Home Search Abstracts Browse Sessions Program Committee E-mail Abstract Author View Session

Session 32 Oral Abstracts
Clinical and Preclinical Vaccine Studies
Wednesday, 4 - 5:30 pm
Presentation Time: 4:45 pm
Room 2008


167
Enhanced Envelope Incorporation in Pseudovirions Produced by Co-transfection with Codon-optimized Envelope Genes
D L Kothe*1, E Chertova2, Y Li1, K P Zammit1, J M Decker1, B T Korber3, G M Shaw1, J D Lifson2, and B H Hahn1
1Univ. of Alabama at Birmingham, USA; 2AIDS Vaccine Prgm., SAIC Frederick Inc., MD, USA; and 3Los Alamos Natl. Lab., Los Alamos, NM

Background:  HIV-1 particles have been shown to contain small amounts of envelope glycoprotein on their surface, which may affect their immunogenicity in vivo. Higher levels of envelope glycoprotein incorporation into particle-based immunogens may improve neutralizing antibody responses in vaccinated individuals.

Methods:  Codon-optimized and rev-dependent 89.6P envelope genes, as well as a codon-optimized subtype B consensus env gene, were co-transfected with an env-minus HIV-1 (SG3) backbone to produce infectious pseudovirions. The 2 halves of an 89.6P infectious molecular clone were also transfected into 293T cells to produce infectious virus particles. Pseudovirions and the 89.6P infectious clone-derived virions were analyzed for envelope content by Western blot and tested for infectivity using the JC53-BL cell assay. gp160, gp120, and p24 levels per particle were analyzed by ELISA and HPLC.

Results:  Western blot analysis of sucrose purified pseudovirions generated by co-transfection of the codon-optimized env gene with an env-minus HIV-1 (SG3) revealed at least 10-fold higher levels of gp160 envelope glycoprotein incorporation compared with pseudovirions generated by co-transfection of the env-minus HIV-1 (SG3) backbone with the rev-dependent wild type env gene. This was confirmed by HPLC analyses that revealed dramatically increased virion envelope content in pseudovirions containing codon-optimized Env compared with transfection or infection derived HIV-1 virion preparations. Finally, optimized Env containing HIV-1 particles were more infectious than wildtype Env-containing HIV-1.

Conclusions: Trans-complementation of env-minus HIV-1 with codon usage optimized env genes generates pseudovirions that contain at least 10-fold more envelope glycoprotein than wild type HIV-1. These pseudovirions are highly infectious suggesting that at least some of the envelope proteins are maintained as trimers on the particle surface. Such pseudovirions (or virus-like particles generated from codon usage optimized env genes) when used as immunogens may elicit markedly improved neutralizing responses.

Keywords: codon-optimized; envelope; HIV-1