Preliminary Study On Immunology Activity And Structure Characterization On Branched Multiple Antigenic Peptides Based On Membrane Proximal Extracellular Region Of HIV-1gp41

The HIV-1gp41membrane-proximal external region (MPER) is a major focus forsuch a vaccine since the epitopes for three potent broadly neutralizing antibodies(bnAbs)4E10,2F5and Z13are located in this region. The MPER is a highly conservedtryptophan-rich region that functions in various viral processes, such as HIV-cellmembrane fusion and gp41oligomerization. Thus, the key aim of MPER-basedvaccines is to mimic the natural pre-hairpin conformation which can stimulatematching B-cells to produce conformation-dependent bnAbs.MPER previously was shown to form a trimeric subdomain in aqueous solution,which was proposed to be a pre-fusion conformation. Multiple antigen peptide system(MAPs) was employed in this thesis for design and synthesis of4E10-and2F5MAP4immunogens. According to previous reports showing that a constrained type-1-turnor α-helical conformation failed to elicit broadly Nabs, the branched peptideimmunogens were developed as the relatively rigid lysine core and flexible epitopes tailwithout additional chemical constraints. Then further studies were characterized onthese immunogens on antigenicity, immunogenicity, neutralizing activity andconformation expecting candidate immunogens against HIV.Branched immunogens were identified first by HPLC, mass spectrum andTricine-SDS PAGE. The PAGE results showed that both immunogen purities couldreach90%and the molecular weights were around10kD. The purities were confirmedby further characterized by HPLC. The molecular weights were also determined as9687.52Da and10056.20Da by mass spectrum in accordance with theoretical values.Western-blotting results showed that4E10-and2F5-MAP4immunogens could bespecifically recognized by corresponding monoclonal antibody4E10or2F5,respectively, suggesting the presence of specific epitopes.After immunizing mice or guinea pigs in adjuvants (oil adjuvant), both4E10-and2F5-MAP4immunogens could induce antibodies in sera that could recognize4E10and2F5epitopes, respectively. And the sera titers could reach103～105by ELISA. Furtherstudy on HIV neutralizing activities showed that both4E10-and2F5-MAP4guinea pigsera could block HIV-1infection compared with those epitopes coupling on KLH. Serafrom4E10-MAP4could neutralize clade BC, C, B and AE which were confirmed to bethe main epidemic strains of HIV-1in southern China. The results showed weakneutralizing ability against clade B, while strong one against clade BC and AE (S91-2). Sera from2F5-MAP4showed strong neutralizing activity against clade BC, and somecertain one against clade B, C and AE.These results are exciting and suggesting the preliminary feasibility for inducingbroadly neutralizing activity against HIV-1by synthetic branched peptide immunogenbased on MPER. While it is still important to improve the immunogens to increaseantigenicity, enhance neutralizing activity and broaden the spectrum according theresults. For further immunogen improvements, first of all, the structure should beunderstood perfectly. So the conformations of4E10-and2F5-MAP4werecharacterized by circular dichroism, Fourier infrared spectrum and simulativecalculation.Compared with linear peptide, the branched structure design drove theconformation complicated. Typical secondary protein structure could be found in both4E10-and2F5-MAP4due to the flexible epitope assembling and the interactionbetween the branches. The results also showed that the ratio of α-helix and β-sheet in4E10-and2F5-MAP4could be various with the change of solvent polarity. It’s alsofurther identified by Fourier infrared spectrum analysis that the presence of secondarystructure in4E10-and2F5-MAP4. According to the publication of MPER crystalstructure of HIV-1gp41,4E10-and2F5-MAP4were modeled by the homology methodand the conformations were predicted by computation module Gromacs or DiscoveryStudio. Complicated tertiary structures could also be found in solution and extendingthe simulation time might provide further evidence for nature conformation. Thestructure studies suggested the secondary structure composition of branched peptidemight play key role in inducing neutralizing antibody which could be the optimizingbasis for immunogen structure.In this thesis, two novel MPER-based MAP immunogen candidates,4E10-and2F5-MAP4, were designed and shown to induce broadly NAbs against HIV-1in sera ofguinea pigs. Typical secondary structures were found in both branched peptides whichmight play important feature in immunogen improvement. The study also provides apreliminary research platform for branched peptide structure investigating and thefoundation for further development.