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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
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LTNPs included in the study
functional orfs for viral regulatory genes tat,
rev, nef, vpr no deletion in cellular ccr5
gene HLA-B5701 protective allele only in one of
the 8 LTNPs
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Generation of recombinant HIV-1 reporter viruses
Not
I
Not
I
5' LTR
5' LTR
ß-Gal
ß-Gal
ß-Lac
ß-Lac
p
UC Ori
BamH
I
p
UC Ori
BamH
I
3' LTR
gag
LTNP env
Pol
gag
nef
3' LTR
p-
TN7 env
D
p
TN7-env
nef
rluc
Ligation
Pol
RT
rluc
Nco
I
vpu
tat
RT
rev
env
Int
tat
Nco
I
vpr
RNaseH
vif
vif
rev
vpu
BstE
II
vpr
Int
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

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Neutralization assay
Preincubation of 100 i.U. virus with serial
dilutions of heat-inactivated plasma, 1 h at
37C, infection of 104 U87-CD4-CCR5/CXCR4 cells
(moi0.01) in triplicates, after 48 hrs cells are
lysed and luciferase avtivity is measured
Inhibition 1-(luc HIV serum/luc virus
only) X 100
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Neutralization of heterologous HIV-1 by LTNP
plasma
controls
MH02 IgG
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LTNP sera neutralize heterologous HIV-1 with
significantly higher IC50 and IC90 values than
sera fom progressors with comparable CD4 number
and viral load.
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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
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Selection of phages with immunodominant epitopes
V3
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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)
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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

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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
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Thanks to
Sascha Antoni
Margot Landersz
Nicole Walz
Michael Humbert
Clinical partners
Boris Brill Dorothee von Laer Georg-Speyer-Haus Fr
ankfurt, 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
DFG DI356-3 German AIDS award (big spender) Dr.
Bodo Sponholz-Stiftung
Schlomo Staszewski Infektionsambulanz,
JWG-University Frankfurt, Germany