Research is focused on studying the events involved in HIV-1 fusion, the development of fusion inhibitors directed against gp41 and the development of monoclonal antibodies with broadly neutralizing activity that are directed against gp41. The HIV-1 Envelope (Env) proteins that mediate membrane fusion represent a major target for the development of new AIDS therapies. Three classes of Env-mediated membrane fusion inhibitors have been described that specifically target the pre-hairpin intermediate conformation of gp41. Class 2 inhibitors bind to the C-terminal heptad repeat (C-HR) of gp41. The single example of a class 3 inhibitor targets the trimeric N-terminal heptad repeat (N-HR) of gp41 and has been postulated to sequestrate the N-HR of the pre-hairpin intermediate through the formation of fusion incompetent heterotrimers. We have now shown that NCCG-gp41, a class 2 inhibitor, and N36Mut(e,g), a class 3 inhibitor, synergistically inhibit Env-mediated membrane fusion for several representative HIV-1 strains (X4 and R5) in both a cell fusion assay (with membrane-bound CD4) and an Env-pseudotyped virus neutralization assay. We have also succeeded in obtaining a monoclonal Fab (Fab 3674) selected from a human non-immune phage library by panning against the chimeric construct NCCG-gp41 (that comprises an exposed coiled-coil trimer of gp41 N-helices fused in helical phase onto the minimal thermostable ectodomain of gp41) that effectively neutralizes diverse laboratory-adapted B-strains of HIV-1 and primary isolates of subtypes A, B and C in an Env-pseudotyped virus neutralization assay, albeit with reduced potency (25x) compared to 2F5 and 4E10. Alanine scanning mutagenesis maps a novel epitope to a shallow groove on the N-helices of gp41 that is exposed between two C-helices in the fusogenic six-helix bundle conformation of gp41. We have shown that bivalent Fab 3674 and the C34 peptide (a potent fusion inhibitor derived from the C-helix of gp41) act at similar stages of the fusion reaction and to neutralize HIV-1 synergistically, providing additional evidence that the epitope of Fab 3674 is new and distinct from the binding site of C34.More recently we have shown that the class 3 inhibitor N36Mut(e,g) prolongs the temporal window during which the virus is susceptible to neutralization by the bivalent Fab-3674, and that bivalent Fab-3674 and N36Mut(e,g) neutralize HXB2 and SF-162 strains of HIV-1, as well as diverse primary B and C clade HIV-1 strains, synergistically in a Env-pseudotyped virus neutralization assay. N36Mut(e,g) also rescues neutralizing activity of the monovalent Fab-3674 against resistant HIV-1 strains and renders a series of related non-neutralizing Fabs neutralizing. Moreover, N36Mut(e,g) exhibits the same effects on the broadly neutralizing 2F5 and 4E10 monoclonal antibodies directed against the membrane proximal extended region of gp41. Very recently, we have subjected Fab 3674 to affinity maturation against the NCCG-gp41 antigen by targeted diversification of the CDR-H2 loop to generate a panel of Fabs with diverse neutralization activity. Three affinity-matured Fabs selected for further study, Fabs 8060, 8066 and 8068, showed significant increases in both potency and breadth of neutralization against HIV-1 pseudotyped with envelopes of primary isolates from the standard subtypes B and C HIV-1 reference panels. The parental Fab 3674 is 10-20 fold less potent in monovalent than bivalent format over the entire B and C panels of HIV-1 pseudotypes. Of note is that the improved neutralization activity of the affinity-matured Fabs relative to the parental Fab 3674 was, on average, significantly greater for the Fabs in monovalent than bivalent format. This suggests that the increased avidity of the Fabs for the target antigen in bivalent format can be partially offset by kinetic and/or steric advantages afforded by the smaller monovalent Fabs. Indeed, the best affinity-matured Fab (8066) in monovalent format (50 kDa) was comparable in HIV-1 neutralization potency to the parental Fab 3674 in bivalent format (120 kDa) across the subtypes B and C reference panels.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2009
Total Cost
$389,430
Indirect Cost
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State
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Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo et al. (2013) Complexes of neutralizing and non-neutralizing affinity matured Fabs with a mimetic of the internal trimeric coiled-coil of HIV-1 gp41. PLoS One 8:e78187
Dogo-Isonagie, Cajetan; Lam, Son; Gustchina, Elena et al. (2012) Peptides from second extracellular loop of C-C chemokine receptor type 5 (CCR5) inhibit diverse strains of HIV-1. J Biol Chem 287:15076-86
Huang, Ying; Cai, Mengli; Clore, G Marius et al. (2011) No interaction of barrier-to-autointegration factor (BAF) with HIV-1 MA, cone-rod homeobox (Crx) or MAN1-C in absence of DNA. PLoS One 6:e25123
Shahzad-ul-Hussan, Syed; Gustchina, Elena; Ghirlando, Rodolfo et al. (2011) Solution structure of the monovalent lectin microvirin in complex with Man(alpha)(1-2)Man provides a basis for anti-HIV activity with low toxicity. J Biol Chem 286:20788-96
Gustchina, Elena; Li, Mi; Louis, John M et al. (2010) Structural basis of HIV-1 neutralization by affinity matured Fabs directed against the internal trimeric coiled-coil of gp41. PLoS Pathog 6:e1001182
Cai, Mengli; Huang, Ying; Craigie, Robert et al. (2010) Structural basis of the association of HIV-1 matrix protein with DNA. PLoS One 5:e15675
Gustchina, Elena; Louis, John M; Frisch, Christian et al. (2009) Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutraliza Virology 393:112-9
Nelson, Josh D; Kinkead, Heather; Brunel, Florence M et al. (2008) Antibody elicited against the gp41 N-heptad repeat (NHR) coiled-coil can neutralize HIV-1 with modest potency but non-neutralizing antibodies also bind to NHR mimetics. Virology 377:170-83
Gustchina, Elena; Bewley, Carole A; Clore, G Marius (2008) Sequestering of the prehairpin intermediate of gp41 by peptide N36Mut(e,g) potentiates the human immunodeficiency virus type 1 neutralizing activity of monoclonal antibodies directed against the N-terminal helical repeat of gp41. J Virol 82:10032-41