One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions. We focus on recreating the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We show that AH peptide-induced vesicle fusion is a reliable and facile technique to form a SLB that contains a high cholesterol content and multiple lipid types. The utility of this technique to form biomimetic SLBs was exemplified by forming SLBs from vesicles that recapitulate the native HIV-1 envelope. Without the use of AH peptides, model HIV-1 vesicles fail to form a complete SLB. The SLB formation was characterized by QCM-D and NR measurements, and AFM imaging. NR results show that the formation of the SLB using AH peptides is complete and that peptides are completely removed from the SLB surface after washing. AFM imaging provided a topographical map of the SLB and revealed three distinct membrane domains. Furthermore, AFM imaging showed that imaged SLB areas did not have major hole defects, did not contain intact vesicles, and did not show AH peptide aggregates. Our high resolution AFM imaging experiments on SLBs demonstrate that NAbs 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area.

Synthesis and characterization of supported lipid bilayer membranes from complex lipid mixtures

253rd National Meeting (2017)

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