14th ICID - Poster Abstracts - International Society for Infectious ...

When citing these abstracts please use the following reference:
Author(s) of abstract. Title of abstract [abstract]. Int J Infect Dis 2010;14S1: Abstract number.
Please note that the official publication of the International Journal of Infectious Diseases 2010, Volume 14, Supplement 1
is available electronically on http://www.sciencedirect.com
Final Abstract Number: 24.008
Session: Arboviruses
Date: Wednesday, March 10, 2010
Time: 12:30-13:30
Room: Poster & Exhibition Area/Ground Level
Type: Poster Presentation
Preparation of antigenically active recombinant yellow fever viral envelope domain III protein
S. Smouse 1 , F. J. BURT 2
1 University of the Free State, Bloemfontein, Free State, South Africa, 2 UNIVERSITY OF THE
FREE STATE, 9324, ZA, South Africa
Background: Yellow fever virus belongs to the genus Flavivirus, of the family Flaviviridae. It is a
mosquito-borne virus endemic in tropical regions of Africa and South America. Although an
effective vaccine is available, the virus remains a major public health threat, particularly in Africa
where vaccination is limited by poverty, civil wars and the inaccessibility of rural areas prone to
outbreaks. It is a re-emerging pathogen with case-fatality rates that can exceed 50%. The
diagnosis of infection and testing of the immune status of vaccinees require reagents that are
prepared in biosafety level 3 and 4 facilities. The viral envelope protein plays an important role in
eliciting antibodies and hence serves as an ideal diagnostic and research tool for the detection of
antibodies. The aim of the study was to compare various bacterial expression systems for the
preparation of a recombinant yellow fever viral envelope protein for the detection of antibodies
against yellow fever in vaccinated individuals.
Methods: The domain III region of the envelope (EDIII) gene was amplified with primers
identified using sequence data retrieved from GenBank. The EDIII gene was cloned into three
different E.coli bacterial expression systems for comparison of protein yield, solubility and
suitablity in an ELISA. Proteins were all expressed with an N+ terminal His tag for purification. A
direct ELISA was developed in which the plates were coated with antigen and reacted with serum
samples from vaccinees.
Results: Each antigen was evaluated using serum samples collected from vaccinees and
serological cross reactivity of the antigen against heterologous flaviviral antibody was determined
using convalescent serum samples from patients with known flaviviral infections, such as West
Nile. Protein yields varied significantly between the different expression systems with higher
yields and increased solubility obtained from expression at lower temperatures. Higher reactivity
was associated with homologous antibody.
Conclusion: Preliminary results suggest that bacterially expressed recombinant EDIII protein is
antigenically active and potentially useful for detecting antibodies against yellow fever virus.