View All Abstracts for Session 22
90 Optimizing the Production of Virus-Like Particles by Regulating HIV-1 Protease in Multi-Gene HIV-1 DNA Vaccine Constructs
D. Ellenberger*1, B. Li1, J. Smith2, H. Robinson2, and S. Butera1
1CDC, Atlanta, GA, USA and 2Vaccine Res. Ctr. of Emory Univ., Atlanta, GA, USA
BACKGROUND: Production of virus-like particles (VLPs) with envelope glycoproteins may increase the immunogenicity of HIV-1 DNA-based vaccine candidates by permitting Env-mediated VLP entry into antigen-presenting cells. While constructing and evaluating subtype B and A/G multi-gene HIV-1 DNA vaccine candidates, we observed that viral proteins expressed in transiently transfected cultures were forming complex aggregates as well as VLPs. An ordered array in the aggregates suggested that they might reflect premature intracellular proteolytic cleavage of gag.
METHODS: An HIV-1-specific protease inhibitor (saquinavir) was added to transfection culture medium. Within the HIV-1 subtype A/G and B backgrounds of our DNA vaccine constructs, directed point mutations in pol protease were introduced. Aggregate and VLP formation were monitored by electron microscopy and other means of quantifying HIV-1 gag production.
RESULTS: The addition of saquinavir to the transfection culture medium dramatically shifted the ratio of gag aggregate formation toward the assembly and budding of immature VLP. Transfection of DNA vaccine constructs with mutated HIV-1 protease showed predominantly immature VLPs.
CONCLUSIONS: These studies define critical parameters such as restricted intracellular HIV-1 protease activity for optimizing VLP formation to potentially derive the greatest immunogenic benefit from HIV-1 DNA-based vaccinations.
Contact Author about this Abstract
