Symposium Oct 2006
Development of a virus neutralization assay using a pseudotype virus
containing a luciferase reporter gene: Implications for vaccine design Joshua Salmans1,
Robert H. Mealey1,
and Susan Carpenter1.
Department of Veterinary Microbiology and Pathology, Washington State
University, Pullman, WA 991631;
Department of Microbiology, University of Iowa, Iowa City, IA 522422
Antigenic variation and immune escape are well-recognized strategies of
persistence for lentiviruses such as equine infectious anemia virus (EIAV) and
HIV-1. In EIAV, however, the development of broadly neutralizing antibodies is
associated with reduced virus replication during long-term infection. Epitopes
that elicit broadly neutralizing antibodies would be a useful component of
effective lentivirus vaccines. To aid in the identification of broadly
neutralizing epitopes, we developed a three-plasmid system that generates a
replication-deficient EIAV pseudotype virus containing a luciferase reporter
gene. The three plasmids include one that encodes for EIAV core and enzymatic
proteins; a second plasmid that encodes the EIAV envelope; and a third plasmid
that expresses a packaged RNA encoding a luciferase reporter gene. Pseudovirions
were produced by co-transfection of the three plasmids in 293T cells.
Supernatant was harvested, and the pseudovirions were titrated by infection of
equine dermal cells and quantification of luciferase activity, as measured in
relative light units. Pseudovirions were tested for susceptibility to
neutralization using sera from EIAV infected horses known to contain high levels
of broadly neutralizing antibodies. Results indicated that neutralizing antibody
response to pseudovirus were comparable to that obtained using infectious virus.
Future studies will use this assay to characterize the development of broadly
neutralizing antibody in EIAV infected horses and identify regions of the EIAV
envelope containing broadly neutralizing epitopes.
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