Desarrollo de una vacuna contra el virus ISA en base a proteĆnas recombinantes
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Fecha
2012
Autores
Profesor/a GuĆa
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Idioma
es
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Universidad AndrƩs Bello
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Licencia CC
Licencia CC
Resumen
La anemia infecciosa del salmĆ³n (ISA) es una enfermedad viral que afecta a peces, especialmente a especies en cultivo como el salmĆ³n del AtlĆ”ntico (Salmo salar). Es causada por el virus ISA que pertenece al gĆ©nero lsavirus de la familia Orthomyxoviridae y es considerado un patĆ³geno emergente que ha causado importantes pĆ©rdidas econĆ³micas a la industria salmonera chilena. En este trabajo se desarrollĆ³ un primer prototipo de vacuna recombinante contra el virus y se estudiĆ³ sus propiedades inmunogĆ©nicas y capacidad de protecciĆ³n. Genes de las cuatro principales proteĆnas estructurales del virus fueron diseƱados y sintetizados con una secuencia optimizada para su expresiĆ³n en bacterias. Mediante esta estrategia, se expresĆ³ en E. colĆ, la proteĆna de matriz (M1 ), un fragmento de la mitad amino terminal de la nucleoproteĆna (N-NP), fragmentos de las mitades amino y carboxilo terminal de la proteĆna hemaglutinina-esterasa (N-HE y C-HE, respectivamente), un fragmento de la mitad amino terminal de la proteĆna de fusiĆ³n (N-FP) y otro fragmento (FP3) que abarca dominios de la regiĆ³n amino y carboxilo de la proteĆna de fusiĆ³n. A excepciĆ³n de la proteĆna de matriz que se expresa tanto en su forma denaturada como en forma nativa, las demĆ”s proteĆnas se producen en la forma de cuerpos de inclusiĆ³n. Mezclas de las proteĆnas recombinantes fueron formuladas y sus propiedades inmunogĆ©nicas se determinaron en conejos y en salmones. Los anticuerpos policlonales desarrollados en conejos fueron evaluados por ELISA, detectĆ”ndose tĆtulos de anticuerpos altos contra las proteĆnas N-NP y M1. Luego se evaluĆ³ la capacidad de estos anticuerpos de neutralizar el efecto citopĆ”tico del virus en cĆ©lulas en cultivo, detectĆ”ndose un grado de neutralizaciĆ³n evidente con el suero de un conejo inmunizado con la mezcla de las proteĆnas recombinantes NNP, NFP2, NHE, CHE y M1 desnaturadas, muy semejante a la neutralizaciĆ³n observada con el suero policlonal contra la proteĆna M1 nativa. Este resultado es concordante con la respuesta inmune humoral de salmones inmunizados con mezcla de las proteĆnas recombinantes NNP, NFP2, NHE, CHE y M1, las que presentan una alta reactividad contra la proteĆna M1. Por otra parte, al analizar la respuesta inmune de peces sobrevivientes a un desafĆo con virus ISA, se detectan tĆtulos de anticuerpos sorprendentemente mĆ”s altos contra la NP que con el resto de las proteĆnas. La alta inmunogenicidad de NP y M1 hacen de ellas interesantes candidatos para vacuna. Basado en estos resultados se evaluĆ³ en salmones la eficacia de protecciĆ³n de vacunas basadas en mezclas de las proteĆnas recombinantes frente a un desafĆo con el virus ISA. Los resultados obtenidos indican que formulaciones con dosis de 5ug o 1 0ug de cada uno de los fragmentos de las 4 proteĆnas estructurales del virus ISA tienen un efecto de protecciĆ³n moderado frente al virus ISA con un porcentaje relativo de sobrevivencia (RPS) entre 60% y 64%. Sin embargo, un interesante efecto potenciador se produce al mezclar las recombinantes con el virus inactivado que forma parte de una vacuna cuĆ”druple incrementando la eficacia de la vacuna inactivada de 57% a 75%.
The infectious salman anaemia (ISA) is a viral disease that affects farmed fish, in particular the Atlantic salman (Salmo salar) amon9 the cultured species. The etiolo9ical a9ent is the ISA virus that belon9s to the 9enus lsavirus of the Orthomyxoviridae family. lt is considered an emer9ent patho9en that has caused si9nificant economic losses to the Chilean salman industry. In this work, a prototype recombinant vaccine was developed a9ainst the virus and different assays were performed to study its immuno9enic and protective properties. To accomplish this, the sequences encodin9 the four majar structural proteins of the virus were desi9ned to optimize their expression in bacteria and were chemically synthesized. By this strate9y it was possible to express in E. coli, the matrix protein (M1), the amino-terminal half of the nucleoprotein (N-NP), the amino-terminal and carboxyl-terminal fra9ments of the hema99lutinin-esterase (N-HE and C-CHE, respectively), the amino-terminal half of the fusion protein (N-Fp), and another fra9ment (FP3) that includes domains of the amino and carboxyl re9ion of the fusion protein. The recombinant proteins were obtained in the form of inclusion bodies, with the exception of the matrix protein that was expressed in both, native and denatured forms. Different mixtures of the recombinant proteins were formulated and their immuno9enic properties were analyzed in salman and rabbits. Hi9h titers of antibodies a9ainst N-NP and M 1 proteins were detected in rabbits by ELISA. The rabbit polyclonal antibodies directed a9ainst the mixture of denatured proteins NNP, NFP2, NHE, CHE and M1 has the ability to partially neutralize the cytopathic effect of the virus on cell culture. A similar neutralization capability was observed with a polyclonal antiserum a9ainst native protein M1. This result is consistent with the hi9h humoral immune response detected a9ainst M1 in salman immunized with a mixture of the recombinant proteins NNP, NFP2, NHE, CHE and M1. On the other hand, when the immune response of the survivin9 fish of challen9e was analyzed, surprisin9ly hi9her titers a9ainst NP were detected in comparison to the rest of the recombinant proteins. This hi9h immuno9enicity of NP and M1 , make them interestin9 candidates for a vaccine. Based on these results, the efficacy of protection of vaccine formulations containin9 mixtures of the recombinant proteins were evaluated in a challen9e a9ainst ISA virus. The results indicate that formulations with doses of 5 1-19 or 1 O 1-19 of each of the fra9ments of the four virus ISA structural proteins have a moderate protective effect a9ainst ISA virus with a relative percent survival (RPS) of 60% to 64%. Moreover, an enhancement of the efficacy of protection of a multivalent vaccine containin9 inactivated ISA virus particles is observed when combined with the mixture of recombinant ISA V anti9ens, increasin9 the efficacy of protection of the inactivated vaccine from 57% to 75%.
The infectious salman anaemia (ISA) is a viral disease that affects farmed fish, in particular the Atlantic salman (Salmo salar) amon9 the cultured species. The etiolo9ical a9ent is the ISA virus that belon9s to the 9enus lsavirus of the Orthomyxoviridae family. lt is considered an emer9ent patho9en that has caused si9nificant economic losses to the Chilean salman industry. In this work, a prototype recombinant vaccine was developed a9ainst the virus and different assays were performed to study its immuno9enic and protective properties. To accomplish this, the sequences encodin9 the four majar structural proteins of the virus were desi9ned to optimize their expression in bacteria and were chemically synthesized. By this strate9y it was possible to express in E. coli, the matrix protein (M1), the amino-terminal half of the nucleoprotein (N-NP), the amino-terminal and carboxyl-terminal fra9ments of the hema99lutinin-esterase (N-HE and C-CHE, respectively), the amino-terminal half of the fusion protein (N-Fp), and another fra9ment (FP3) that includes domains of the amino and carboxyl re9ion of the fusion protein. The recombinant proteins were obtained in the form of inclusion bodies, with the exception of the matrix protein that was expressed in both, native and denatured forms. Different mixtures of the recombinant proteins were formulated and their immuno9enic properties were analyzed in salman and rabbits. Hi9h titers of antibodies a9ainst N-NP and M 1 proteins were detected in rabbits by ELISA. The rabbit polyclonal antibodies directed a9ainst the mixture of denatured proteins NNP, NFP2, NHE, CHE and M1 has the ability to partially neutralize the cytopathic effect of the virus on cell culture. A similar neutralization capability was observed with a polyclonal antiserum a9ainst native protein M1. This result is consistent with the hi9h humoral immune response detected a9ainst M1 in salman immunized with a mixture of the recombinant proteins NNP, NFP2, NHE, CHE and M1. On the other hand, when the immune response of the survivin9 fish of challen9e was analyzed, surprisin9ly hi9her titers a9ainst NP were detected in comparison to the rest of the recombinant proteins. This hi9h immuno9enicity of NP and M1 , make them interestin9 candidates for a vaccine. Based on these results, the efficacy of protection of vaccine formulations containin9 mixtures of the recombinant proteins were evaluated in a challen9e a9ainst ISA virus. The results indicate that formulations with doses of 5 1-19 or 1 O 1-19 of each of the fra9ments of the four virus ISA structural proteins have a moderate protective effect a9ainst ISA virus with a relative percent survival (RPS) of 60% to 64%. Moreover, an enhancement of the efficacy of protection of a multivalent vaccine containin9 inactivated ISA virus particles is observed when combined with the mixture of recombinant ISA V anti9ens, increasin9 the efficacy of protection of the inactivated vaccine from 57% to 75%.
Notas
Tesis (Doctor en BiotecnologĆa)
Palabras clave
SalmĆ³n, Enfermedades, Industria del SalmĆ³n, Vacunas, Chile, Virus ISA