Background:  HIV-1 vaccine immunogens that elicit broad neutralizing antibodies remains an elusive goal. The highly conserved hydrophobic 30-amino acid membrane proximal region (MPR) of HIV gp41 region contains the epitopes for 2 rare HIV-1 broadly neutralizing antibodies, 2F5 and 4E10. Both of these antibodies demonstrate enhanced binding to their epitopes by the intimate juxtaposition with a lipid membrane environment. Hepatitis B surface antigen (HbsAg (HepB) particles can be utilized both as an immunogenic array and to provide the lipid environment needed for 2F5 and 4E10 binding.

Methods:  Recombinant HepB-MPR fusion proteins were either transiently expressed in HEK 293 cells or using the baculovirus expression system. Biochemical analysis was done using Western blot, MPR sandwich ELISA, or lipid/detergent MPR capture ELISA. HIV⁺sera were analyzed in a pseudovirus based neutralization assay.

Results:  We show that recombinant HepB-MPR particles with MPR at the C-terminus bind 2F5 and 4E10 with nanomolar affinities. C-terminal addition of hydrophobic residues from the Env transmembrane region further increase relative binding affinity. We show that HepB-MPR particles can be depleted of lipids, which decreases 2F5 and 4E10 binding, and that reconstitution with synthetic lipids restores binding. Immunization of these particles in mice raised sera that cross-reacted with YU2 and JR-FL gp160 envelope glycoproteins expressed on the cell surface. The HIV neutralizing activity of the mice sera was limited and the particles are currently being evaluated in guinea pigs and rabbits. A set of sera from HIV-infected individuals was screened for binding to the HepB-MPR particles and to the MPR in a lipid/detergent-based ELISA. Several sera possessed MPR-binding antibodies but the presence of the antibodies did not correlate with neutralization potency against difficult to neutralize primary isolates, although a correlation was observed with lab adapted isolates, suggesting availability of low titer, 2F5- or 4E10-like neutralizing antibodies that can neutralize easy viruses.

Conclusions:  We have characterized novel HepB-MPR immunogens to potentially elicit antibodies against the gp41 MPR region. Immunogenicity studies using these particles themselves and in prime boost combination with gp160 proteoliposomes displaying the MPR in a native context are ongoing. We show the presence of MPR binding antibodies in selected patient sera indicating that this region is immunogenic during the natural in vivo infection process.