Georg-Speyer-Haus Institute for Biomedical Research Frankfurt, Germany Identification of vaccine-relevant mimotopes for neutralizing IgG present in plasma from long-term non-progressors (LTNP) by phage display Dr. Ursula Dietrich

Long-term non-progressors (LTNP) infected for more than 8 years low viral load high stable CD4 cell number no disease progression in the absence of any antiretroviral therapy Multiple reasons for LTNP status defects in viral regulatory genes (nef, vpr, vif,…) 32 bp deletion in cellular ccr5 coreceptor gene strong CTL response certain HLA-alleles (HLA-B*5701) Role of HIV-1 neutralizing antibodies in containment of the infection?

LTNPs included in the study patient Infected since CD4+ [cells/µl] viral RNA [cps/ml] viral subtype LTNP MH01 1990 522 2,388 B MH02 962 9,920 MH03 <1986 616 <50 MH04 <1987 558 2,288 MH05 <1985 540 66 MH06 1,320 4,936 MH07 1,023 52 MH08 1988 437 19,000 C functional orfs for viral regulatory genes tat, rev, nef, vpr no deletion in cellular ccr5 gene HLA-B*5701 protective allele only in one of the 8 LTNPs

Generation of recombinant HIV-1 reporter viruses Not I Not I 5' LTR 5' LTR ß-Lac ß-Gal ß-Lac ß-Gal p UC Ori p UC Ori BamH I BamH I LTNP env 3' LTR gag gag nef Pol 3' LTR p- TN7 env D p nef TN7-env Pol Ligation rluc rluc RT Nco I vpu tat RT rev Nco I env Int vpr tat RNaseH BstE II vif vpu rev vif Int vpr RNaseH Nde I Nde I Nde I BstE II pTN7∆env contains an env deleted HIV-1 NL4-3 genome and a renilla luciferase reporter gene instead of nef amplification of LTNP virus´ env genes by RT-PCR and cloning into pTN7∆env (MH01, MH02, MH03, MH04, MH06, MH08) control viruses: D117III, JR-CSF, YU2, 89.6, NL4-3 and 7 viruses from progressing patients with comparable CD4 count and VL transfection into 293T cells to generate recombinant virus stocks, titration on tzm-bl cells sequence analyses and neutralization assays

Neutralization assay Preincubation of 100 i.U. virus with serial dilutions of heat-inactivated plasma, 1 h at 37°C, infection of 104 U87-CD4-CCR5/CXCR4 cells (moi=0.01) in triplicates, after 48 hrs cells are lysed and luciferase avtivity is measured Inhibition [%]= [1-(luc HIV+ serum/luc virus only)] X 100

Neutralization of heterologous HIV-1 by LTNP plasma Neutralization assay with purified MH002 IgGs 20000 40000 60000 80000 100000 120000 140000 160000 180000 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:25600 1:51200 HIV neg. IgGs Medium only no Virus but MH002 HR010 virus, no YU 2 Dilution Luciferase activity YU2 MH02 IgG controls IC50 IC90

Which are the target epitopes for the HIV-1 neutralizing antibodies LTNP sera neutralize heterologous HIV-1 with significantly higher IC50 and IC90 values than sera fom progressors with comparable CD4 number and viral load. Which are the target epitopes for the HIV-1 neutralizing antibodies present in LTNP sera?

Biopanning immobilize anti-human Fc-antibody for capturing of plasma IgG positive selection: LTNP plasma negative selection: HIV-neg plasma pool three selection rounds titering of selected phages specificity-test by phage ELISA sequencing of positive phage clones linear and 3D-alignment of selected peptides to HIV-1 proteins and published structures

Selection of phages with immunodominant epitopes: V3 gp120 K R I H G P A F Y T 1-c.4 18x L 2-c.21 3x W 2-c.22 3-7.1 5x V S 3-12.22 Q 4-c.12 6x 4-12.3 2x E 13-12.5 4x M 14-12.1 10x 15-12.10 20-12.2 20-12.4

Summary of phage dipslay screenings with LTNP sera 8 LTNPs (MH01-MH08 used) for biopannings 1400 phage clones analyzed by ELISA for specificity 700 clones sequenced Immunodominant epitopes (V3, KLIC) were selected frequently Analysis for the capacity of linear peptide sequences to encode conformational epitopes on the surface of the gp120 structure (3DEX program: Humbert et al., J Comp Chem 2005, 26 (9),879-887)

Immunization of mice with selected phage groups immunized and boosted s.c. + adjuvant with 2.5x1012 phages grouped for linear or conformational similarity bleeding ten days after sixth boost (80. day) Test mice sera by ELISA and for neutralization of HIV-1

Summary A group of LTNPs was characterized in which HIV neutralizing antibodies very likely contribute to the absence of disease progression LTNP sera contain broadly neutralizing antibodies against HIV-1 We could select peptide ligands for HIV-specific antibodies present in the LTNP sera, some of which mimic conformational epitopes on the surface of gp120 according to 3DEX-analysis Sera from mice immunized with groups of the selected phages could neutralize primary HIV-1 in vitro, proving that the selected phages indeed present HIV-specific epitopes for neutralizing antibodies on their surface

Thanks to… Clinical partners: DFG DI356-3 Michael Humbert Sascha Antoni Margot Landersz Nicole Walz Clinical partners: Boris Brill Dorothee von Laer Georg-Speyer-Haus Frankfurt, Germany Vicente Soriano Berta Rodes Instituto de Salud Carlos III Madrid, Spain Matthias T. Dittmar Department of Virology University of Heidelberg Heidelberg, Germany Heribert Knechten Praxiszentrum Blondelstr. Aachen, Germany Schlomo Staszewski Infektionsambulanz, JWG-University Frankfurt, Germany DFG DI356-3 German AIDS award (big spender) Dr. Bodo Sponholz-Stiftung