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185
Blocking of gp41-Mediated Fusion by Antibodies
C D Weiss, Y He, R Vassell, E de Rosny, L King, M Zaitseva, P Wingfield, and H Golding
Food and Drug Admin, Bethesda, MD
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Background: Gp120 binding to cellular receptors causes gp41 to undergo conformational changes, involving 2 heptad-repeat regions in the ectodomain of gp41that fold into a 6-helix bundle (hairpin) structure. Bundle formation facilitates virus entry in part by bringing viral and cellular membrane close together for membrane fusion. Peptides corresponding to these heptad-repeat domains are potent inhibitors of HIV infection. They appear to bind the prehairpin fusion intermediate and interfere with formation of the 6-helix bundle in a dominant-negative fashion. We tested whether antibodies against prehairpin and 6-helix bundle structures could bind gp41 and inhibit fusion.
Methods: Based on the highly conserved, heptad-repeat segments of gp41, we designed peptides and recombinant proteins that mimic the prehairpin and 6-helix bundle conformations of gp41. All constructs were characterized for inhibitory activity and ability to bind gp41, and used to immunize rabbits. Antibodies were analyzed in a variety of neutralization assays, including syncytia, infectivity, and reduced-temperature assays to slow down the fusion process.
Results: All constructs were highly immunogenic and elicited antibodies that bind gp41 preferentially after receptor activation. Antibodies against either prehairpin intermediate or 6-helix bundle could be shown to bind at the interfaces of target and effector cells. Importantly, these antibodies could also inhibit fusion and infectivity under certain conditions.
Conclusions: Antibodies have access to prehairpin and 6-helix bundles conformations of gp41 and are capable of inhibiting gp41-mediated fusion. The high degree of conservation of the heptad-repeat regions of gp41make them attractive targets for drug and vaccine development.
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