RESUMO
The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/prevenção & controle , Vírus da Diarreia Viral Bovina Tipo 1/imunologia , Vírus da Diarreia Viral Bovina Tipo 2/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Baculoviridae , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/patologia , Bovinos , Cobaias , Insetos , Masculino , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Resultado do Tratamento , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Infection with Bovine Viral Diarrhea Viruses (BVDV) in cattle results in a wide range of clinical manifestations, ranging from mild respiratory disease to fetal death and mucosal disease, depending on the virulence of the virus and the immune and reproductive status of the host. In this study 30 Argentinean BVDV isolates were characterized by phylogenetic analysis. The isolates were genotyped based on comparison of the 5' untranslated region (5' UTR) and the E2 gene. In both phylogenetic trees, 76% of the viruses were assigned to BVDV 1b, whereas BVDV 1a, 2a and 2b were also found. Eight of the BVDV 1b isolates were further characterized by cross-neutralization tests using guinea pig antisera and sera from bovines vaccinated with two different commercial vaccines. The results demonstrated the presence of a marked antigenic diversity among Argentinean BVDV isolates and suggest the need to incorporate BVDV 1b isolates in diagnostic strategies.
Assuntos
Variação Antigênica/imunologia , Doença das Mucosas por Vírus da Diarreia Viral Bovina/imunologia , Vírus da Diarreia Viral Bovina/imunologia , Filogenia , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Variação Antigênica/genética , Argentina , Sequência de Bases , Doença das Mucosas por Vírus da Diarreia Viral Bovina/genética , Bovinos , Vírus da Diarreia Viral Bovina/genética , Cobaias , Dados de Sequência Molecular , Testes de Neutralização/veterinária , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Bovine Viral Diarrhea Virus (BVDV) is the causative agent of a worldwide disease. The virus infects bovines of all ages, causing reproductive problems and contaminating biological products of high commercial value. The large-scale production of BVDV vaccines presents the challenge of processing antigenic proteins that are highly susceptible to the processing environment. Potency testing requires the immunization of cattle in order to determine the neutralizing antibodies titers induced by the vaccine. An alternative to the in vivo test is an in vitro measurement of key viral antigens. This paper describes the development and validation of a sandwich-type indirect ELISA that is able to detect and quantify BVDV E2 glycoprotein in live and inactivated BVDV. Validation parameters such as repeatability, intermediate precision, and reproducibility indicated that the developed ELISA constitutes an advanced tool for evaluating the BVDV antigen throughout manufacturing and vaccine release testing.