Oral Abstract Session 01 - Global HIV Vaccine Enterprise

Topic 9: Non-Vaccine Prevention
P09.11
Conformation-Dependent Recognition of HIV Gp120
by DARPins Provides Novel Possibilities to Develop
Distinct HIV Entry Inhibitors
A.M. Mann 1 , N. Friedrich 1 , A. Krarup 1 , P. Rusert 1 , J. Weber 1 ,
B. Dreier 2 , P. Pugach 3 , M. Robbiani 3 , J.A. Robinson 4 ,
A. Pluckthun 2 , A. Trkola 1
1 University of Zurich, Zurich, Switzerland; 2 Institute of
Biochemistry, University of Zurich, Zurich, Switzerland; 3 Center
for Biomedical Research, Population Council, New York, NY,
USA; 4 Institute of Organic Chemistry, University of Zurich,
Zurich, Switzerland
Background: Designed Ankyrin Repeat proteins (DARPins) are
a novel type of binding protein scaffold designed as antibody
alternative for biomedical applications. DARPins share many
properties with antibodies, most noteworthy a high target
specificity and affinity, but they differ from antibodies in size,
structure and binding pattern and, importantly, favorable
biophysical properties such as exceptional stability. This
together with high-yield prokaryotic production renders DARPins
promising candidates for microbicide development.
Methods: DARPin DNA libraries encoding either two or three
internal ankyrin repeats were subjected to ribosome display
selections and panned against recombinant gp120 proteins.
Wild type, CD4 induced, V1V2 truncated and deglycosylated JR-
FL gp120, as well as structural V3 loop mimetics were probed
as target. Obtained DARPin clones were sequenced, mapped
for reactivity with HIV gp120 by ELISA and probed for inhibitory
activity using the TZM-bl pseudotype virus inhibition assay.
Results: DARPin selection proved more successful when
conformationally arrested targets were used for panning.
Overall, gp120-specific DARPins recognizing a variety of epitopes
including the CD4bs, CD4i and the V3 loop were obtained.
Gp120 mutant binding analysis revealed that DARPin molecules
depended to a higher degree on a structural conservation of
the envelope protein than gp120 specific antibodies recognizing
overlapping domains. Most noteworthy, V3 loop specific clones
were selected, which unlike V3 loop antibodies, recognized
the V3 loop in a conformation-dependent manner and thus do
not efficiently bind linear V3 loop peptides. In contrast to V3loop
directed antibodies these DARPins proved to bypass the
envelope shielding by the V1V2 domain and thus were capable
of neutralizing TIER-2 viruses.
Conclusion: Gp120 proved a challenging target for selection
of DARPin binders against gp120. Nonetheless, by combining
different selection strategies we were able to derive a variety of
gp120-specific DARPin molecules, including some with unique
HIV entry blocking activity.
P09.12
AIDS Vaccine 2012
Posters
Alcohol and Illicit Drug Use Among Potential HIV
Vaccine Efficacy Trial Volunteers Along Lake Victoria,
Uganda
I. Ssekandi 1 , A. Ssetaala 1 , J. Mpendo 1 , A. Nanvubya 1 ,
L. Nielsen 2 , N. Kiwanuka 3
1 UVRI-IAVI HIV Vaccine Program, Kampala, Uganda;
2 International AIDS Vaccine Initiative, New York, USA;
3 Makerere College of Health Sciences School of Public Health,
Kampala, Uganda
Background: HIV has adversely affected fishing communities
(FC), with prevalence ranging between 25-30% on Lake Victoria.
To better characterize the HIV prevalence among FC and explore
potential for HIV vaccine efficacy trials, we conducted a pilot
study on HIV prevalence, sexual risk behavior, alcohol and drug
use in these communities.
Methods: A cross-sectional HIV prevalence survey was conducted
in 8 FC over 3 months. Census and mapping to determine the
average population size in each community were conducted
prior to the study. After obtaining informed consent, 2,200
individuals aged 18-49 years from randomly selected households
were tested for HIV and responded to questionnaires on sexual
behavior, alcohol consumption and drug use.
Results: Fifty three percent (53.0%) had consumed alcohol
in the past three months with males consuming more alcohol
than females [59.0% vs. 46.8%, p