Study On Immunogenicity Of Multiple Antigen Peptide Immunogens Containing Epitopes From MPER And Infection Enhancing Activity Of Peptides Derived From MPER

Stimulating protective antiviral antibody responses is prerequisite for a humanimmunodeficiency virus type1(HIV-1) vaccine to successfully curb the globalacquired immunodeficiency syndrome (AIDS) epidemic. Thus in this study, weinvestigated novel multiple antigen peptide mimetics containing gp41membrane-proximal external region who could elicit broad neutralizing antibodiesagainst human immunodeficiency virus type1in guinea pigs. Furthermore, efficientgene transfer is a critical goal in retroviral transduction. Based on the MPER sequence,we further developed novel modified peptide derivatives significantly enhanceretrovirus infection.1．Study on peptide immunogens based on HIV-1MPERThe HIV-1gp41membrane-proximal external region (MPER) is a major focusfor such a vaccine since the epitopes for three potent broadly neutralizing antibodies(NAbs)4E10,2F5and Z13are located in this region. The MPER functions inHIV-cell membrane fusion and is believed to undergo functional changes in itsconformation during the process of membrane fusion, in which it is exposedtransiently to the immune system to elicit broadly NAbs. Thus the task of constructingsuch a potent HIV vaccine based on MPER is challenging because it is a structurallycomplex and changing region, and whose confirmation yet undefined. As revealed inco-crystal structures of2F5or4E10in complex with a linear peptide, these antibodiesrecognize their core epitopes via a type-1-turn and an-helical secondary structure,respectively; however, conformationally constrained and totally flexible2F5and4E10immunogens both have not been shown to induce broadly NAbs. In the last twodecades, only few breakthrough studies on chimeric MPER vaccines have reportedachieving very low neutralizing activity, and no effective MPER-based peptideimmunogens have been reported to nduce bNAbs against HIV-1.In this study, we employed the MAP system without additional chemicalconstraints based on the hypothesis that interactions among each epitope monomer, the relatively rigid lysine core and flexible6-Ahx linker and antigenic tail in the MAPcan reach a balance between rigidity and flexibility of conformation to effectivelyinduce broadly NAbs. Specifically, the immunogens were synthesized with MAPs togive an appropriate aggregation of epitopes in one molecule and relatively highmolecular weights which may elicit antisera with high binding activities. And thesynthesis and application of peptide vaccines are relatively simple compared withprotein-based or virus-based vaccines.Four-branched multiple antigenic peptide were solid-phase synthesized andidentified antigenicity by ELSIA and absorption neutralization assay, and were furtherconfirmed the ability to induce gp41-specific binding and cross-neutralizing activityimmulsified with oil-phased adjuvant ADO-1. Analyzed by gp41specific end-pointELISA,4E10-MAP4,2F5-MAP4,4E10-KLH and2F5-KLH were all found tospecifically bind to gp41, with comparatively high endpoint titers observed(1104-105). Purified IgGs from pooled antisera of guinea pigs with these fourimmunogens were found to specifically bind to gp41.Results of the HIV-1pseudovirus neutralization assay showed that4E10-MAP4and2F5-MAP4both elicited broadly NAbs, albeit at moderate levels, against HIV-1pseudoviruses in serum of individually tested guinea pigs. While4E10-KLH and2F5-KLH both elicited high titered antibodies with gp41binding activity in guineapigs, they showed no neutralizing activity. Ten strains from four clades of HIV-1pseudoviruses were tested in total.4E10-MAP4elicited neutralizing activity againstviruses of clades BC (73%positive), C (93%positive), B (20%positive) and AE(30%positive); whereas2F5-MAP4elicited neutralizing activity against viruses ofclade BC (100%positive) and B (10%positive). Neutralization potencies of purifiedIgG from pooled sera of guinea pigs immunized with MPER peptide mimetics werealso tested against these ten HIV-1strains with broadly neutralization activities.The peptide absorption neutralization assay was performed to confirm at thecellular level whether anti-MAP IgGs could specifically recognize monomer epitopeof4E10/2F5. Results showed that the neutralizing activity of anti-MAP IgGs could beinhibited by the linear peptides in which epitopes of4E10/2F5located (4E10-LP, or2F5-LP), suggesting that anti-MAP IgGs can specifically recognize monomer epitopeof4E10/2F5.Other than broadly neutralizing antibodies against HIV-1, non-neutralizing antibodies also contribute to HIV-1vaccine development by inducing ADCC. In thepresent study, we also confirmed the weak ADCC depending guinea pig seraimmunized by4E10-and2F5-MAP4.In conclusion, two novel MPER-based MAP immunogens,4E10-and2F5-MAP4, were designed and shown to induce broadly NAbs against HIV-1in seraof guinea pigs. Although their potency and breath of neutralization were notcomparable to those of the corresponding mAbs, this is the first report of broad,although modest, neutralization elicited by HIV-1MPER-based synthetic MAPvaccination, laying the foundation for further development.2. Study on enveloped virus infection enhancing activity induced by peptidederivatives of MPERImproving the efficiency of gene transfer is a major principal for developingretrovirus-based gene transfer systems. Many infection-enhancing agents arecommonly used to increase retroviral transduction, such as diethylaminoethyl (DEAE)dextran, polybrene and protamine sulfate. Several amyloid-forming andinfection-enhancing peptides were discovered and investigated for their highefficiencies and low cytotoxicities, including two semen-derived peptides SEVI (a39-residue semen-derived infection enhancing peptide) and SEM, an A peptideassociated with Alzheimer’s disease, as well as an HIV-1gp120-derived peptide EF-CSEVI have been demonstrated the ability to boost retroviral gene delivery.Here, a13-residue peptide P13(Ac-NWFDITNWLWYIK-NH2; also known as4E10-LP in the first chapter) derived from the MPER, together with its16-residuepeptide derivative P16(Ac-NWFDITNWLWYIK-KKK-NH2) were found to enhanceHIV-1infection significantly, and P16exhibited infection enhancing activity in otherenveloped virus not limited to HIV-1. Amyloid fibril structures similar to that of SEVIwere also found in these two peptides by ThT binding assay and TEM, and it wasconfirmed that P13and P16form amyloid fibrils to facilitate retroviral infection.In order to achieve higher viral infection enhancing activity, four strategies wereneeded:1) low-speed agitation to form mature amyloid fibrils;2) virion and peptidepreincubated at high concentration to easily form complex;3) low ionic strength andlow pH value in infection buffer;4) infection without FBS in4h.As commonly observed with SEVI and the typical cell-penetrating Tat peptide,cationic amino acids such as Arg, His and Lys can eliminate electrostatic repulsion and thus facilitate subsequent attachment between negatively charged viral and targetcell membranes, thereby aiding fusion events. The increasing infection enhancementwith P13(1,840Da; pI=7, charge=0) mutated to P16(2,225Da; pI=10.48,charge=+3) have pointed to the functional contribution of cationic amino acid Lys.Next, ionic strength, pH value, and heparin inhibition assays have all confirmed thatpositively charged property plays an important role in the P16-mediated enhancementof viral infection.To investigate factors influencing the P16-mediated enhancement of viralinfection, we analyzed sequence characteristics of the MPER domain. MPER is ahighly conserved and Trp-rich domain in HIV-1gp41, possessing membrane lipidmixing and perturbing properties, which are important in viral fusion. Here, weattempted to determine if this characteristic of Trp enrichment plays a role inpeptide-mediated enhancement of HIV-1infection. Ala substitutions of the aromaticTrp residues in P16resulted in reduction of the enhancement of viral infectivity. Thisreduction of infection enhancement is partially with decreased amyloid fibrilformation.In summary, P16-mediated enhancement of HIV-1infection may occur throughformation of amyloid fibrils, whether soluble or insoluble, which serve as a scaffold.The positively charged fibril scaffolds help negatively charged virions to aggregate onthe target cell membrane, and the presence of Trp facilitates the embedding of fibrilsinto the cell membrane interface, resulting in improved viral infection rates. Ourexamination of the amyloid-forming and HIV-1infection-enhancing activities of P16and its derivatives, as well as the contribution of their electropositive property,existence of aromatic Trp residues and cationic Lys residues to these activities, hasrevealed possible benefits for the development of more effective gene transferenhancers. Notably, P16could enhance viral infection more potently than othercommonly used agents, such as DEAE dextran, polybrene and SEVI, enlightening uson its potential application in retroviral infection and gene therapy clinical trials.