o tamanho das microparticulas. As formula,coes foram preparadas pelo metodo de emulsao multipla e evapora,cao do solvente. A formula~ao que melhor se enquadrou nos objetivos da vacina de dose unica foi preparada com PLGA 75:25, solu,cao 3% de PVA como estabilizante, agita,cao de 11000 rpm para forma,cao da emulsao primaria e de 800 rpm para forma,cao da emulsao multipla e evapora,cao do solvente. As particulas assim obtidas apresentaram um tamanho medio de 25 ,um, uma taxa de encapsula,cao maior que 90% e aproximadamente 50% da proteina foi liberada in vitro em 60 dias. Analises por SDS-PAGE e Westem Bloning revelaram que a proteina se manteve integra apos encapsula,cao. Os resultados da avalia,cao da imunogenicidade em bovinos mostraram que a formula,cao baseada em microesferas polimericas biodegradaveis e habil a conseguir, com uma unica dose, uma resposta imune similar aquela conseguida com tres doses das formula,coes convencionais da vacina de Bm86.
Title in English
Development of vaccine based system of sustained release containing recombinant protein
Keywords in English
Bm86
Microspheres
Recombinant protein
Vaccine
Abstract in English
In Brazil, and in others tropical countries, the ticks have become a huge economic problem since the industry of livestock has developed. Ticks and tick-borne diseases affect animal and human health and are the cause of significant economic losses. The cattle tick Boophilus microplus is one of the most important arthropods in veterinary. This tick species causes both direct effects, such as blood sucking, and indirect effects, such as transmission of a wide variety of pathogens, which usually result in lethal infections. The gene vaccines based on Bm86 antigen, a midgut membrane-bound protein of the cattle tick B. microplus, represent a good alternative to control tick infestations, compared to chemicals. However, due to these vaccine formulations need 4 doses over the first year with booster at each 6 months to be effective, they are not suitable for countries with extensive cattle raising, like Brazil. Aiming a sustained release of Bm86 antigen, in this work we developed a single shot vaccine based on Bm86 loaded polymeric microspheres. In order to obtain desired release patterns, different formulations and processing parameters were varied, for example, the composition of the polymer, the monomer ratio lactic acid:glycolic acid and the size of the microparticles. The formulations were prepared by solvent evaporation method based on double emulsion. The formulation that presented better result as single shot vaccine was prepared with PLGA 75:25, solution 3% of PVA as stabilizer, agitation of 11000 rpm to form the primary emulsion and 800 rpm to obtain the double emulsion and solvent evaporation. The particles thus obtained presented an average size of 25 m, encapsulation ratio greater than 90% and approximately 50% of the protein was released in vitro in 60 days. Analysis by SDSPAGE and Western Blot showed that the integrity of the protein remained after encapsulation. The immunogenic studies showed that the formulation based onbiodegradable polymeric microspheres is able to elicit, with a single dose, an immune response and protection similar to that attained with 3 doses of conventional Bm86 vaccine formulations.
 
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Dissertacao.pdf (608.89 Kbytes)
Publishing Date
2009-09-25
 
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