A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

Abstract

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Mechanism of HIV-1 Neutralization by Antibodies Targeting a Membrane-Proximal Region of gp41

“…In our study, the postfusion gp41 construct containing the intact MPER showed relatively high-affinity binding to both 10E8 and 4E10. Because of its stability, it might be a better vaccine antigen than those studied so far (34)(35)(36). Buzon and colleagues reported that 4E10 did not interact with gp41 including the MPER in the postfusion conformation, in agreement with clashes revealed by molecular docking (37).…”

“…It is possible that the presence of the fusion peptide and transmembrane segment in our gp41 construct has some impact on the local conformation of the 4E10 epitope, as we found that both 4E10 and 10E8 also show binding to our gp41 protein expressed on the insect cell surface (data not shown). Likewise, liposomes with a cholesterol-rich, HIV-1-like lipid composition might be more effective for inducing potent MPER-specific bNAbs than any previously tested lipid-based immunogens (35,38,39). We note that a singlechain antibody (VHH), 2H10, recently isolated from llamas immunized with a liposome-based gp41-MPER immunogen, requires a hydrophobic CDR3 for neutralization, but not for antigen recognition (35).…”

“…Likewise, liposomes with a cholesterol-rich, HIV-1-like lipid composition might be more effective for inducing potent MPER-specific bNAbs than any previously tested lipid-based immunogens (35,38,39). We note that a singlechain antibody (VHH), 2H10, recently isolated from llamas immunized with a liposome-based gp41-MPER immunogen, requires a hydrophobic CDR3 for neutralization, but not for antigen recognition (35). The mode of neutralization used by this vaccination-induced antibody appears to be consistent with the general mechanism of HIV-1 neutralization by MPER-specific antibodies we propose here.…”

Proteoliposomal formulations of an HIV-1 gp41-based miniprotein elicit a lipid-dependent immunodominant response overlapping the 2F5 binding motif

“…Anti-MPER bNAbs share a common neutralization mechanism in which interactions of CDRH3 hydrophobic residues with membrane lipids seem to be essential325556575859. Additionally, MPER structure is influenced by membrane lipid composition2930.…”

“…Additionally, MPER structure is influenced by membrane lipid composition2930. Therefore, it is widely assumed that the generation of a robust anti-MPER response should require its presentation within a membrane environment to properly present neutralizing determinants and to implement lipid cross-reactivity5559606162.…”