Structure and immunogenicity of a peptide vaccine, including the complete HIV-1 gp41 2F5 epitope: implications for antibody recognition mechanism and immunogen design

Abstract

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund's adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.

Keywords: Fourier Transform IR (FTIR); HIV-1; Liposomes; Nuclear Magnetic Resonance; Peptide Conformation; Vaccine Development.

FIGURE 1.

Design of MPER-derived peptide vaccine. A, scheme describing the HIV-1 gp41 organization and the sequence of the MPER peptide vaccine used in this study (HIV-1 Env residues 656–683, numbering and sequence derived from the prototypic HXBc2 isolate). The gp41 ectodomain regions designated in the top diagram include the following abbreviations: FP, fusion peptide; NHR and CHR, N- and C-terminal helical regions, respectively; Cyt, cytosolic domain. The MPER sequence below highlights the five Trp residues in green and the core epitope residues recognized by 2F5 antibody underlined. The line on top spans the extended 2F5 epitope as defined by proteomic analyses (34). Blue asterisks denote residues implied in secondary binding by CDR-H3 loop (25) and the box an aromatic rich anchor to the membrane interface. B, structures adopted by MPER-derived peptides. PDB accession numbers indicated in the panel designate structures in solution (1LCX and 1MZI) or in contact with DPC micelles (1JAV and 2PV6). Lateral side chains of Trp residues are depicted in green to align the structures with the MPER amino acid sequence.

FIGURE 2.

NMR parameters for MPERp. A, bar graphics showing the Δδ_Hα (Δδ_Hα = δ_Hα^observed − δ_Hα^RC, ppm) values as a function of sequence in 20 mm DPC (black bars) or 25% HFIP (gray bars) at pH 7.0 and 25 °C. Dashed lines indicate the random coil (RC) ranges. Random coil values for C_αH protons were taken from Wishart et al. (66). The N- and C-terminal residues are excluded because of charged end effects. B and C, NOE summaries for the peptide in 20 mm DPC and 25% HFIP. The intensities of the sequential NOEs, classified as strong, medium, and weak, are indicated by the thickness of the lines.

FIGURE 3.

Selected NOESY spectral regions of MPERp in DPC or HFIP conditions (left and right panels, respectively). The depicted regions show intraresidue α-β and nonsequential α-β (i, i+3) NOES. The nonsequential NOEs are boxed.

FIGURE 4.

Structures adopted by MPERp in 25% HFIP or DPC 20 mm and classical vaccine adjuvants. A, calculated NMR structures and corresponding IR spectra. Lateral side chains of Trp residues are depicted in green and aligned with the amino acid sequence as in the caption for Fig. 1. Both structures are continuous helices. Additional structural signatures common to all calculated models are highlighted (see text). The corresponding IR spectra displayed on the left disclose in red the absorption band components arising from the helical conformation (Table 2). B, IR absorption in the amide I region by MPERp in D₂O-based buffer mixed with solutions of alum and Freund's adjuvant (FA) following the procedure used for preparing vaccines. In all panels the IR spectra were decomposed into different band components (numerical values are disclosed in Table 3). Red dotted lines correspond to α-helix components.

FIGURE 5.

Correlation between 2F5 antibody function and binding to liposomal vaccines. A, cell entry inhibition assay. Left, pseudoviruses were preincubated with MAb2F5 or the recombinant 2F5 Fab constructs, and single cell entry events were monitored by FACS after incubation with TZM-bl target cells. Fab2F5 WT inhibited cell entry (blue), albeit with lower potency than the bi-functional mAb (black). In contrast Fab2F5 ΔCDR-H3 was almost completely unable to inhibit the process (red). Right, displays specificity controls for the HIV-1 Env-mediated cell entry. bNAbs (2F5 and 4E10) and T-20 were applied at 2 and 50 μg/ml, respectively. Means ± S.D. of six measurements in three independent experiments are displayed. B–E, vesicle flotation experiments in sucrose gradients. Rhodamine-labeled liposomes were collected in the 1st and 2nd fractions (i.e. floating fractions) (B). C, MPERp (30 μm) was incubated in solution in absence (top) or presence of liposomes (peptide-to-lipid ratio of 1:100, bottom panels) for 15 min before centrifugation. The presence of the peptide in the different fractions was probed with MAb2F5 in Western blot. Virtually all input peptide co-floated with liposomes indicating quantitative partitioning into membranes. D, MAb2F5 (15 μg ml⁻¹) was incubated for 15 min with MPERp-containing or empty liposomes before centrifugation (top and bottom panels, respectively). Consistent with antibody binding to membrane-inserted MPERp, MAb2F5 was recovered from the floating fractions upon incubation with peptide-containing liposomes. Similarly, Fab2F5 WT co-floated with both types of MPERp-containing liposomes (E, top panels). In contrast, in the case of POPG-MPERp vesicles, Fab2F5 ΔCDR-H3 was predominantly recovered from pellets (E, bottom panels).

FIGURE 6.

Combined IR and molecular dynamics simulations of MPERp interacting with POPC/Chol/PA (A) or POPG (B) lipid bilayers. Left panels, IR absorption in the amide I region by MPERp in D₂O-based buffer mixed with solutions of POPC/Chol/PA (2:1.5:0.2 mol/mol) liposomes or POPG liposomes, following the procedure used for preparing vaccines. In both panels, the IR spectra were decomposed into different band components (numerical values are disclosed in Table 3). Red dotted lines correspond to α-helix components. Right panels, snapshots of MPERp were taken at times 215 and 233 ns (top and bottom, respectively). Side views of the peptides display in space-filling representation residues Lys-665/Trp-666 in red and Leu-669/Trp-672/Phe-673 in blue. Phospholipids are shown in stick representation. Residues depicted in green have at least one atom within <3 Å from the phospholipid molecules.

FIGURE 7.

Immunogenicity of MPERp formulated with different adjuvants. A, midpoint IgG titers in sera from rabbits immunized with MPERp in the different adjuvants. Sera were titrated in ELISA using 1.4 μm MPERp. Experimental values were adjusted to sigmoid dose-response curves, and midpoint titers were determined as EC₅₀ values (i.e. the dilutions giving 50% response between minimum and maximum). Values displayed in panels correspond to 1/dilution × 10³ ± S.E. B, affinity for the 2F5 epitope of rabbit IgG purified from sera with 2F5ep-Cys (NEQELLELDKWASLWN-C) peptide immobilized onto a beaded agarose support. Competitive ELISAs were performed using plates coated with MPERp (1.4 μm). Prior to adding to the plates, 0.1 μg/ml of 2F5-specific IgG was preincubated for 30 min with serial dilutions of soluble 2F5ep (NEQELLELDKWASLWN) peptide. Percentages of binding inhibition were adjusted to saturation curves, which were subsequently used to infer the IC₅₀ values ±S.E. displayed in the panels. C, inhibition of cell entry. In these assays HIV-Env pseudoviruses were preincubated with increasing amounts of purified antibodies, and infection of TZM-bl target cells was subsequently monitored by flow cytometry as in Fig. 5A. Plotted inhibition percentage values are means of four experimental determinations. The red columns correspond to the level of neutralization exerted on VSV-G-pseudotyped viruses used as negative control.

FIGURE 8.

Recovered responses after vaccination with the POPG/lipid A-MPERp formulation. A, midpoint IgG titers (negative log EC₅₀ dilution values) and inhibition of cell entry by 2F5-specific antibodies in the sera of four different rabbits (R1–R4). Antibodies purified with 2F5ep-Cys were used at 100 μg/ml in the latter assay. B, levels of inhibition of cell entry mediated by HIV-1-Env or VSV-GP controls. Rabbits were classified in two groups according to significant differences in the levels of inhibition (R1/R4 versus R2/R3). Significant inhibition of Env-HIV-1 versus VSV-GP was nonetheless observed for both groups (**, p < 0.005; *, p < 0.05). MAb2F5 (2 μg/ml) was included as positive control. Means ± S.D. of six determinations are shown.

FIGURE 9.

2F5 epitope organization in MPERp and putative mechanism of antibody recognition. A, 2F5 epitope in HFIP and DPC structures. Core epitope residues ELDKWA are shown in red, and downstream residues putatively implied in secondary interactions with CDR-H3 loop are depicted in blue. Trp-666 and Leu-669/Trp-672/Phe-673 are displayed on the same side of the helix. B, comparison of 2F5 epitope structure in Fab′ complex (PDB code 3D0L) and MPERp. The chain portion spanning residues Leu-661–Trp-670 is shown in gray in the three structures, with projecting side chains of Asp-664 (left) or Lys-665 (right) and Trp-666 in red. Side chain of Leu-669 is displayed in blue to establish the relative position of the downstream helix. The comparison suggests that the 3₁₀-helix observed in DPC might include an intermediate of the conformational change required for positioning Asp-664 side chain into the 2F5 paratope. Lys-665 accommodation into the paratope would not require by comparison major conformational changes of the peptide backbone. C, fitting of the MPERp helix into Fab′-bound peptide. The Fab paratope structure (PDB code 3D0L) is displayed in ribbon representation. The base of the flexible loop of the heavy chain (not solved in the crystal) is marked by the yellow side chains of residues Pro-98 and Arg-100B. The MPER residues Trp-666 and Leu-669 in the bound peptide are displayed in red and blue, respectively. In the right panel, the helix turn of MPERp (DPC structure) containing Leu-669 (displayed in blue) has been fitted into the Fab-bound structure. The dotted lines mark the estimated position of the loop relative to the MPERp helix.