title: Optimization of virus yield as a strategy to improve rabies vaccine production by Vero cells in a bioreactor.
abstract: To improve rabies vaccine production by Vero cells, we have developed a strategy based on high cell density culture and optimization of virus yield. We have first optimized cell growth in spinner flask using a Taguchi's L8 experimental design. We analyzed the effects of the following factors: initial glucose and glutamine concentrations, Cytodex 1 concentration and the regulation of glucose level at 1 g l(-1). We have also investigated the effect of the following factor interactions: Cytodex 1 concentration/glutamine concentration, Cytodex 1 concentration/glucose concentration and glucose concentration/glutamine concentration. Statistical analysis of the collected data pointed to the initial glucose concentration, the regulation of glucose level at 1 g l(-1) and the interactions between Cytodex 1 concentration/initial glucose concentration and Cytodex 1 concentration/initial glutamine concentration as the parameters that affected cell growth. Using the optimal conditions determined earlier, we have studied Vero cell growth in a 7-l bioreactor and in batch culture, and obtained a cell density level equal to 3.6 +/- 0.2 x 10(6) cells ml-1. Cell infection with rabies virus (LP 2061/Vero strain) at a multiplicity of infection (MOI) of 0.3 using M199 medium supplemented with 0.2% bovine serum albumin (BSA), yielded a maximal virus titer equal to 8 +/- 1.6 x 10(7) Fluorescent Focus Units (FFU) ml-1. We have also studied Vero cell growth in a 7-l bioreactor using recirculation as a perfusion culture mode during cell proliferation step and perfusion for virus multiplication phase. In comparison to batch culture, we reached a higher cell density level that was equal to 10.1 +/- 0.5 x 10(6) cells ml-1. Cell infection under the conditions previously indicated, yielded 14l of virus harvest that had a virus titer equal to 2.6 +/- 0.5 x 10(7) FFU ml-1. The activity of the inactivated virus harvest showed a protective activity that meets WHO requirements.
authors.affiliation: Viral Vaccines Research and Development Unit, Institut Pasteur de Tunis 13, Place Pasteur, BP 74, 1002 Belvédère, Tunis, Tunisia.
authors.forename: Khaled
authors.initials: K
authors.lastname: Trabelsi
authors.forename: Samia
authors.initials: S
authors.lastname: Rourou
authors.forename: Houssem
authors.initials: H
authors.lastname: Loukil
authors.forename: Samy
authors.initials: S
authors.lastname: Majoul
authors.forename: Héla
authors.initials: H
authors.lastname: Kallel
mesh_terms: D000818:Animals; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods; D002522:Chlorocebus aethiops; D003470:Culture Media / Q000737:chemistry; D011819:Rabies Vaccines / Q000096:biosynthesis*; D011820:Rabies virus / Q000254:growth & development*; D014709:Vero Cells; D038301:Virus Inactivation
chemical_list: D003470:Culture Media; D011819:Rabies Vaccines