This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.This project seeks to evaluate the adjuvant effects of CD40L to enhance the human immunodeficiency virus (HIV)-specific humoral and cellular immunity elicited by a DNA/MVA vaccine using a macaque/simian immunodeficiency virus (SIV) model. The macaque CD40L was cloned and expressed in cis (on the same plasmid) and in trans (on a separate plasmid) with respect to the SIV 239 vaccine DNA. In the cis form, the protein was expressed as a membrane bound form that is incorporated into SIV virus-like particles. In the trans form, the protein was expressed as a soluble secreted form. Intracellular expression of the CD40L protein was confirmed by flow cytometry and extracellular protein was confirmed by Western blotting.The expressed proteins were highly active in stimulating DC from SIV-infected macaques. These activated DC upregulated CD80 and produced TNF-a and IL-12. In addition, these proteins were active in stimulating macaque B cells. On going experiments are testing the adjuvant effects of these constructs in macaques.
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