Virus influenza equina: aislamiento y estudio de genes con potencial para el desarrollo de una vacuna
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Fecha
2013
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Un prototipo de una vacuna contra el virus de influenza equina (VIE), se desarrolló
escogiéndose como antígenos candidatos la hemaglutinina (HA) y la
neuroaminidasa (NA) , proteínas elegidas por ser las más inmunogénicas y de
mayor variabilidad. También se incluyó la nucleoproteína (NP), por ser una
proteína interna conservada que caracteriza a todos los virus influenza del tipo A.
En una primera aproximación , se diseñó una vacuna de ADN en base a vectores
que expresan HA, NA y NP de los dos aislados existentes en Chile (A 1-H7N7 y
A2-H3N8). Sueros de ratones inmunizados con esta formulación , dieron, por
E LISA, títulos superiores a 1/1024. Estudios de neutralización del virus en células
MDCK y en huevos embrionados de pollo indicaron que estos sueros son
protectivos. En células MDCK se obtuvo neutralización hasta en la dilución 1/1000
y en huevo se obtuvo un índice neutralizante capaz de evitar la infección de un
millón de virus por ml.
También se formuló una vacuna con las proteínas recombinantes HA, NA y NP.
En ratones se obtuvieron sueros con títulos en ELISA de 1/64 cuando se usaron
proteínas del virus A1 y de 1/32 con proteínas del virus A2.
Estos títulos fueron mejorados al hacer una inmunización mixta, es decir, primero
con ADN y seguido de un refuerzo con las proteínas. En este caso, se obtuvieron
títulos de 1/128 para el virus A1 y de 1/64 para el virus A2 y absorbancias
significativamente mayores.
Estas formulaciones fueron evaluadas en cobayos , donde además se comparó
con una vacuna comercial. El análisis por ELISA con el virus completo de los
sueros indica títulos de 1/32 para el virus A 1 y de 1/64 para el virus A2. En el
análisis por ELISA con las proteínas recombinantes , la vacuna de ADN muestra
títulos superiores a 1/512 para HA 1 y 1/1024 para HA2, y la vacuna comercial da
un título de 1/64 comparable a los sueros preinmunes. Por Western blot con los
virus y el líquido alantoídeo, la vacuna de ADN reconoce una proteína cercana a
los 55 kDa que puede corresponder a la NP. El análisis por Western blot con las
proteínas recombinantes muestra que la vacuna de ADN reconoce las 3 proteínas
de cada aislado y la vacuna comercial sólo la HA del virus A 1, repitiéndose lo
observado en ratones.
Análisis de la capacidad neutralizante de los sueros de cobayos en huevos
embrionados indicó índices de neutralización superiores al título del virus , lo que
significa que se pudo neutralizar todo el virus existente. Lo otro destacable es que
la vacuna de ADN fue capaz de neutralizar el aislado nuevo del virus A2L, efecto
que no se observó en la vacuna comercial desarrollada con los mismos aislados
del virus de influenza equina . Lo mismo se observó en ensayos de neutralización
en células MDCK. Con las vacunas recombinantes y mixta no se obtuvo buenos
índices neutralizantes. Análisis de los sueros por el ensayo de inhibición de la hemoaglutinación indican
que la vacuna de ADN presenta los títulos más altos (> de 1 /8000), mejores que la
vacuna comercial (1/2048). En cambio, las vacunas recombinantes y mixta no
presentan actividad en este ensayo.
Habiendo definido la mejor formulación , la dosis apropiada y comprobada la
inocuidad de dicha formulación, se procedió a ensayar la vacuna de ADN en
caballos. Análisis por ELISA con el virus, de sueros de animales inmunizados dió
títulos de 1/512, sin reconocimiento de las proteínas del líquido alantoídeo. Ahora,
si el análisis por ELISA se realiza con las proteínas recombinantes, la vacuna de
ADN da origen a sueros que reconocen las proteínas recombinantes de los
aislados A1 y A2, en cambio la vacuna comercial da origen a sueros que
reconocen sólo las proteínas del aislado A 1. Análisis por Wesfern blof con virus
detecta una banda de aproximadamente 55kDa , observada en cobayos, y si el
análisis se hace por Wesfern blof con las proteínas recombinantes, las seis
proteínas son detectadas.
Los sueros de caballos inmunizados mostraron un efecto protectivo en células
MDCK y en huevos embrionados de pollo. El efecto se observó claramente con el
virus A 1. Debido a que se trabajó con un pool de los sueros y algunos caballos
habían estado en contacto con el virus A2 y A2L, no se observó diferencias
significativas entre los sueros preinmunes y los sueros de los mismos caballos
inmunizados con las distintas formulaciones. Estudios de inhibición de la
hemoaglutinación de sueros individuales de cada caballo, dieron títulos de 1/1024
para el virus A 1, de 1/4096 para A2 con preinmunes de 1/16 de título y de 1/4096
para A2L con título de 1/512 para los preinmunes, los que fueron más altos que
los obtenidos con sueros obtenidos con la vacuna comercial.
Estos resultados permiten concluir que la vacuna de ADN prototipo desarrollada
es capaz de entregar una respuesta inmune humoral medible en todos los
animales de experimentación ensayados y que los sueros correspondientes tienen
un efecto protector en dos modelos distintos de evaluación.
A prototype vaccine against equine influenza virus (EIV) was developed using hemagglutinin (HA) and neuraminidase (NA) as candidate antigens , chosen for their higher immunogenicity and variability. The nucleoprotein (NP) was also included as a conserved interna! protein that characterizes all influenza type A viruses. In a first approach, a DNA vaccine based on vectors expressing HA, NA, NP from the two isolates that exist in Chile (A1-H7N7 and A2-H3N8) was designed. Sera from m ice immunized with this formulation yielded , by ELISA, titers over 1/1024. Studies of virus-neutralizing capacity in MDCK cells and in embryonated chicken eggs indicated that the sera were protective. In MDCK cells, neutralization was achieved with dilutions up to 1/1000 and in eggs, a neutralizing index capable of preventing infection of 1 million viruses per ml. Another vaccine was formulated with recombinant proteins HA, NA, and NP. Mice sera with EL ISA titers of 1/64 were obtained when we u sed virus A 1 proteins and 1/32 with virus A2 proteins. These titers were improved with mixed immunization , i.e. , first with DNA followed by a boost with protein. In this case, the titers obtained were 1/128 for the A 1 virus and 1/64 for the A2 virus and significantly higher absorban ces. These formulations were also assessed by immunization of guinea pigs and compared in parallel to a commercial vaccine. ELISA analysis of whole virus titers indicated 1/32 for the A 1 virus and 1/64 for the A2 virus. ELISA using recombinant proteins determined that DNA vaccination yielded titers of over 1/512 for HA 1 and 1/1024 for HA2, while the commercial vaccine yielded a 1/64 titer, comparable to preimmune sera. In Western blot analysis of allantoic fluid and viruses, the DNA vaccine recognizes a protein of around 55 kDa that might correspond to the NP. Western blot with the recombinant proteins shows that the DNA vaccine recognizes all 6 proteins and the commercial vaccine only detects HA from virus A 1, as observed in m ice. Analysis of the neutralizing capacity of sera from guinea pigs in embryonated eggs indicated neutralization indexes above the viral titer, which means that all the virus present was neutralized. Also noteworthy is that the DNA vaccine was able to neutralize the new virus isolate A2L, an effect not observed in the commercial vaccine. The same was observed in neutralization assays in MDCK cells. Recombinant and mixed vaccines did not achieve acceptable neutralization indexes. Analysis of the sera by the hemagglutination inhibition assay indicated that the DNA vaccine had the highest titers (> 1 /8000), better than the commercial vaccine (1/2048). In contrast, recombinant and mixed vaccines were negative in this assay. Having defined the best formulation, the appropriate dosage and safety of the tested formulation, we proceeded to test the DNA vaccine in horses. ELISA analysis with the virus of sera from immunized animals resulted in titers of 1/512, without recognition of allantoic fluid proteins. In ELISA done with the recombinant proteins, the DNA vaccine produced serum which recognized recombinant proteins from the A 1 and A2 isolates, whereas the commercial vaccine gives rise to serum which recognizes only proteins from the A 1 isolate. Western blot analysis detects a band at approximately 55kDa , as observed in guinea pigs, and if the analysis is done by Western blot with the virus, all six recombinant proteins are detected . Sera from immunized horses showed a protective effect in MDCK cells and in embryonated chicken eggs. The effect was clearly observed with the A 1 virus because we tested a pool of sera and some horses had been in contact with viruses A2 and A2L there was no significant difference between the pre-immune sera and sera from the same horses immunized with different formulations. In the hemagglutination inhibition assay of serum of individual horses, we observed titers of 1/1024 for the A1 virus, 1/4096 for A2 with 1/16 for preimmune sera and 1/4096 for A2L with 1/512 for preimmune sera . These titers were higher than those obtained with the commercial vaccine. These results suggest that the DNA vaccine prototype developed is capable of delivering a measurable humoral immune response in all experimental animals tested and that corresponding sera are protective in two different models of evaluation.
A prototype vaccine against equine influenza virus (EIV) was developed using hemagglutinin (HA) and neuraminidase (NA) as candidate antigens , chosen for their higher immunogenicity and variability. The nucleoprotein (NP) was also included as a conserved interna! protein that characterizes all influenza type A viruses. In a first approach, a DNA vaccine based on vectors expressing HA, NA, NP from the two isolates that exist in Chile (A1-H7N7 and A2-H3N8) was designed. Sera from m ice immunized with this formulation yielded , by ELISA, titers over 1/1024. Studies of virus-neutralizing capacity in MDCK cells and in embryonated chicken eggs indicated that the sera were protective. In MDCK cells, neutralization was achieved with dilutions up to 1/1000 and in eggs, a neutralizing index capable of preventing infection of 1 million viruses per ml. Another vaccine was formulated with recombinant proteins HA, NA, and NP. Mice sera with EL ISA titers of 1/64 were obtained when we u sed virus A 1 proteins and 1/32 with virus A2 proteins. These titers were improved with mixed immunization , i.e. , first with DNA followed by a boost with protein. In this case, the titers obtained were 1/128 for the A 1 virus and 1/64 for the A2 virus and significantly higher absorban ces. These formulations were also assessed by immunization of guinea pigs and compared in parallel to a commercial vaccine. ELISA analysis of whole virus titers indicated 1/32 for the A 1 virus and 1/64 for the A2 virus. ELISA using recombinant proteins determined that DNA vaccination yielded titers of over 1/512 for HA 1 and 1/1024 for HA2, while the commercial vaccine yielded a 1/64 titer, comparable to preimmune sera. In Western blot analysis of allantoic fluid and viruses, the DNA vaccine recognizes a protein of around 55 kDa that might correspond to the NP. Western blot with the recombinant proteins shows that the DNA vaccine recognizes all 6 proteins and the commercial vaccine only detects HA from virus A 1, as observed in m ice. Analysis of the neutralizing capacity of sera from guinea pigs in embryonated eggs indicated neutralization indexes above the viral titer, which means that all the virus present was neutralized. Also noteworthy is that the DNA vaccine was able to neutralize the new virus isolate A2L, an effect not observed in the commercial vaccine. The same was observed in neutralization assays in MDCK cells. Recombinant and mixed vaccines did not achieve acceptable neutralization indexes. Analysis of the sera by the hemagglutination inhibition assay indicated that the DNA vaccine had the highest titers (> 1 /8000), better than the commercial vaccine (1/2048). In contrast, recombinant and mixed vaccines were negative in this assay. Having defined the best formulation, the appropriate dosage and safety of the tested formulation, we proceeded to test the DNA vaccine in horses. ELISA analysis with the virus of sera from immunized animals resulted in titers of 1/512, without recognition of allantoic fluid proteins. In ELISA done with the recombinant proteins, the DNA vaccine produced serum which recognized recombinant proteins from the A 1 and A2 isolates, whereas the commercial vaccine gives rise to serum which recognizes only proteins from the A 1 isolate. Western blot analysis detects a band at approximately 55kDa , as observed in guinea pigs, and if the analysis is done by Western blot with the virus, all six recombinant proteins are detected . Sera from immunized horses showed a protective effect in MDCK cells and in embryonated chicken eggs. The effect was clearly observed with the A 1 virus because we tested a pool of sera and some horses had been in contact with viruses A2 and A2L there was no significant difference between the pre-immune sera and sera from the same horses immunized with different formulations. In the hemagglutination inhibition assay of serum of individual horses, we observed titers of 1/1024 for the A1 virus, 1/4096 for A2 with 1/16 for preimmune sera and 1/4096 for A2L with 1/512 for preimmune sera . These titers were higher than those obtained with the commercial vaccine. These results suggest that the DNA vaccine prototype developed is capable of delivering a measurable humoral immune response in all experimental animals tested and that corresponding sera are protective in two different models of evaluation.
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Vacuna Contra la Influenza, Caballos, Enfermedades