European Journal of Medical Research - Deutsche AIDS ...

June 27, 2007 EUROPEAN JOURNAL OF MEDICAL RESEARCH
117
Results: In several rounds of bio-panning we selected the
highest-affinity SH3-binders of Sam68 from the SH3-proteome-pool.
These were in part members of the Src-family,
consistent with earlier observations by several groups. 2 of
the top-three-binders were identified as Sam68-binders for the
first time. The specificity of the interactions was confirmed
by pull-down assays with recombinant proteins and the binding-affinities
were determined by a phage-ELISA.
Conclusions: Using phage-display we could identify new
high-affinity SH3-binders of Sam68 and confirm known ones,
showing the general usability of the library as a tool for identifying
SH3-interactions. Further detailed characterisation of
these interactions will allow us to assess their importance for
the role of Sam68 in the export of late viral transcripts, being
the basis for design of novel therapeutic vaccines directed
against cellular structures.
E.9 (Poster)
Is the transmembrane envelope protein gp41
involved in HIV induced immunopathogenesis?
Results of cytokine arrays and microarrays
Denner J. 1 , Behrendt R. 1 , Lau M. 1 , Schmidt C.-M. 1 ,
Kurth R. 1
1 Robert Koch Institut, Berlin, Germany
Objective: Although the human immunodeficiency viruses
(HIV) have long been known to be the cause of AIDS, the
precise mechanism of disease induction by these viruses is
still unclear. Other retroviruses such as the feline leukemia
virus (FeLV) and the Koala retrovirus (KoRV) also induce
immunodeficiencies in infected animals, which are clinically
similar to AIDS. Reports showing inhibition of mitogen-induced
lymphocyte proliferation by inactivated retroviral particles
including HIV suggest that a viral protein may be involved
in the retroviral pathogenesis.
Methods: To study the influence of the viral transmembrane
envelope (TM) protein on cytokine production by normal human
lymphocytes, PBMCs from healthy blood donors were
incubated with recombinant TM proteins of different retroviruses
including HIV. In addition, synthetic peptides corresponding
to a highly conserved domain in the TM proteins
and designated immunosuppressive (isu-) peptides were also
used. Cytokine release was measured using different methods,
in addition a microarray analysis (Human Genome Survey
Microarray V2.0, 29098 genes) was performed.
Results: The expression of cytokines such as IL-10, IL-6, IL-
8, RANTES, MCP-1, MCP-2, TNF-alpha, MIP-1alpha, MIP-
1beta, MIP-3 increased upon exposure to the TM proteins and
the corresponding isu-peptides. In contrast, the expression of
IL-2 decreased and the expression of about 90 other cytokines
measured remained unchanged. The extent of changes in the
cytokine expression varied from donor to donor. The cytokine
data were confirmed by a microarray analysis.
Conclusion: These data indicate that retroviral TM proteins
modulate the cytokine production of normal PBMCs and
therefore may play an significant role in retrovirus-induced
immunopathogenesis. It is important to note that elevated IL-
6, IL-10, IL-8, gro-alpha and TNF-alpha and decreased IL-2
values have been regularly observed in HIV-1 infected individuals.
E.10 (Poster)
Isolation of RNA aptamers directed against HIV-1
envelope-derived surface epitopes
Müller I. 1 , Bunka D. 2 , Schult-Dietrich P. 1 , Brauer F. 1 ,
Adler A. 3 , Katinger H. 4 , Stockley P. 2 , von Laer D. 1
1 Georg-Speyer-Haus, Frankfurt am Main, Germany, 2 Astbury
Centre for Structural Molecular Biology, Leeds, United
Kingdom, 3 Darmstadt University of Technology, Darmstadt,
Germany, 4 Institute for Applied Microbiology, Vienna,
Austria
Aim: As the limitations of anti-HIV drug therapy become evident,
alternative therapeutic strategies are gaining interest.
Peptides derived from the heptad repeats of the HIV-1 gp41
envelope glycoprotein have shown a strong potential to inhibit
HIV-1 entry. To overcome the several disadvantages excluding
a broad application, we have isolated RNA molecules,
which were found to interact with the HIV-1 gp4, thus blocking
membrane fusion. As another possibility RNA aptamers
were selected against HIV-1 gp120.
Method: The isolation of RNA aptamers was performed using
the SELEX technology. Several different HIV-1 envelope
peptides served as targets in manual or robot-driven selections.
The starting DNA libraries contained a 30(50)-nucleotide
random region flanked by defined primer binding
sites. To ensure the selection of nuclease-resistant RNA aptamers,
2’-NH2 or -F-modified pyrimidines were included
during all in vitro transcription reactions.
Results: 10 rounds of selection were performed, followed by
an affinity maturation step using neutralizing antibodies for
binding competition. The most potent anti-gp41 RNA pool,
435UU, was able to bind to its target with a Kd » 750 nM.
Moreover, the 435UU RNA pool demonstrated specific HIV
neutralization with IC50 values approx. 250 nM. The selection
of anti-gp120 aptamers using HIV pseudoviruses coupled
to paramagnetic beads revealed an individual aptamer with an
IC50 of about 500 nM. Analysis of individual sequences revealed
for all selections still heterogenous pools. Furthermore,
secondary structure prediction using the MFold algorithm
displayed only a few defined structural motifs.
Conclusion: In order to increase the selection stringency, new
selection and maturation strategies are currently being established.
E.11 (Poster)
Investigation of HOCl-modified proteins which
exhibit a high activity towards HIV-1 gp120 in
vitro by inhibition of virus attachment
van Yperen M. 1 , Schreiber M. 1
1 Bernhard-Nocht-Institut for tropical medicine (BNI),
Virologie, Hamburg, Germany
The high-affinity interaction of the HIV-1 envelope glycoprotein
(gp120-gp41) and its receptor CD4 is important for viral
entry into host cells. We have generated a novel viral envelope
binding protein based on hypochlorite-modified HSA,
BSA, MSA and ß-Gal which exhibit an antiviral activity directed
towards NL4-3wt. Hypochlorous acid (HOCl) is a potent
oxidant produced by myeloperoxidase that causes modifications
in several amino acids. Antiviral tests were performed
by X-Gal staining and counting of infected cells. The compounds
revealed an inhibitory potential of rel. IC50 > 5 g/ml
mHSA, > 3 g/ml mBSA, > 20 g/ml mMSA, and > 14 g/ml