Background: The lack of immunogencity of the conserved HIV-1 gp41 membrane proximal region (MPER) may be due to transient exposure of the pre-hairpin intermediate form of gp41, to MPER mimicry of self epitopes, or to masking of neutralizing MPER epitopes by virion lipids. The long CDRH3 loops of MPER neutralizing mAbs 2F5 and 4E10 have a hydrophobic face, postulated to interact with virion lipids. Our initial studies with nominal gp41 MPER peptide-lipid conjugates highlights the importance of lipid binding and showed that binding of 2F5 and 4E10 follows a 2-step “encounter-docking” model. We have now studied the role of the CDRH3 loops in lipid binding and HIV-1 neutralization and designed lipid based immunogens for induction of neutralizing antibodies.

 

  

Methods: We designed mutations in the CDHR3 loops to interfere either with peptide binding or with lipid interaction. We have also designed and constructed liposome based immuogens that present both viral lipid composition and gp41 neutralizing MPER epitopes. Antibody binding was measured by surface plasmon resonance (SPR) and neutralization activity in the TZM/bl pseudovirus neutralization assay.

 Results: Alanine substitutions at positions on the hydrophobic faces of the CDR H3 loops (2F5: L100aA/F100bA and 4E10:W100A/W100bA/L100cA), led to much weakened or loss of binding to phosphatidylserine exposed viral liposomes and to liposomal peptide-lipid complexes. These mutations also weakened binding to nominal epitope MPER peptides. Mutations of hydrophobic residues in the CDRH3 loop of 2F5 and 4E10 that disrupted binding to lipids abrogated HIV-1 neutralization.

 

  

Conclusions: Our results provide support to our model that the binding of 2F5 and 4E10 occurs sequentially and is initiated by binding of mAb to viral membrane lipids. This finding has been exploited to design several liposome based immunogens that present gp41 neutralizing epitopes in a viral lipid environment and conjugated to different adjuvants.