Recombinant HIV type 1 Glycoprotein 41 (a.a. 466-753) [Beta-galactosidase]

Induction of broadly neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development. Antibody 10E8, reactive with the distal portion of the membrane-proximal external region (MPER) of HIV-1 gp41, is broadly neutralizing. However, the ontogeny of distal MPER antibodies and the relationship of memory B cell to plasma bnAbs are poorly understood. HIV-1-specific memory B cell flow sorting and proteomic identification of anti-MPER plasma antibodies from an HIV-1-infected individual were used to isolate broadly neutralizing distal MPER bnAbs of the same B cell clonal lineage. Structural analysis demonstrated that antibodies from memory B cells and plasma recognized the envelope gp41 bnAb epitope in a distinct orientation compared with other distal MPER bnAbs. The unmutated common ancestor of this distal MPER bnAb was autoreactive, suggesting lineage immune tolerance control. Construction of chimeric antibodies of memory B cell and plasma antibodies yielded a bnAb that potently neutralized most HIV-1 strains.

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naive B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually reintroduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking Nglycans at residue N276 (natural or engineered), indicating that PNGS deletion of well- ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant.