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  Student Research Symposium Oct 2006  

Development of a virus neutralization assay using a pseudotype virus containing a luciferase reporter gene: Implications for vaccine design

Joshua Salmans1, Sandy Taylor1, Matt Littke1, Melinda Brindley 2, Wendy Maury2, 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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