Figure 1.
HIV-1 envelope glycoprotein gp41 schematic and structure of the 2F5 antibody-peptide complex.
(A) The 2F5 (red) and 4E10 (blue) contiguous epitopes lie within the membrane proximal external region (MPER) of the HIV transmembrane envelope glycoprotein, gp41. (B) Crystal structure (PDB: 1TJI) of the antibody 2F5 heavy chain (black) and light chain (gray) in complex with the gp41 peptide (red). The third complementarity-determining region of the 2F5 heavy chain (CDRH3) is colored in green and magenta. Residues colored green (98PTT100 and 100EPIA100G) were removed to decrease the length of the CDRH3 loop. Insertions were also engineered into this region to elongate the length of the loop. Residues at the apex of the CDRH3 in magenta (100ALFGV100D) were not altered. The residue V100D is marked by the blue circle as this residue is substituted for by a W in several of the 2F5 variant antibodies.
Figure 2.
HIV neutralization values of the CDRH3 length-altered antibodies.
The top and middle panels display the CDRH3 sequences of the length-altered CDRH3 2F5 variants and their HIV neutralization activities. Residues colored in magenta (LFGV) were conserved while flanking residues colored in green (PTT and PIA) were removed sequentially to produce the shorter loop antibodies. In brown are the glycine (G) residues inserted to elongate the CDRH3 length. The W and Y substitutions in the altered-CDRH3 are colored in blue and orange, respectively. The bottom panel shows neutralization values achieved by selected CDRH3-altered 2F5 variants against a larger set of HIV isolates.
Figure 3.
CD analysis and binding kinetics of the CDRH3 length-altered antibodies to selected MPER probes.
(A) CD spectra of 10 µM wt MPER peptide and linked peptide in H2O. (B) Shown on the vertical axis are the affinity constants (KD), of the wt 2F5 Mab and the CDRH3 length-altered variants (r2, r4, r6, e2 and e4) for the selected MPER probes. Gray colored bars indicate antibody interactions with the MPER peptide analyte, white bars represent interactions with the linked peptide, and black bars indicate interactions with the ES2 2F5-epitope scaffold. The dotted line indicates an arbitrary cut off which segregates low affinity interactions (KD>100 nM) from high affinity interactions (KD<100 nM). For a complete description of binding kinetic constants see Table 1. (C) The Octet binding curves of wt 2F5 Mab to the three probes in solution are shown for the wt MPER peptide, the linked peptide, and the ES2 2F5-epitope scaffold. The experimental binding curves are shown in blue and the applied Languir 1∶1 model fitting of the curves are shown in red. Representative binding curves of the CDRH3-length altered 2F5 variants r4 and e4 are shown below. For the binding curves of all variants, see supplementary figures 1S and 2S.
Table 1.
Antibody binding kinetic constants to selected MPER probes.
Figure 4.
Schematic representation of the 2F5 wt and loop-shortened CDRH3 and approximate location of the MPER.
Left, the wt 2F5 CDRH3 apex is shown as a schematic representation based upon the mAb structure. Residues P98 to A100G are shown with each residue represented by a colored cylinder. On the right, a schematic of the length-altered version, where residues T99,T100, P100E and I100F , represented by the green cylinders with the discontinuous border, were deleted. The red residues, L100A, F100B, G100C and V100D, were maintained for the length alterations as described in the text. The W and Y substitutions are noted next to the original residue in the variant schematic depiction (Rosetta modeling of a variant CDRH3 was performed, but clashes with peptide binding were assessed to not be compatible with the predicted conformations). The letters a, b and c represent the locations of the second W substitutions to generate antibody variants r4W2a, b and c, respectively.
Figure 5.
MPER Binding and HIV neutralization of mutant antibodies after W/Y substitutions.
(A) Antibody affinity constants corresponding to antibody binding to MPER peptide before and after the W/Y substitutions, in white and shaded columns, respectively. (B) Statistical analysis comparing the means of the binding measurements to the MPER peptide from the antibodies before and after the W substitutions. The affinity constant (KD), the on-rate (kon) and the off-rate (koff) are shown on the vertical axis with each dot representing antibody binding to the MPER peptide. Closed circles represent antibodies with no Ws in the CDRH3 while open circles represent antibodies with W substitutions. Arrows indicate paired antibodies before and after the substitutions. The p values were obtained by subjecting the data to a paired t test statistical analysis. (C) Correlation between antibody KD to MPER peptide, left panel, or KD to ES2 scaffold, right panel, and HIV neutralization. Antibody IC50 values are shown on the vertical axis and the affinity constant (KD) to the MPER peptide in the horizontal axis. Red circles represent antibody IC50 values against the sensitive viruses (HXBc2, MN and 7312a-C3) and blue squares are IC50 against the more resistant isolates. Above the plots, in the gray boxes, are the names of the antibodies whose measurements are found directly underneath. The data were subjected to a Pearson correlation test, two-tailed resulting in a statistically significant p-value (p = 0.0052) and a positive correlation r-value (r = 0.695 ) for the wt MPER peptide and a non-significant p-value for the ES2 scaffold (p = 0.669 and r = 0.125).
Figure 6.
Model of the 2F5 antibody interacting with the HIV spike and MPER alanine scanning mutagenesis.
(A) Left, top view of the trimeric MPER crystal structure (PDB: 3G9R) as it may appear in the HIV unliganded primary isolate trimer prior to receptor engagement. Middle, following receptor engagement there is a putative torsional movement of the individual protomers within the spike, exposing the MPER, as indicated by the blue arrows. Right, when the 2F5 antibody is finally bound to its epitope. (B) Model of the HIV spike with gp120 (green) bound to the antibody 17b (orange), the primary virus receptor CD4 (yellow) (PDB:2NXY) and the MPER (modified from PDB:3G9R) (red [2F5 eptitope], blue [4E10 epitope] and yellow) bound to the antibody 2F5 (black) with the wt CDRH3 (magenta) positioned within the groove between two of the MPER helices. The aromatic residues of the MPER are shown. (C) Alanine scanning of the 4E10 MPER region 669–673 and effects on recognition of 4E10, wt 2F5 and the CDRH3 and W/Y variants are shown. The bars represent fold-increase or -decrease in the binding constant (KD) of the Mabs to the alanine-substituted peptides compared to KDs for the wt MPER peptide. A positive bar indicates an affinity increase and a negative bar indicates an affinity decrease. The bars are color-coded for each ligand Mab. For context, the MPER residues comprising the 2F5 and 4E10 epitopes are shown and the residues 669–673 scanned by the alanine substitutions are as indicated.
Figure 7.
Bars represent ELISA optical density readings at 450 nm wavelength corresponding to the binding of antibodies (2F5 in black, 4E10 in green, 11F10 in gray and the CDRH3-altered variants in blue, violet, yellow, brown and orange) to CL (top panel), CL:B2G1 complex (middle) and B2G1 (bottom). Light blue color designates CDRH3 altered antibodies with no W substitutions, violet with one W substitution, yellow with two W substitutions, brown with three W substitutions, and orange with one W and one or two Y substitutions. The red and dark blue bars represent the positive and negative binding levels of control IgG included in the assay. The dotted line indicates an arbitrary “cut-off” between the negative and positive values.