Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10

Abstract

SUMMARY Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

“…The lack of ADCC activity for the MPER bNAbs is probably due in part to the lower affinity of these antibodies for Env on virus-infected cells, which is consistent with their specificity for an epitope consisting of gp41 sequences that are transiently exposed during fusion and phospholipids that are preferentially enriched in viral membranes (77)(78)(79)(80)). Yet these antibodies still bound to HIV-1 NL4-3 -and HIV-1 JR-FL -infected cells, as indicated by levels of Env staining similar to other antibodies with ADCC against these viruses, such as the V2 apex bNAbs PG9 and PG16.…”

“…1). The reason for this discrepancy is unclear, but it may be related to the limited accessibility of these antibodies for engagement by Fc␥Rs on NK cells when bound to virus-infected cells due to their specificity for an epitope consisting of phospholipids and sequences exposed at the base of gp41 (77)(78)(79)(80). For SHIV AD8-EO -infected cells, there was almost complete correspondence between antibody binding and susceptibility to ADCC ( Fig.…”

Surface-Matrix Screening Identifies Semi-specific Interactions that Improve Potency of a Near Pan-reactive HIV-1-Neutralizing Antibody

“…Diverse studies ranging from electron paramagnetic resonance (EPR) analysis (Song et al, 2009; Sun et al, 2008) to mutational and partitioning analyses (Ofek et al, 2010; Rujas et al, 2017), to crystallographic analysis of Fab in complex with phospholipid headgroups (Irimia et al, 2016, 2017) indicate antibodies 10E8 and 4E10 to co-recognize membrane and MPER peptide. In particular, the orientation suggested by the structure of 10E8 with 1,2 dihexanoyl- sn -glycerol-3-phospho-(1’- rac -glycerol) and scaffolded MPER (Irimia et al, 2017) would position the indole ring of Trp100c HC to extend into the viral lipid membrane (Figure 4A).…”

“…In particular, the orientation suggested by the structure of 10E8 with 1,2 dihexanoyl- sn -glycerol-3-phospho-(1’- rac -glycerol) and scaffolded MPER (Irimia et al, 2017) would position the indole ring of Trp100c HC to extend into the viral lipid membrane (Figure 4A). Examination of the structure of 4E10 with 1,2 dihexanoyl- sn -glycerol-3-phosphate and MPER peptide (Irimia et al, 2016) suggested that a Trp substitution in 4E10 at position 100a HC would be similarly positioned to interact with the viral lipid membrane (Figure 4B). Indeed, Trp substitutions of different positions at the apex of the third heavy chain complementarity-determining region (CDR H3) of 4E10 showed only the substitution at position 100a HC to increase neutralization potency (Figure 4C).…”

Identification and specificity of broadly neutralizing antibodies against HIV

“…The minority of B cells which circumvent this checkpoint are anergic, but yet can still be activated by an MPER immunogen 86. The more recently isolated MPER bnAb 10E8 is highly potent and does not display any autoreactivity nor does it bind lipids as has been reported for other MPER Abs 87. This may be because 10E8 approaches MPER from an altered angle and uses a different binding mode to 4E10 36.…”