The membrane proximal region (MPR) of HIV-1 gp41 is a desirable candidate for development of a vaccine that elicits neutralizing antibodies since it is targeted by three of the most potent broadly neutralizing monoclonal antibodies isolated from infected patients. Structural studies of these antibodies and their epitopes suggest that MPR immunogens may be presented in a lipid membrane environment. In this thesis, I report the synthesis and evaluation of MPR lipid-peptide conjugates for membrane presentation of the MPR and elicitation of neutralizing antibodies to HIV-1.

In Chapter 2, I hypothesized that covalent attachment of lipid anchors would enhance the humoral immune response to MPR-derived peptides presented in liposomes. In a comparison of eight lipids conjugated to MPR peptides, cholesterol hemisuccinate promoted the strongest anti-peptide titers in mice. Lipid conjugation was employed to manipulate the biophysical properties and antibody inducing capability of MPR peptides, and this lipopeptide toolkit will be useful for interrogating the role of structure in the immune response to the MPR.

In Chapter 3, MPR lipopeptide immunogens were further utilized to study the cause of weak immune responses to the MPR. We initially hypothesized that the antibody response against the MPR is restricted by immunologic tolerance, but a comparative assessment of antibody responses to MPR lipopeptides in two mouse models of defective immune tolerance indicated that tolerance mechanisms are insufficient to explain the poor antibody responses to the MPR.

I also hypothesized that MPR immunogenicity could be increased through phosphorylation of amino acid side chains. Evaluation of modified MPR immunogens revealed a modification that increased anti-MPR antibodies by an order of magnitude. Importantly, the sera of rabbits immunized with these modified constructs neutralized a laboratory-adapted HIV-1 strain in vitro, providing a foundation for further work on this strategy.

Finally, supplementary studies were conducted involving delivery systems and novel molecular adjuvants for MPR peptide immunogens. In Chapter 4, metal chelation through lipid-anchored multivalent nitrilotriacetic acid was investigated as a non-covalent strategy to attach peptide and protein antigens to liposomal vaccine carriers. Also, in Chapter 5 retinoids were explored as candidate liposomal adjuvants for enhancing the antibody response to MPR immunogens. These studies may lead to improved formulations for delivery of MPR peptide antigens to elicit neutralizing antibodies against HIV-1.