1. 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

2. 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?

3. 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

4. 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

5. 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

6. 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

7. 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?

9. 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

10. 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 Q
4-c.12 6x
4-12.3 2x E 4x M
10x

11. 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), )

12. 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

14. 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

15. 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