Design of Novel gp41-based Immunogens for HIV-1 Vaccine Development

Degree awarded: Ph.D. Biology. The Catholic University of AmericaThe HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates viral entry by binding to host receptors whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been determined, the detailed mechanism is poorly understood. The goal of this study is to design gp41 immunogens that mimic the prehairpin structure, an intermediate potentially vulnerable to neutralizing antibody interactions that can block virus entry. A combination of molecular genetics, structural modeling and biochemical approaches was used to produce soluble near full-length gp41 trimers that mimic the prehairpin intermediate. The two long heptad repeat helices, HR1 and HR2, of gp41 ectodomain were mutated to disrupt the intra-molecular HR1-HR2, but not the inter-molecular HR1-HR1, interactions. Attachment of a 27-residue "foldon" containing a trimerization motif to the C-terminus and slow refolding channeled gp41 into trimers. The trimers were stabilized in a prehairpin-like structure, as evidenced by binding of a HR2 peptide to the exposed HR1 grooves, lack of binding to six-helix bundle-specific mAb NC-1, and inhibition of virus neutralization by the broadly neutralizing antibodies 2F5 and 4E10. By fusing with the bacteriophage T4 small outer capsid protein, Soc, and displaying on the capsid, the gp41 trimers were converted into nanoparticle-arrayed immunogens. Various gp41 formulations, including a powerful T4-based vaccine delivery platform that simultaneously delivers both DNA and protein were tested for immunogenicity in mouse and rabbit models. Delivery of gp41 DNA packaged inside T4 heads induced as high gp41-specific antibodies as the protein antigens. The specificity of antibody responses depended on the conformation of the immunogen, the vaccine formulation, and the usage of a dendritic cell targeting ligand. The DNA vaccines induced robust gp41 antibody titers with broad specificity, including those for neutralization epitopes in the membrane proximal external region, whereas the protein vaccines induced antibodies with greater specificity to the prehairpin structure. These results provided insights into the mechanism of HIV-1 entry and generated novel gp41 immunogens and delivery tools that in future could lead to the design of efficacious HIV-1 vaccine formulations.Made available in DSpace on 2013-06-25T14:58:51Z (GMT). No. of bitstreams: 1 Gao_cua_0043A_10417display.pdf: 2511048 bytes, checksum: f660aa396ccfba9d46f1f8807b6846ec (MD5