Study on the expression of human lysozyme in oviduct bioreactor mediated by recombinant avian adeno-associated virus.

Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 µg/mL, reached a maximum of 683 ± 16.4 µg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins.

Efficient production of an avian adeno-associated virus vector using insect cell/baculovirus expression system.

Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene. After demonstration of the correct expression of AAAV genes, rAAAV-GFP was produced by triple infection of insect cells or triple transfection of HEK293 cells for comparison purpose. Electron microscopy revealed the formation of typical AAAV particles in the insect cells. Western blotting showed the correct assembly of rAAAV particles with a VP protein ratio similar to that of AAAV. Quantitative PCR showed that the insect cell-produced rAAAV yield was almost 25-fold higher than that produced by HEK293 cells. Fluorescent microscopy showed that the insect cell-produced rAAAV could transfer GFP reporter gene into two avian cell types with similar transfer efficiency to that of HEK293 cell-produced rAAAV. These data suggest that insect cell/baculovirus expression system could be used for scalable production of rAAAV, and the viral vector produced could be used as the gene transfer vehicle for avian cells.

A recombinant avian adeno-associated virus as a vector factor for infectious bursal disease vaccination

El virus de la enfermedad infecciosa de la bolsa es una enfermedad de distribución mundial que afecta a aves jóvenes que debe ser controlada mediante vacunación y constituye una de las preocupaciones principales de la industria avícola mundial. Los virus adeno-asociados aviares son virus no patogénicos capaces de dar cabida a porciones relativamente largas de ADN y de infectar una amplia variedad de tipos celulares. Un miembro de esta familia, el virus adeno-asociado aviar ha sido caracterizado por completo y utilizado como un vector para entrega de genes en células y tejidos de embriones de pollo. En el presente estudio se demostró mediante inmunohistoquímica y microscopia electrónica la factibilidad de generar virus adeno-asociados recombinantes expresando la proteína viral 2 del virus de la enfermedad infecciosa de la bolsa. Luego de la inoculación in ovo o intramuscular de aves libres de patógenos específicos con el virus recombinante, se observó evidencia serológica de la expresión de la proteína VP2. La utilización de virus adeno-asociado aviar recombinantes para la entrega de genes es una opción interesante para la vacunación de aves domésticas.

Infectious bursal disease is a worldwide distributed immunosuppressive disease of young chickens that need to be controlled by vaccination; it represents one of the main concerns for the poultry industry. The adeno-associated viruses are non-pathogenic viruses, capable of accommodating relatively long pieces of DNA, and of infecting a wide variety of cell types. A member of this family, the avian adeno-associated virus has been fully characterized and successfully used for gene delivery in chicken embryo tissues and cells. In this study, it was demonstrated by electron microscopy and immunocitochemistry the feasibility of generating recombinant avian adeno-associated virus (rAAAV) virions expressing the immunogenic viral protein 2 of infectious bursal disease virus (IBDV). Serological evidence of VP2 protein expression measured as IBDV specific antibody response after in ovo or intramuscular inoculation of the recombinant virus in specific pathogen free (SPF) chickens was observed. The use of rAAAV virions for gene delivery in poultry is a promising approach to poultry vaccination.