RESUMO
Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. 'Pei chiao' (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium-mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%-0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2-week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination-dependent gradational increase in the elicitation of serum and saliva anti-PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.
Assuntos
Antígenos Virais/biossíntese , Musa/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem , Administração Oral , Animais , Anticorpos Neutralizantes/sangue , Antígenos Virais/genética , Imunoglobulina A/análise , Imunoglobulina G/sangue , Plantas Geneticamente Modificadas/metabolismo , Saliva/química , Saliva/imunologia , Suínos , Transformação Genética , Proteínas do Envelope Viral/metabolismo , Carga Viral/veterinária , Viremia/prevenção & controleRESUMO
Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Enterotoxinas/genética , Enterotoxinas/imunologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Nicotiana/genética , Nicotiana/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Adjuvantes Imunológicos/administração & dosagem , Administração Oral , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/metabolismo , Toxinas Bacterianas/administração & dosagem , Sequência de Bases , Primers do DNA/genética , Enterotoxinas/administração & dosagem , Escherichia coli/genética , Escherichia coli/imunologia , Proteínas de Escherichia coli/administração & dosagem , Ativação Linfocitária , Masculino , Folhas de Planta/imunologia , Plantas Geneticamente Modificadas , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Saliva/imunologia , Sus scrofa , Suínos , Vacinas de Plantas Comestíveis/administração & dosagem , Vacinas de Plantas Comestíveis/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Proteínas do Envelope Viral/administração & dosagem , Carga Viral , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
The aim of the study was to evaluate the immunogenicity of the ORF5-encoded major envelop glycoprotein 5 (GP5) of porcine reproductive and respiratory syndrome virus (PRRSV) expressed in tobacco plant as a potential pig oral vaccine in protection against PRRSV infection. Six-week-old PRRSV-free pigs were fed four times orally with 50g of chopped fresh GP5 transgenic tobacco leaves (GP5-T) (GP5 reaching 0.011% of total soluble protein) or wild-type tobacco leaves (W-T) each on days 0, 14, 28, and 42. Samples of serum, saliva, and peripheral blood mononuclear cells (PBMCs) were collected on days -1, 6, 13, 20, 27, 34, 41, and 48 after the initial oral vaccination. A similar vaccination-dependent gradual increase in the responses of serum and saliva anti-PRRSV total IgG and IgA, respectively, and in the levels of PRRSV-specific blastogenic response of PBMCs was seen in GP5-T-treated pigs; all statistically significant elevations occurred after the 2nd vaccination and were revealed after 20 days post-initial oral vaccination (DPIOV). Pigs fed on GP5-T also developed serum neutralizing antibodies to PRRSV at a titer of 1:4-1:8 after the 4th vaccination by 48 DPIOV. No detectable anti-PRRSV antibody responses and PRRSV-specific blastogenic response were seen in W-T-treated pigs. The present study has demonstrated that pigs fed on GP5-T could develop specific mucosal as well as systemic humoral and cellular immune responses against PRRSV. The results also support that transgenic plant as GP5-T can be an effective system for oral delivery of recombinant subunit vaccines in pigs.