Enhancement of α-Helix Mimicry by an α/β-Peptide Foldamer via Incorporation of a Dense Ionic Side-Chain Array

We report a new method for preorganization of α/β-peptide helices, based on the use of a dense array of acidic and basic side chains. Previously we have used cyclically constrained β residues to promote α/β-peptide helicity; here we show that an engineered ion pair array can be comparably effective, as indicated by mimicry of the CHR domain of HIV protein gp41. The new design is effective in biochemical and cell-based infectivity assays; however, the resulting α/β-peptide is susceptible to proteolysis. This susceptibility was addressed via introduction of a few cyclic β residues near the cleavage site, to produce the most stable, effective α/β-peptide gp41 CHR analogue identified. Crystal structures of an α- and α/β-peptide (each involved in a gp41-mimetic helix bundle) that contain the dense acid/base residue array manifest disorder in the ionic side chains, but there is little side-chain disorder in analogous α- and α/β-peptide structures with a sparser ionic side-chain array. These observations suggest that dense arrays of complementary acidic and basic residues can provide conformational stabilization via Coulombic attractions that do not require entropically costly ordering of side chains

Cross-boosting of clade A and clade B autologous NAb responses by clade C trimers.

<p>NAb titers at the indicated time points for all five animals in the listed groups are compared. Bars show arithmetic means + s.e.m. The clade C trimer boosts occurred at weeks 36 and 48. <b>A</b> and <b>C</b>, BG505.T332N titers; <b>B</b> and <b>D</b>, B41 titers.</p

Mapping autologous NAb specificities using BG505.T332N, B41 and CZA97 virus mutants.

<p><b>A</b>, Neutralization of BG505.T332N virus mutants. The values recorded for the various mutant viruses are the percentage neutralization at a dilution of 1/50, relative to the BG505.T332N parental virus (labeled WT and defined as 100%), and are the averages of 2 replicates ± s.e.m. Red boxes highlight fully or substantially resistant viruses (<25% neutralization); yellow, moderately resistant viruses (25–75% neutralization); green, sensitive viruses (>75% neutralization). The Q130N, S241N and P291T changes introduce N-linked glycans at positions 130, 241 and 289, respectively. The S241N+P291T double mutant contains glycans at both positions 241 and 289. The MG505 cl.A2 and cl.H3 viruses differ from BG505.T332N at several positions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s002" target="_blank">S1 Fig</a>). Of note is that MG505 cl.A2 has a lysine residue at position-241 (i.e., as per the S241K mutant), whereas cl.H3 has a glycan site (i.e., as per the S241N mutant). A glycan is present at position-289 in both MG505 clones. The K241S change in MG505 cl.A2 restores the Ser residue that is present at position-241 in the BG505.T332N virus. Full titration curves for the sera showed only a single example of reduced neutralization of the glycan knock-in mutants, compared with BG505.T332N, that was not evident at the standard test dilution of 1/50 (rabbit #5739 at week 62; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s005" target="_blank">S4 Fig</a>). Overall, the extent of neutralization of the glycan knock-in mutants did not correlate well with the titers against the wild-type BG505.T332N virus (Spearman rank correlation for the 241-glycan knock-in: r = 0.30, p = 0.099; for the 289-glycan knock-in: r = 0.21, p = 0.25; for the double mutant: r = 0.27, p = 0.15). <b>B</b>, Neutralization of B41 virus mutants. The organization is the same as in panel-A. The B41 parental virus is labeled WT and its neutralization defined as 100%. The N132T and A291T changes into this virus introduce N-linked glycans at positions 130 and 289, respectively. The CH01 and VRC01 bNAbs were used as control reagents for assessing overall neutralization sensitivity (only shown for B41 mutants for which a partial effect on CH01 neutralization was observed). <b>C</b>, Neutralization of CZA97 virus mutants. The organization is the same as in panel-A. The serum dilution used was 1/60. The CZA97 cl.12 parental virus is labeled cl.12 and its neutralization defined as 100%. The CZA97 cl.29 virus differs from cl.12 at several positions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s002" target="_blank">S1 Fig</a>) but of note is that it contains a glycan at position-411. The CZA97 cl.12-D411N and cl.29-N411D mutants contain and lack glycans at position-411, respectively.</p

Individual rabbits immunized with three different trimers can develop moderate to strong autologous NAb responses to each of them^a.

<p>Individual rabbits immunized with three different trimers can develop moderate to strong autologous NAb responses to each of them<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#t001fn001" target="_blank">^a</a>.</p

Correlations between trimer-binding and neutralizing antibody titers.

<p>The scatterplots show neutralization titers on the y-axes and the trimer-binding antibody titers on the x-axes. Within each plot the symbols corresponding to neutralization of the BG505.T332N and B41 viruses are color-coded as indicated on the figure panels. Spearman correlation coefficients (r-values) and the corresponding significances (p-values) are color-coded analogously. Correlation analyses were performed for both BG505 and B41 NAb and binding antibody titers for sera from the early monovalent immunogen regimens during weeks 4–36 (top panel, group 1; lower panel, group 3).</p

<i>De novo</i> cross-neutralization of BG505.T332N (clade A) induced by clade C trimers after priming with clade B trimers.

<p>BG505.T332N NAb titers at the indicated time points for the five animals in group 1, which received only clade B and C trimers are shown. Bars show arithmetic means + s.e.m. The NAb titers were compared with the cut-off value of 20 by the Wilcoxon signed rank test (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#sec011" target="_blank">Results</a>).</p

Autologous NAb responses to sequentially delivered clade A and clade B trimers.

<p>The neutralization titers (IC₅₀) against Tier-2 Env-pseudotyped viruses are plotted on the y-axis as a function of time after the first immunization on the x-axis (weeks). The scales are kept constant within each panel to facilitate comparisons among groups. The schedule is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.g001" target="_blank">Fig 1C</a> with immunizations at weeks 0, 4, 20, 24, 36, 48 and 60, and for rabbits #5734–5, #5735–5 and #5736–5 also at week 73. Blood was taken for analysis immediately before and 2 weeks after each immunization. The test viruses were as follows: <b>A</b> and <b>C</b>, BG505.T332N; <b>B</b> and <b>D</b>, B41. The curves connecting the reciprocal neutralization titers are color-coded for each rabbit as per the key associated with each group. Thus, changes in titers can be monitored over time both for individual animals and on a group-wide basis.</p

Neutralization of heterologous Tier-1 viruses.

<p>The arithmetic mean neutralization titers (IC₅₀) against the Env-pseudotyped Tier-1 viruses MN.3 (clade B) and MW965.26 (clade C), derived at DUMC, are plotted over time for the rabbit groups denoted by the key to the right.</p

Neutralization of heterologous Tier-2 viruses.

<p>Neutralization of heterologous Tier-2 viruses.</p

A glycan hole in the BG505 trimer.

<p>Left panel: The BG505 SOSIP.664 trimer and the BG505.T332N virus lack glycans at positions 241 and 289, which results in a large exposed peptide surface on the side of gp120 near the gp120/gp41 interface. For clarity one protomer of the BG505 SOSIP.664 trimer has been colored, with the polypeptide chain in blue ribbons and glycans denoted by green spheres. Right panel: The restored glycans at positions 241 and 289 (as in the BG505 S241N+P291T double mutant virus) now mask the underlying peptide surface. The individual glycans at positions 241 and 289 both have a substantial masking effect, accounting for the similar phenotypes of the single and double mutant knock-in viruses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.s009" target="_blank">S3 Table</a>).</p