Cholesterol Constrains the Antigenic Configuration of the Membrane-Proximal Neutralizing HIV-1 Epitope

Abstract

The envelope glycoprotein (Env) enables HIV-1 cell entry through fusion of host-cell and viral membranes induced by the transmembrane subunit gp41. Antibodies targeting the C-terminal sequence of the membrane-proximal external region (C-MPER) block the fusogenic activity of gp41 and achieve neutralization of divergent HIV-1 strains and isolates. Thus, recreating the structure that generates broadly neutralizing C-MPER antibodies during infection is a major goal in HIV vaccine development. Here, we have reconstituted a peptide termed CpreTM-TMD in a membrane environment. This peptide contains the C-MPER epitope and the minimum TMD residues required for the anchorage of the Env glycoprotein to the viral membrane. In addition, we have used antibody 10E8 variants to gauge the antigenic configuration attained by CpreTM-TMD as a function of the membrane cholesterol content, a functional determinant of the HIV envelope and liposome-based vaccines. Differential binding of the 10E8 variants and the trend of the IgG responses recovered from rabbits immunized with liposome-peptide formulations, suggested that cholesterol may restrict 10E8 accessibility to the C-MPER epitope. Our data ruled out the destabilization of the lipid bilayer architecture in CpreTM-TMD-containing membranes, and pointed to the perturbation of the helical conformation by lipid packing as the cause of the antigenic configuration loss induced by cholesterol. Overall, our results provide additional insights into the structural basis of the Env complex anchoring to membranes, and suggest new approaches to the design of effective immunogens directed against the near pan-neutralizing HIV-1 epitope C-MPER.

African Swine Fever Virus Gene B117L Encodes a Small Protein Endowed with Low-pH-Dependent Membrane Permeabilizing Activity

“…In contrast, samples treated with PB117L-2 showed low levels of permeabilization at neutral pH that became significant at pH 5.0, whereas the peptide PB117L-3 appeared to induce low levels of permeabilization that were comparable at both pHs. An increase in membrane permeability is not observed in GUVs that contain non-lytic transmembrane domains of class II viroporins (53,54) or other TMHs (56,57) and, therefore, the data displayed in Figures 8C and D support the potential pore-forming activity of B117L TMH.…”

A two‐step mechanism for the binding of the HIV‐1 MPER epitope by the 10E8 antibody onto biosensor‐supported lipid bilayers

“…To get further insights into the mechanism involved in the molecular recognition of ctMPER helix at membrane surfaces, here we use biosensor slides coated with carboxymethylated 6 kDa dextran that promoted the formation of Supported Lipid Bilayers (SLBs) [22]. In addition, we use ctMPER-TMD epitope peptides reconstituted in the SLBs as transmembrane helices [21,23]. We carry out a quantitative surface plasmon resonance (SPR) analysis and provide equilibrium dissociation constants and association/dissociation rate constants measured in peptide-containing SLBs.…”

“…Next, the multilamellar vesicles were bath sonicated (1 h, 55 °C) and subjected to 15 freeze and thaw cycles to obtain unilamellar vesicles. The helical secondary structure adopted by the peptides upon reconstitution in lipid bilayers was verified by infrared spectroscopy as described [23,27].…”

“…For assessing Fab binding to single vesicles, NBD-labeled Giant Unilamellar Vesicles (GUVs) were prepared as previously described [23,29]. GUVs were added to the bovine serum albumin (BSA)-blocked microscope chamber that already included 250 nM of 10E8-based Fabs conjugated with the KK114 probe at residue C216 HC and were incubated for 15 min prior to imaging.…”

Neutralizing Antibodies Targeting HIV-1 gp41

“…This may be a particular problem as the MPER sequence has a high tendency to fold back and interact itself with the membrane thereby occluding its access. Fifth, the lipid composition including cholesterol seems to be important as it may constrain the antigenic conformation of the MPER epitope [ 136 ].…”