In previous studies, our lab has characterized a number of highly mutated antibodies against structural epitopes of the Human immunodeficiency virus (HIV) envelope protein. These antibodies were first isolated from long-term non-progressors (LTNPs). We have previous mapped 6F5 to a novel structural epitope that encompasses areas in both heptad repeats of GP41, mapping to amino acids of 557, 654 and 657 of reference sequence HXB2. In these studies, three other antibodies that were less than 90% homologous to 6F5 also resolved amino acid 657. On sequence analysis, 6F5 and its relatives had the same gene usage and general structure. These similarities and the similar epitope mapping implied these were once distantly related to a single B cell lineage. As fusion of the viral membrane to the target cell depends on these heptad repeat regions associating and forming a six-helix post-fusion bundle, antibodies that can interfere in this may be highly useful.
Methods: (see results)
Because 6F5 maps to 557 and 654/657 which are widely separated on the primary sequence, we explored if there was differential binding to the post-fusion six-helix-bundle form. Two peptides (N36 and C34) each containing one of the heptad repeats can form the post-fusion six-helix-bundle in vitro. On sandwich ELISA testing, 6F11 and 7B6 did not bind any form. Interestingly, 4E4 specifically captured both peptides alone, but not the six-helix-bundle and 6F5 only bound the six-helix-bundle but not the other peptide.
A small number of samples were obtained to assess the prevalence of these responses in LTNPs. Antibodies that compete 6F11 are much more prevalent in LTNPs than normal progressors (75% vs. 20%). Functionally, we found that despite being mapped to a similar portion of Gp41 (657), only 6F5 is shown to have significant ADCC activity, however relative 6F11 does not.
If targeting these epitopes correlates with the LTNP state, then these sites may be highly significant as targets of therapeutics or in vaccine strategies. Further studies on a larger cohort of LTNPs are ongoing. Additionally, deep sequencing of antibody sequences are being done to explore the development of structural epitope targeting by this family of antibodies.
J. Hoffman, None
M. Hicar, None