Introduction: Anti-HIV-1 gp41 envelope (Env) antibodies (Abs), which bind to linear and/or conformational epitopes, are induced in HIV-infected individuals. A subset of anti-HIV-1 gp41 Abs with binding specificities within gp41 cluster II that react with the membrane proximal external region (MPER) (aa644-667) have been described. Anti-gp41 Abs are largely non-neutralizing and only rarely are broadly neutralizing (2F5, 4E10, Z13). 2F5 and 4E10 Abs also show reactivity towards phospholipids and other autoantigens. Thus, we have probed the relationship between phospholipid reactivity and neutralization and compared peptide epitope, phospholipid and peptide-lipid conjugate binding of 2F5 to gp41 cluster II Abs.

 

 

Methods: Anti-gp41cluster II Abs (98-6, 167-D, 126-6) were derived from HIV-infected individuals and murine anti-MPER Abs (5A9, 13H11) were made following immunization with group M consensus Env ConS gp140 oligomer. 2F5 peptide-lipid conjugates were prepared by incorporating the 2F5 nominal epitope peptide into synthetic liposomes via a lipophilic linker. Binding kinetics and antibody cross-blocking were measured using a surface plasmon resonance binding assay. Neutralization assays were performed with pseudoviruses in TZM/bl cells or mitogen-activated peripheral blood mononuclear cells (PBMCs).

 

 

Results: In comparison to 2F5 Ab, both human cluster II and murine MPER Abs bound to the 2F5 nominal epitope peptides with faster dissociation kinetics. In crossblocking experiments, binding of 2F5 was blocked partially by cluster II Abs, while 2F5 completely blocked the binding of cluster II Abs. 98-6 and 13H11 did not reciprocally inhibit each other’s binding to gp41 epitopes. However, 98-6 and 13H11 acted synergistically to completely block 2F5 binding. 98-6 showed strong reactivity with cardiolipin and bound to 2F5 peptide-lipid conjugates. In contrast, 5A9 and 13H11 did not bind to phospholipids nor to 2F5 peptide-lipid conjugates. None of the human cluster II Abs or murine MPER Abs had neutralizing activity against the HIV-1 primary isolate B.6535 pseudovirus in the TZM/bl assay. However, in the PBMC assay, human Ab 98.6 neutralized the primary isolate B.6535 (ID50= 3.5μg/mL).

 

 

Conclusions: HIV-1 neutralizing activity is rare among cluster II anti-gp41 mAbs. Only anti-gp41 Abs (2F5, 98-6) that bound with high avidity to both MPER epitopes and phospholipids neutralized HIV-1.