RESUMO
Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious and devastating disease that affects the pig industry worldwide. The glycoprotein E2 of CSFV is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. Several CSFV genotypes, including 1.1, 2.1, 2.2, and 2.3, have been identified in Mainland China. The glycoprotein E2 of genotypes 1.1 and 2.1 was expressed by using a baculovirus system and tested for its protective immunity in rabbits to develop novel CSF vaccines that elicit a broad immune response. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with E2 of genotypes 1.1 (CSFV-1.1E2), 2.1 (CSFV-2.1E2), or their combination (CSFV-1.1 + 2.1E2). A commercial CSF vaccine (C-strain) and phosphate-buffered saline (PBS) were used as positive or negative controls, respectively. All animals were challenged with CSFV C-strain at 4 weeks and then boosted with the same dose. All rabbits inoculated with CSFV-1.1E2, CSFV-2.1E2, and CSFV-1.1 + 2.1E2 elicited high levels of ELISA antibody, neutralizing antibody, and lymphocyte proliferative responses to CSFV. The rabbits inoculated with CSFV-1.1E2 and CSFV-1.1 + 2.1E2 received complete protection against CSFV C-strain. Two of the four rabbits vaccinated with CSFV-2.1E2 were completely protected. These results demonstrate that CSFV-1.1E2 and CSFV-1.1 + 2.1E2 not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits. Therefore, CSFV-1.1E2 and CSFV-1.1 + 2.1E2 are promising candidate subunit vaccines against CSF.
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/prevenção & controle , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Baculoviridae/genética , Proliferação de Células , China , Peste Suína Clássica/imunologia , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/genética , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Vetores Genéticos , Injeções Intramusculares , Linfócitos/imunologia , Testes de Neutralização , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
Classical swine fever (CSF) is a devastating infectious disease caused by classical swine fever virus (CSFV). The screening of CSFV-specific ligands is of great significance for diagnosis and treatment of CSF. Affinity selection from random peptide libraries is an efficient approach to discover ligands with high stability and specificity. Here, we screened phage ligands for the CSFV E2 protein from f8/8 landscape phage display library by biopanning and obtained four phage clones specific for the E2 protein of CSFV. Viral blocking assays indicated that the phage clone displaying the octapeptide sequence DRATSSNA remarkably inhibited the CSFV replication in PK-15 cells at a titer of 10(10) transduction units, as evidenced by significantly decreased viral RNA copies and viral titers. The phage-displayed E2-binding peptides have the potential to be developed as antivirals for CSF.
Assuntos
Antivirais/farmacologia , Vírus da Febre Suína Clássica/efeitos dos fármacos , Biblioteca de Peptídeos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/fisiologia , Avaliação Pré-Clínica de Medicamentos , Ligantes , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Suínos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious swine disease leading to significant economic losses worldwide. Vaccines are widely used to control the disease, and no CSFV-specific antivirals are currently available. To facilitate anti-CSFV molecule discovery, we developed a reporter virus CSFV-N(pro)Fluc stably expressing the firefly luciferase (Fluc) gene in the N(pro) gene. The reporter virus enabled more sensitive and convenient detection of the N(pro) protein expression and the viral replication by luciferase reporter assay than by traditional methods. The CSFV N(pro) protein was detectable as early as 4.5h post-infection. As a proof-of-concept for its utility in rapid antiviral screening, this reporter virus was used to quantify anti-CSFV neutralizing antibodies of 50 swine sera and to assess 12 small interfering RNAs targeting different regions of the CSFV genome. The results were comparable to those obtained by traditional methods. Taken together, the reporter virus CSFV-N(pro)Fluc represents a useful tool for rapid and quantitative screening and evaluation of antivirals against CSFV.
Assuntos
Antivirais/farmacologia , Vírus da Febre Suína Clássica/efeitos dos fármacos , Vírus da Febre Suína Clássica/genética , Peste Suína Clássica/virologia , Avaliação Pré-Clínica de Medicamentos/métodos , Luciferases de Vaga-Lume/genética , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Peste Suína Clássica/tratamento farmacológico , Peste Suína Clássica/imunologia , Vírus da Febre Suína Clássica/imunologia , Vírus da Febre Suína Clássica/fisiologia , Vaga-Lumes/genética , Genes Reporter , Luciferases de Vaga-Lume/metabolismo , Suínos , Replicação Viral/efeitos dos fármacosRESUMO
The use of attenuated classical swine fever virus (CSFV) strains as live vaccines is no longer allowed for the control of classical swine fever in Europe, due to the inability to differentiate between infected and vaccinated animals (Differentiating Infected from Vaccinated Animals; DIVA), except as emergency vaccines or as bait vaccines for wild boars. Thus, the establishment of a DIVA vaccine(s) is of pivotal importance for the control of this infectious disease. In this study, recombinant versions of the live-attenuated vaccine strain CSFV Riems were generated by replacing parts of the E2 gene with the corresponding sequence of border disease virus strain Gifhorn. Three cDNA clones were constructed: pRiems-ABC-Gif, pRiems-A-Gif and pRiems-BC-Gif. Infectious particles were obtained from clones pRiems-ABC-Gif and pRiems-BC-Gif only, whereas transfected RNA from clone pRiems-A-Gif behaved like a replicon. Based on its ability to be differentiated in vitro from wild-type CSFV by mAbs, vRiems-ABC-Gif was assessed for immunogenicity and protection against challenge infection in pigs. Before challenge, no CSFV-specific anti-E2 antibodies could be detected with commercial E2-blocking ELISAs in vRiems-ABC-Gif-vaccinated animals, whereas vRiems-vaccinated pigs developed high titres of anti-E2 antibodies, confirming the marker properties of this vaccine candidate. After oral vaccination, only partial protection against challenge infection was observed in the vRiems-ABC-Gif vaccinees, whereas all intramuscularly vaccinated animals and all vRiems-vaccinated animals were fully protected. These experiments suggest that the strategy of exchanging specific antigenic epitopes among pestiviruses is a promising tool for the development of new CSFV marker vaccines.