Peptide Targeted by Human Antibodies Associated with HIV Vaccine-Associated Protection Assumes a Dynamic α-Helical Structure

<div><p>The only evidence of vaccine-induced protection from HIV acquisition in humans was obtained in the RV144 HIV vaccine clinical trial. One immune correlate of risk in RV144 was observed to be higher titers of vaccine-induced antibodies (Abs) reacting with a 23-mer non-glycosylated peptide with the same amino acid sequence as a segment in the second variable (V2) loop of the MN strain of HIV. We used NMR to analyze the dynamic 3D structure of this peptide. Distance restraints between spatially proximate inter-residue protons were calculated from NOE cross peak intensities and used to constrain a thorough search of all possible conformations of the peptide. α–helical folding was strongly preferred by part of the peptide. A high-throughput structure prediction of this segment in all circulating HIV strains demonstrated that α–helical conformations are preferred by this segment almost universally across all subtypes. Notably, α–helical conformations of this segment of the V2 loop cluster cross-subtype-conserved amino acids on one face of the helix and the variable amino acid positions on the other in a semblance of an amphipathic α–helix. Accordingly, some Abs that protected against HIV in RV144 may have targeted a specific, conserved α–helical peptide epitope in the V2 loop of HIV’s surface envelope glycoprotein.</p></div

Reactivity of RV144 subject sera with MN peptide.

<p>Positive or negative reactivity with the MN peptide, as determined by ELISA titers, for week 26 serum immunoglobulin (Ig) samples from the RV144 case–control study (Haynes et al) with participants stratified according to HIV infection status and treatment assignment. ELISAs were performed as described in Haynes et al. Positive reactivity of a subject’s serum Ig with the MN peptide is defined as a titer (1/serial dilution) of greater than 15, which is the limit of detection for this specific ELISA assay. Values below the limit of detection (LOD) are negative and set to half the LOD. A) Data for vaccinated subjects stratified by HIV infection status. The denominator in the proportion at the top indicates the number of vaccinated subjects (e.g. 205 total subjects included in the RV144 case control study who were vaccinated were not infected with HIV at the end of study follow-up). The numerator in the proportion at the top is the number of subjects whose serum Ig exhibited a positive reaction with the MN peptide. B) Data for both vaccinated and placebo subjects displayed as a dot plot. Sex and immune-response categories are indicated by the color and shape of the points.</p

Quantification of the structural preference of the α-helical V2^167-179 conformation for the β-sheet conformation.

<p>Quantification of the structural preference of the α-helical V2^167-179 conformation for the β-sheet conformation.</p

Sequence conservation in the context of alpha-helical structure.

<p>The sequence logo of the V2 loop of circulating HIV strains showing positional conservation of residues in the loop. The most highly conserved residues in the V2 loop map to the same face of the alpha-helical structure in 3D space as shown by the red arrows.</p

Computational folding of HIV V2 MN peptide shows a preference for the alpha-helical secondary structure.

<p>(A). Shows the lowest energy conformation of the <i>ab</i>-initio folding (top left) and the top ten conformations of the NMR-restrained ICM folding. Segments of the peptide that adopt alpha-helical structure are depicted in red. (B). Sequence alignment of the top ten NMR-restrained folding conformations with their corresponding energy scores. Red color indicates segments of the peptide that have adopted an α–helical conformation.</p

Distribution of the helicity degree of the folded V2^155-176 variants.

<p>3D structure of each unique V2^155-176 sequence recorded in the LANL HIV database was predicted using the <i>ab initio</i> folding protocol as described in the Methods section, and the helicity degree (in %) was calculated. The higher the helicity degree is, the more residues of a given sequence tend to accept an α-helical conformation.</p

NMR structural analysis of HIV V2 MN peptide supports a propensity for alpha-helical structure.

<p>(A) Representative amide proton to side-chain correlations from a 2D homonuclear NOESY spectrum, (B) Inter-residue H^N-H^N correlations from 2D NOESY, and (C) 2D natural abundance C¹³-edited HSQC spectrum of the Calpha region. The residue specific annotations are displayed in panels (B) and (C).</p

Distribution of the energy difference between the α-helical and β-sheet V2^167-179 conformations on the informatics of all V2^167-179 variants.

<p>For each unique V2^167-179 sequence recorded in the LANL HIV database two homology models were built as described in the Methods section, and the difference between the energies of these two models (dEnergy) was calculated by subtracting the energy of the β-sheet model from the energy of the α-helical model. Thus, negative dEnergy scores suggest that the α-helical conformation is energetically favorable in comparison to the β-sheet conformation.</p