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15 Fusion Proteins of gp120 and gp41 Joined by Flexible Linkers as Potent Inhibitors of HIV-1 Entry and Candidate Vaccines
Y.-H. Chow*1, O. Wei1,2, C. Broder2, and D. Dimitrov1
1NCI-FCRDC, NIH, Frederick, MD, USA and 2Uniformed Services Univ. of the Hlth. Sci., Bethesda, MD, USA
Background: HIV-1 entry into cells involves a series of conformational changes of its envelope glycoprotein (Env) initiated by binding to receptor molecules. We hypothesized that by joining gp120 and gp41 by flexible linkers of different length, Env and its subunits may not only be stabilized, but also the extent of conformational changes that gp41 would undergo after receptor engagement would be limited and impart structural constraints of Env conformation changes and accumulation of fusion intermediates of sufficient concentration for elicitation of unique or desired immune responses.
Methods: 89.6 gp120 and truncated gp41 (without transmembrane domain and cytoplasmic tail) were inserted in pEF1/Myc-His and the resulting plasmid used for transfection. The protein was purified and used for inhibition of HIV-1 entry and HIV-1 Env-mediated fusion measured by reporter gene assays.
Results: Three different fusion proteins were developed and tested: without linker and with 13 and 23 aa residue linkers. The fusion proteins with the linker inhibited about 100-fold more potently entry of several different R5, X4, and R5X4 HIV-1 isolates (50% inhibitory activity on the order of nanograms per milliliter) than the fusion protein without linker obtained under the same conditions. Immunizations of mice are in progress.
Conclusions: The finding that fusion gp120-gp41 proteins, which were developed to obtain stable Envs and entry intermediates induced by receptor molecules, exhibit very potent inhibitory activity against a variety of HIV-1 isolates suggests the existence of structurally relevant and broadly conserved elements that were exposed even in the absence of receptor-mediated activation. These findings open new perspectives for development of novel inhibitors and Env-based vaccines.
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