Inducción y evaluación de la respuesta inmune humoral del Salmón del Atlántico (Salmo salar L.) frente a variantes de las proteínas recombinantes E1 y E2 de Alfavirus de salmónidos (SAV)
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Date
2013
Authors
Soto Garcés, Melissa Viviana
Profesor/a Guía
Facultad/escuela
Idioma
en
Journal Title
Journal ISSN
Volume Title
Publisher
Universidad Andrés Bello
Nombre de Curso
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Abstract
Las enfermedades causadas por Alfavirus de Salmón (SAV) son patologías
de gran importancia en la industria del cultivo de salmónidos, para las cuales
existen actualmente pocas medidas de profilaxis y el desarrollo de estas en países
libres de estos patógenos, como Chile, representa un gran desafío. Con el fin de
desarrollar una vacuna contra SAV, en ausencia del agente viral en cuestión y que
permita su uso libremente a nivel comercial, se diseñaron antígenos modificados
de SAV, que pudieran inducir una respuesta inmune humoral. Los antígenos se
expresaron en forma recombinante y se diseñaron 3 formulaciones de vacunas,
las cuales se inyectaron en Salmones del Atlántico y se evaluó la respuesta
inmune en el tiempo, junto con el desarrollo de efectos adversos en los peces
vacunados. Los resultados muestran que la vacuna es segura, pues no entrega
niveles importantes de melanosis y adherencias. Adicionalmente, las fórmulas
inducen la producción de lgM que fue detectada mediante ELISA, de grado
variable según la formulación aplicada y el tiempo post inoculación de la vacuna.
Estos anticuerpos son específicos, ya que por western blot se demostró que
reconocen principalmente las proteínas recombinantes. Paralelamente se diseñó
una plataforma para la evaluación de la respuesta inmune conformacional contra
SAV inducida por la vacunación, mediante el desarrollo de VLP (Virus like
particles) de SAV-3 en el sistema de baculovirus en células de insecto, las cuales
fueron detectadas mediante microscopía electrónica de transmisión en
expresiones a baja temperatura. De esta manera se generó una vacuna con
antígenos de Alfavirus que es capaz de inducir una fuerte respuesta humoral
sostenida en el tiempo con bajos índices de efectos adversos, y fue posible el
desarrollo de VLP de SAV-3 las cuales podrán ser utilizadas en estudios futuros
para la evaluación de la respuesta inmune contra los epítopos conformacionales.
Diseases caused by Salmonid Alphavirus (SAV) corresponds to pathologies of major importance in the salmen farm industry, for which currently exist few prophylaxis measures, and the development of preventive solutions in countries where those pathogens are absent, such as in Chile, is a major challenge. In order to develop a vaccine against SAV, in absence of the viral agent and with a real potential commercial use, we designed SAV-modified antigens which could induce a humoral immune response. The antigens were expressed in a recombinant plataform, and 3 vaccine alternatives were formulated. Those vaccines were injected in Atlantic salmen and the immune response was evaluated over time, together with the development of side effects in the vaccinated fish. Results show that these vaccines were safe, as they don't induced significant levels of melanosis and adhesions. Additionally, the formulations elicited the production of lgM, as detected by ELISA, in variable degree depending on the time after vaccination and antigenic composition. The vaccine-induced antibodies were specific, because they recongnized mainly the recombinant SAV-derived antigens demonstrated by western blot. In parallel to vaccine formulation and evaluation, we designed a platform intended to evaluate the conformational immune response against SAV induced for the vaccínation, through the development of VLPs (virus like particles) in baculovirus system. SAV-VLPs were successfully detected by transmission electron microscopy, in cultures evaluated at different host cells and expression conditions, giving the initial expression platform for future optimization studies. Thus, our vaccíne SAV-modified vaccine was capabe to induce a strong humoral response in time, with minimal levels of side effects, and the development of SAV- 3 VLPs was possible, enabling our laboratory to perform future studies aimed to evaluate the immune response against the conformational epitopes.
Diseases caused by Salmonid Alphavirus (SAV) corresponds to pathologies of major importance in the salmen farm industry, for which currently exist few prophylaxis measures, and the development of preventive solutions in countries where those pathogens are absent, such as in Chile, is a major challenge. In order to develop a vaccine against SAV, in absence of the viral agent and with a real potential commercial use, we designed SAV-modified antigens which could induce a humoral immune response. The antigens were expressed in a recombinant plataform, and 3 vaccine alternatives were formulated. Those vaccines were injected in Atlantic salmen and the immune response was evaluated over time, together with the development of side effects in the vaccinated fish. Results show that these vaccines were safe, as they don't induced significant levels of melanosis and adhesions. Additionally, the formulations elicited the production of lgM, as detected by ELISA, in variable degree depending on the time after vaccination and antigenic composition. The vaccine-induced antibodies were specific, because they recongnized mainly the recombinant SAV-derived antigens demonstrated by western blot. In parallel to vaccine formulation and evaluation, we designed a platform intended to evaluate the conformational immune response against SAV induced for the vaccínation, through the development of VLPs (virus like particles) in baculovirus system. SAV-VLPs were successfully detected by transmission electron microscopy, in cultures evaluated at different host cells and expression conditions, giving the initial expression platform for future optimization studies. Thus, our vaccíne SAV-modified vaccine was capabe to induce a strong humoral response in time, with minimal levels of side effects, and the development of SAV- 3 VLPs was possible, enabling our laboratory to perform future studies aimed to evaluate the immune response against the conformational epitopes.
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Tesis (Magister en Biotecnología)
Keywords
Salmón del Atlántico, Enfermedades