June 6-11, 2010
Here we report the utilization of Drosophila melanogaster Schneider 2 (S2) cells and the Semliki Forest Virus (SFV) expression system to produce the rabies virus glycoprotein (RVGP). Although they represent different expression systems, they offer similar advantages in generating high-level expression of functional membrane proteins. They are both relatively easy and safe to handle and are scalable. RVGP synthesized by S2 cells and SFV carrying an mRNA coding for RVGP (SFV-RVGP) were assayed in mouse protection studies. We have constructed S2 gene vectors with the hygromycin selection gene (H) in which the RVGP gene is inserted under the control of the metalothionein (MT) promoter. A S2MTRVGP-H cell population was selected and the expression of RVGP was evaluated. Cell cultures in bioreactor were performed and RVGP were produced. The data showed a high RVGP expression level. RVGP mRNA analysis enlightened the relationship between cell growth and specific productivity. Parameters for storage, lysis and concentration of cells bearing the RVGP were studied for productivity evaluation and purification. A protocol for cell preparation including cell freezing as dry pellet, cell thawing at 4ºC with Tris, NaCl, MgCl2, PMSF and cell lysis was developed, Regarding the SFV expression system, the RVGP gene was cloned into a modified pSFV2genC expression vector. BHK-21 cells were electroporated with expression and helper RNA vectors and SFV-RVGP particles were obtained. These were titrated by qPCR and used to infect BHK-21 cells. RVGP expression was confirmed by qPCR, western blotting, ELISA and cell immunofluorescence. The ability of S2 derived RVGP and SFV-RVGP particles to produce both humoral and cellular immune response and protect mice against an experimental rabies virus challenge were investigated. High levels of antibodies against RVGP were found in both groups of immunized mice. A higher cellular immune response, as measured by the CD4+/CD8+ cell quantification was observed in SFV-RVGP immunized mice. Preliminary data show that immunization protocols were capable of inducing a high degree of protection against rabies experimental challenge. Taken together our data show several optimization steps for high-level RVGP expression in stably transfected S2 cells, which were shown to be biologically active by protecting mice against a rabies experimental infection. On the other hand, the promising SFV system constituted by recombinant viral particles carrying the mRNA coding for RVGP was shown to elicit a cellular immune response and protect mice against an experimental infection Supported by FAPESP, CNRS, CNPq, Fundação Butantan.
Carlos Pereira, "RABIES VIRUS GLYCOPROTEIN (RVGP) EXPRESSION IN DROSOPHILA S2 CELLS AND BY SEMLIKI FOREST VIRUS (SFV). SYNTHESIS AND PROTECTION STUDIES" in "Vaccine Technology III", John G. Auniņš,Merck, USA; Barry C. Buckland, BiologicB, USA; Kathrin U. Jansen, Pfizer, USA; Paula Marques Alves, IBET, Portugal Eds, ECI Symposium Series, (2010). http://dc.engconfintl.org/vaccine_iii/22