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Background: The monoclonal antibodies 2F5 and 4E10 have broadly neutralizing activity against HIV-1, recognize epitopes in the ectodomain of gp41, and are models for HIV-1 vaccine design.  We hypothesized that gp41 modified to mimic the pre-hairpin conformation might elicit neutralizing antibody responses when expressed as part of an HIV-1 viral-like particle (VLP).

Methods: We modified the HIV-1 subtype B consensus gp41 sequence intending to present the membrane-proximal region in its pre-hairpin state.  HIV gag and modified gp41 genes were inserted into a dual promoter plasmid for co-expression and for use as the priming component of a DNA prime-VLP boost vaccine. We analyzed gene expression by 2F5 specific Western blot (WB) and p24 ELISA following transfection of 293T cells.  Surface expression of gp41 in 293T cells was assessed via flow cytometry using 2F5.

We employed a dual promoter baculovirus expression system for large-scale VLP production. Following insertion of gp41 and Gag into the baculovirus system, we assessed gene expression by WB, p24, and flow-cytometry.  VLP production in both 293T and SF9 cells was confirmed via electron microscopy and WB following sucrose cushion purification.

Results: Both HIV-1 Gag and modified gp41 designs are well expressed in 293T cells as assessed via WB.  Flow cytometric analysis readily detects envelope proteins on the cell surface.  Production of VLPs was observed using electron microscopy. Antigenic analysis following sucrose cushion purification demonstrates the presence of both gp41 and Gag.

Baculoviruses expressing modified gp41 and gag in SF9 cells achieve high titers following triple-plaque purification. Flow cytometric analysis demonstrated 50% of infected cells co-expressing gp41 and Gag.  In a sucrose step purification system, gene expression corresponds to time of optimal VLP production.  Electron microscopy of infected cells demonstrates immature VLPs.  Both modified gp41 envelope and Gag are detected in VLPs following sucrose cushion purification.  VLP yield as measured by p24 approaches 1mg/ml. In a pilot experiment, antibody responses to HIV Gag are readily detected 10 days following either a single injection of DNA or purified VLPs in mice.

Conclusion: These dual promoter systems provide a strategy for expression of modified HIV gp41 in the context of a VLP.  This approach may present gp41 in a conformation capable of neutralizing antibody induction.  Immunogenicity studies are ongoing.

Keywords: gp41; viral-like particle; baculovirus