Background:  The development of vaccine immunogens that can elicit high-titer, potent, broadly HIV-neutralizing antibodies remains a major challenge. Only several such antibodies have been identified—including 2F5, 4E10, and Z13—that can bind peptides from the gp41 subunit of the HIV envelope glycoprotein (Env). However, immunogens based on these peptides have not elicited broadly neutralizing antibodies. To identify other cross-reactive antibodies that may bind to gp41 structures with potential as HIV vaccine immunogens, we have developed a competitive antigen panning (CAP) method, selecting for Env-interacting antibodies in the presence of excess gp120, which facilitates the identification of gp41-reactive antibodies in the context of native Env.

Methods:  We used an immune antibody library constructed from the bone marrow of 3 HIV-1-infected long-term non-progressors and CAP, where labeled gp140 is mixed with excess of unlabeled gp120 and used for panning of the library.

Results:  By using CAP, 5 antibodies (m43, m44, m45, m47, and m48) were selected from an immune human antibody phage library derived from long-term non-progressors with high concentrations of broadly neutralizing antibodies. In both IgG1 and Fab formats, they neutralized HIV-1 primary isolates from different clades with on average higher potency than 4E10 and Z13 in peripheral blood mononuclear cell (PBMC), replication-competent virus assays, but exhibited on average weaker activity in cell line pseudovirus assays than did 2F5, and comparable or higher activity than Z13. They bound with high affinity to soluble Env (gp140) from various primary isolates (except a clade A isolate), but not to denatured gp140 and antigen-mapping gp41 peptides indicating a conformational nature of their epitopes. They do not bind to N36/C34-formed 6-helix bundles and 5-helix bundles except that m44 and m45 bind to 5-helix bundles. All antibodies competed strongly with the cluster IV antibody T3, variably with one another, and variably and weaker with 2F5, 4E10, Z13, and the cluster V antibody D3. Alanine scanning mutants of gp41 are being generated and used to map the epitopes of new anti-gp41 antibodies.

Conclusions:  Our results suggest the existence of new conserved conformational neutralization epitopes on gp41 that may have potential as HIV vaccine immunogens and as targets for therapeutics.