Abstract
We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.
Highlights
The surface envelope (Env) glycoproteins of HIV-1, gp120 and gp41, mediate fusion of the viral and cell membranes [1]
In the case of 5-helix, a single chain pre-hairpin mimetic that presents a single antibody binding site in which the N-heptad repeat (N-HR) trimer is surrounded by two C-HR helices, the binding affinities differ by,20 (#10 vs,200 nM) [35]; for N35CCG-N13, an N-HR trimer construct stabilized by disulfide bridges [19] that presents three antibody binding sites, the binding affinities differ by,8 (,30 vs,250 nM; Fig. S1 in File S1); and for CCIZN36, an N-HR trimer construct stabilized by a disulfide-linked leucine zipper [37], which presents three antibody binding sites, the binding affinities differ by a factor of only 2 (,5 versus,10 nM) [36]
This variation might be attributable to differences in conformational flexibility of the N-HR trimer between the various pre-hairpin mimetics that may facilitate accommodation of the conformational differences within the CDR-H2 binding loops of Fab8066 and Fab8062 [36]
Summary
The surface envelope (Env) glycoproteins of HIV-1, gp120 and gp, mediate fusion of the viral and cell membranes [1]. The initial events in the fusion process involve the binding of CD4 and the chemokine co-receptor to gp120 triggering a series of conformational changes in both gp120 and gp that culminate in fusion of the viral and cell membranes [2,3,4,5,6,7] Steps in this process, representing a possible ‘‘activated’’ state of gp120/ gp, have recently been visualized by crystallography and cryoelectron microscopy of a soluble cleaved HIV-1 Env trimer [8,9]. Terminal heptad repeat (C-HR, residues 623–663) do not interact with one another and both structural elements are solvent accessible This structure approximates the postulated pre-hairpin intermediate in which the viral and cell membranes are bridged via the C- and N-termini of gp, respectively [4,10,11]. Various constructs have been devised to mimic both the pre-hairpin intermediate [17,18,19] and six-helix bundle conformations of gp (Figs. 1A and D) [12,16,18]
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