This document summarizes research on producing yellow fever virus in Vero cells grown in serum-free medium. The researchers were able to obtain very high virus titers of 108 pfu/mL using the best conditions for cell propagation and viral infection in serum-free medium. Analysis showed no difference in antigenic properties between virus cultivated in serum-containing and serum-free medium. SDS-PAGE analysis confirmed a drastic reduction in protein content for yellow fever virus cultivated in serum-free medium. Developing a cell culture process for propagating yellow fever virus in serum-free Vero cells could be an important step towards producing a new inactivated yellow fever vaccine.

1. Vaccine Technology II - Algarve, Portugal 01/06/2008 – 06/06/2008
(Poster Number 19)
PRODUCTION OF YELLOW FEVER VIRUS IN VERO CELLS GROWN
IN SERUM-FREE MEDIUM
Leda R. Castilho, Federal University of Rio de Janeiro - COPPE - Cell Culture Engineering
Laboratory
Cx. Postal 68502, Ilha do Fundao, Rio de Janeiro, RJ, 21941-972, Brazil T: +55 21 25628336, F:
+55 21 25628300, leda@peq.coppe.ufrj.br
Marta C. O. Souza, Erica A. Schulze, Marlon F. Silva, Luciane P. Gaspar, Marcos S. Freire,
FIOCRUZ/BioManguinhos, Viral Technology Laboratory
The attenuated vaccine against yellow fever (YF) virus produced in embryonated eggs at FIOCRUZ
(Brazil) has been available for decades. This vaccine has been used for human immunization with
an excellent history of efficacy and safety. However, in the latest years, the occurrence of adverse
events associated with the 17D and 17DD substrains indicated the need for developing
technologies for the production of an inactivated vaccine. With this aim, the use of continuous cell
lines for production of the antigen offers advantages over viruses grown in embryonated eggs. A
major advantage is the production of virus in large scale under controlled conditions (pH,
temperature, dissolved O2).
Vero is a continuous, adherent cell line, which has been recommended by the World Health
Organization for the production of human vaccines. Commercial processes using this cell line for
vaccine production are based on its culture on microcarriers in stirred systems. The aim of this
study was the development of an efficient cell culture process and an adequate infection strategy
for propagating the yellow fever virus in Vero cells using stirred bioreactors.
Using the best conditions determined for cell propagation and viral infection, employing a
serumfree medium, very high virus titers (108
pfu/mL) were obtained, indicating that the
methodology developed in this work is efficient and could be employed in a process for the
production of yellow fever virus. Analysis of the antigenic properties of the 17DD virus by an
enzyme-linked immunoassay showed no difference between the virus cultivated in serum-
containing and in serum-free medium. In addition, SDS-PAGE analysis of the supernatant obtained
from infected cell cultures confirmed a drastic reduction in the protein content of samples of the YF
virus cultivated in serum-free medium. Thus, the development of a cell culture process for
propagating the yellow fever virus in Vero cells grown in serum-free conditions can represent an
important step towards the production of a new, inactivated vaccine against yellow fever.