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Equid alphaherpesvirus 1 (EHV-1) infection causes abortion, respiratory disease, perinatal deaths and neurological disorders in horses. The natural infection and available vaccines provide only partial and short-lived protection against reinfections. In the present study, we analyzed the ability of purified baculovirus-expressed glycoprotein D (gD) administered by different routes to induce protective immunity in BALB/c mice after challenge with the EHV-1 AR8 strain. Clinical signs varied among the different groups of mice immunized by parenteral routes, and, although gD induced a specific serum IgG response, it did not prevent the virus from reaching the lungs. Intranasally immunized mice showed no clinical signs, and virus isolation from lungs, histological lesions and antigen detection by immunohistochemistry were negative. In addition, by this route, gD did not stimulate the production of serum IgG and IgA. However, a specific IgA response in the respiratory tract was confirmed in intranasally immunized mice. Thus, we conclude that the mucosal immune response could reduce the initial viral attachment and prevent the virus from reaching the lungs. Our findings provide additional data to further study new immunization strategies in the natural host. La infección con alfaherpesvirus equino 1 (EHV-1) causa abortos, enfermedad respiratoria, muertes perinatales y desórdenes neurológicos en equinos. La infección natural y las vacunas disponibles solo proporcionan protección parcial y de corta duración contra las reinfecciones. En el presente estudio se analizó la inducción de inmunidad protectiva de la glicoproteina D (gD) expresada en baculovirus y purificada al ser administrada por diferentes rutas en ratones BALB/c desafiados con la cepa AR8 de EHV-1. Los signos clínicos fueron variables entre los grupos de ratones inmunizados por rutas parenterales y, aunque la gD indujo respuesta especifica de IgG en suero, no logró prevenir la llegada del virus al pulmón. En los ratones inmunizados intranasalmente no se observaron signos clinicos ni lesiones histopatológi-cas, y el aislamiento viral y la detección de antigenos por inmunohistoquímica en pulmón fueron negativos. Además, por esta ruta la gD no estimuló la producción de IgG y de IgA en suero. Sin embargo se confirmó la respuesta de IgA especifica en el tracto respiratorio de ratones inmunizados intranasalmente. Esta respuesta inmune mucosal podría haber reducido la unión inicial del virus a la célula huésped y, de este modo, prevenir la llegada del virus al pulmón. Nuestros hallazgos proporcionan un aporte para continuar estudiando nuevas estrategias de inmunización en el huésped natural.

El virus vaccinia Ankara modificado (MVA) es un virus altamente atenuado que se utiliza eficientemente como vector viral no replicativo para el desarrollo de nuevas vacunas. En este trabajo de Tesis se desarrolló un nuevo inmunógeno basado en MVA que expresa como antígeno de interés la glicoproteína D (versión secretada, gDs) del virus herpes bovino tipo I (BoHV-1), un agente infeccioso ampliamente distribuido en Argentina. Primeramente, se diseñó y construyó el vector de transferencia para obtener MVA recombinantes utilizando como sitio blanco de inserción el gen no esencial MVA086R. Se obtuvieron virus MVA-gDs recombinantes y se demostró la correcta expresión, antigenicidad y N-glicosilación de la glicoproteína D expresada a partir de dichos virus. Posteriormente, se evaluó la capacidad inmunogénica de los virus MVA-gDs en animales de experimentación (ratones y conejos). En el modelo de ratón, se observó que los virus MVA-gDs desencadenaron una respuesta inmune específica, de tipo humoral, que se mantuvo en niveles similares durante un período de siete meses (luego de la aplicación de dos dosis del inmunógeno). Además, se demostró que la síntesis de novo de la glicoproteína D a partir del vector viral no replicativo MVA-gDs fue la responsable de la respuesta inmune específica. La inoculación de conejos con virus MVA-gDs (por vía intramuscular o subcutánea) desencadenó una respuesta inmune específica de tipo humoral, observándose la máxima respuesta a los 9 días post dosis de refuerzo. En un ensayo de desafío de conejos con el virus BoHV-1, se observó que la inmunización por vía intramuscular de los animales con virus MVA-gDs disminuyó la excreción del virus de desafío. Finalmente, se demostró que la inmunización de ratones por vía intranasal con virus MVA-gDs adicionados con toxina colérica indujo respuestas inmunes humorales específicas de tipo IgA en muestras de lavados nasales y broncopulmonares y de tipo IgG en muestras de suero. De esta forma, se obtuvo, caracterizó y evaluó un nuevo inmunógeno denominado MVA- gDs que, en el futuro, podría ser evaluado como candidato a vacuna contra el virus BoHV-1 en bovinos. Modified vaccinia virus Ankara (MVA) is a highly attenuated virus. During the past years, non-replicative recombinant MVA vectors have gained major interest in the development of new veterinary vaccines. Bovine herpesvirus 1 (BoHV-1) infection is widely distributed in Argentina and there are no efficacious vaccines to prevent the diseases caused by this virus. In order to develop a candidate vaccine for BoHV-1, the secreted version of glycoprotein D (gDs) was selected as the antigen to be expressed in new recombinant MVA non-replicative based vectors. First, the design and construction of the transference vector necessary to obtain recombinant MVA viruses was achieved using the non-essential gene MVA086R. Recombinant MVA-gDs virus was obtained and molecularly characterized, confirming the correct expression, antigenicity and N-glycosilation of glycoprotein D. Then, immunogenicity of recombinant MVA-gDs viruses was assessed in experimental animals (mice and rabbits). Mice immunized with MVA-gDs viruses developed a specific humoral immune response that was maintained for seven months and was dependent on the booster dose. In addition, mice immunized with MVA-gDs virus demonstrated that MVA-gDs non-replicative viral vector was responsible for the de novo synthesis of glycoprotein D and, consequently, for the immune response induced in animals immunized with not inactivated MVA-gDs virus. Intramuscular and subcutaneous rabbit inoculation with MVA-gDs virus was immunogenic and induced a specific humoral immune response. In a rabbit BoHV-1 challenge assay, diminished viral excretion was observed in those animals intramuscularly immunized with recombinant MVA-gDs. Finally, in order to evaluate MVA-gDs virus as a mucosal vaccine, two doses of MVA-gDs supplemented with cholera toxin were delivered by intranasal immunization of mice. These immunizations induced anti-gD IgA humoral immune responses in nasal and bronchopulmonar lavages as well as anti-gD IgG antibodies in serum samples. In conclusion, the new immunogen designated MVA-gDs was developed, characterized and evaluated in experimental animals. In the future, it could be evaluated in cattle as a candidate bovine vaccine for BoHV-1. Fil: Ferrer, María Florencia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Made available in DSpace on 2014-08-20T13:32:53Z (GMT). No. of bitstreams: 1 dissertacao_luana_alves_dummer.pdf: 909354 bytes, checksum: d2af8cc0772177718829cf7f15391d43 (MD5) Previous issue date: 2008-06-12 Outbreaks of fatal meningoencephalitis caused by Bovine Herpesvirus type 5 (BoHV-5) cause important economic losses in national trade of bovine beef. Commercial vaccines developed against Bovine Herpesvirus type 1 can protect cattle of neurological disease caused by BoHV-5. These cross-reactions are due to its genetic and antigenic similarities of both BoHV-1 and 5. However, these vaccines can not prevent latent infection of BoHV-5, even viral spread to healthy animals. This way, new vaccines strategies are based on envelope glycoproteins and are focusing safety, economic viability and on prevention of BoHV-5 latency and spread. This glycoprotein acts in initial steps of viral infection and glycoprotein D has drawn the attention in studies carried out with BoHV-1 and other homologous herpesvirus. The gD acts on viral membrane fusion with permissive cells through glycoproteins and host receptors interactions, so it is essential for viral entry. A vaccine that is capable to stimulate specific humoral and cellular immunity against BoHV-5 must be developed. The aim of this work was the production of a recombinant truncated form of gD of BoHV-5 in yeast Pichia pastoris and its antigenicity and immunogenicity evaluation. Data show that yeast P. pastoris can express the truncated form of BoHV-5 gD to the supernatant due to its secretion of ~190mg/L of recombinant protein, simplifying protein purification. The recombinant protein was successfully recognized by antibodies of animals immunized with BoHV-5, suggesting its antigenicity and immunogenicity and that native protein characteristic are conserved. The data presented herein allow the design of future studies aiming at expression optimization and further immunogenicity evaluation, as well as its capacity to neutralize the virus in biological models and on cattle. Surtos de meningoencefalites fatais causadas pelo Herpesvírus Bovino tipo 5 (BoHV-5) ocasionam importantes perdas econômicas no comércio nacional de carne bovina. Vacinas comerciais destinadas ao Herpesvírus Bovino tipo 1 são capazes de impedir o aparecimento de sintomatologia clínica do BoHV-5. Estas reações cruzadas se devem a existência de semelhanças genéticas e antigênicas existentes entre ambos os vírus. No entanto, estas vacinas não impedem o estabelecimento da infecção latente e a disseminação do BoHV-5 para animais não imunizados. Desta forma, estratégias que visem à produção de vacinas seguras, economicamente viáveis e que possam ser capazes de impedir a latência do BoHV-5 e sua disseminação estão focadas para glicoproteínas localizadas no envelope viral e que atuam nas etapas iniciais da infecção. Dentre estas, a glicoproteína D tem se destacado em estudos realizados com BoHV-1 e outros herpesvírus homólogos. A gD atua na fusão do envelope viral com a membrana da célula permissiva no hospedeiro através de interações com receptores celulares e com outras glicoproteínas virais, sendo essencial para a entrada do capsídeo na célula. Assim, uma vacina que seja capaz de estimular respostas humorais e celulares contra esta glicoproteína deve ser desenvolvida. Os objetivos deste trabalho foram à produção da forma truncada da gD do BoHV-5 em levedura Pichia pastoris e a avaliação desta quanto a sua antigenicidade e imunogenicidade. Os resultados demonstram que a Pichia pastoris expressou a forma truncada da gD de BoHV-5 secretando para o meio de cultivo ~190mg/L da proteína recombinante, facilitando a purificação da mesma. Testada quanto a sua a sua antigenicidade e imunogenicidade, a proteína recombinante foi reconhecida por anticorpos de animais imunizados com o BoHV-5, sugerindo que características da proteína nativa foram conservadas na proteína recombinante. Os dados apresentados irão permitir o desenvolvimento de estudos 6 futuros com o objetivo de aperfeiçoar a expressão da proteína recombinante e avaliar a sua capacidade de neutralizar o vírus na espécie-alvo.