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218 Efficiency and Stability of gp120 Incorporation on the Surface of HIV-1 Pseudovirion Particles
J. Hammonds*, X. Chen, and P. Spearman
Vanderbilt Univ., Nashville, TN, USA
Background: Antibodies raised by immunization with soluble, monomeric forms of the HIV-1 envelope glycoprotein (Env) have failed to elicit neutralizing antibodies against primary HIV-1 isolates. HIV-1 pseudovirions, or virus-like particles, incorporate envelope glycoproteins in native, oligomeric form. HIV-1 pseudovirions represent a means of eliciting antibodies directed against potential neutralizing determinants on particle-bound gp120.
Methods: We investigated factors determining Env incorporation and stability on HIV-1 pseudovirions. The incorporation and stability of Env on pseudovirions was quantitated using an ELISA for gp120.
Results: Expression of Gag-Pro and Env from separate expression vectors resulted in incorporation of Env into pseudovirions in quantities equivalent to that of infectious HIV-1 particles. Truncation of the Env cytoplasmic tail or replacement of the tail by that of the vesicular stomatitis glycoprotein (VSV-G) failed to enhance Env incorporation. The stability of gp120 retention on the pseudovirion surface following exposure to heat or to increasing concentrations of soluble CD4 (sCD4) was next investigated. Full-length Bal Env was not dissociated by incubation with sCD4 and was resistant to dissociation by heating, whereas significant amounts of NL4-3 gp120 were dissociated by sCD4 and heat. The use of uncleaved Gag rather than Gag-Pro for the core components of the pseudovirions enhanced retention of gp120.
Conclusions: Pseudovirion strategies utilizing primary isolate Env and uncleaved Gag may be ideal for the production of pseudovirion-based HIV-1 immunogens. CD4-dependent changes in Env conformation can be induced on the pseudovirion surface without substantial loss of gp120.
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