DESIGN OF SYNTHETIC gp41 MIMETICS AS HIV VACCINE CANDIDATES
Marie Florence Brunel, Scripps Research Institute
Eliciting broadly neutralizing antibodies (nAbs) is a major goal of HIV vaccine design. Such Abs recognize highly conserved regions of the HIV envelope spikes, and to date, six of them have been described. Four of these broadly nAbs, 2F5, 4E10, Z13 and D5 recognize epitopes found on the transmembrane gp41. We have already fully characterized the 4E10 peptide epitope, and, based on a crystal structure of a peptide bound to 4E10, we have designed a first generation of constrained peptides to develop a more efficient antigen.
The goal of this proposal is to increase our knowledge about the epitopes of the antibodies that recognize conserved regions on gp41, and to develop antigens that would elicit neutralizing antibodies. Our first goal is to study the membrane proximal external region, where the epitopes for 4E10 and Z13 are found. The Zwick laboratory, at The Scripps Research Institute (TSRI), developed a new, more potent analog of Z13: Z13e1, which recognizes a region N-terminal to the epitope for 4E10. We will isolate the minimal core epitope of Z13e1, and once the epitope is identified, we will try to obtain a crystal structure of a peptide bound to Z13e1 in collaboration with the Wilson group at TSRI. Preliminary results suggest that this peptide could also adopt a helical conformation, and a first generation of antigens will be engineered by introducing backbone structural constraints thioether tethers and aminoisobutyric acid) in the permissive sites. For both 4E10 and Z13e1, we ill present the peptides in a membrane-like environment as a membrane could be required to select against Abs that would have unfavorable interactions with the HIV membrane. We will use nanodiscs, "nano-scale" structures consisting of a phospholipid bilayer "disc" surrounded by a membrane Scaffold Protein belt. For immunogenicity assays in rabbits, as an alternative to the usual protein carrier, we will also load the peptides on CPMV (the cow pea mosaic virus). The information gained from each experiment (insolution affinity, BIAcore), and potentially from the X-ray structures will be used in an iterative process to guide rational improvements for engineering subsequent generations of antigens.
A new nAb that binds a region found on the N-heptad repeat (NHR) of gp41 was recently isolated by the Zwick laboratory. A second goal of this proposal is to fully characterize this more complex epitope. This project will require the synthesis of helical trimers. We propose to use a trimerizing domain, and to attach portions of the NHR onto it to identify the position and length of the epitope. An Alanine-scan will then be performed. The presence of other nAbs that bind this region of gp41 is probable, and our peptides will be used as bait for sequencing and subsequently characterizing these antibodies when they get isolated. Once the epitope is fully characterized, immunizations will be performed. Several ways to optimize the antigens will be investigated as the position of the trimerizing domain and the inclusion of disulfide bonds to lock the peptide conformation.
Our work will result in an increase in knowledge of nAbs with epitopes found on gp41. It will also help us to understand the structure of gp41 during the fusion mechanism, as well as the potential role of the membrane for the elicitation of nAbs. Each round of syntheses will generate more effective antigen candidates, and constitute important steps towards the development of an HIV vaccine.