Abstract Book Vol. 1 [International Conference on AIDS (16th: 2006: Toronto, Canada)]

Uoda

Poster Exhibition
Track A
MOPEO001
The road to finding potent HIV-1 entry inhibitors:
lessons learned from requirements for BMS-806
binding to HIV-1 envelope glycoprotein
N. Madani', A. Hubicki2, D. Ng3, A. Smith', J. Sodroski'. 'Dana-Farber Cancer
Institute, Harvard Medical School, Cancer Immunology and AIDS, Department
of Pathology, Boston, United States, 2Dana-Farber Cancer Institute, Cancer
Immunology and AIDS, Boston, United States, 3University of Pennsylvania,
Department of Chemistry, Philadelphia, United States
Background: BMS-806 is a novel compound that binds HIV-1 envelope
glycoprotein gp120 and broadly inhibits both X4 and R5 viral isolates.
Previously we identified a series of mutations in gpl20 resulting in resistance
to the antiviral effects of BMS-806.
Methods: BMS-806 was synthesized and its inhibitory effects were examined
using a single-round infection of recombinant HIV-1 encoding firefly luciferase.
In parallel, direct binding assays were performed using [3H]BMS-806.
Results: Based on the CD4-bound conformation of gpl20, the amino
acid residues implicated in the resistance to BMS-806 were located in the
"phenylalanine 43 cavity" and a water-filled channel that extends from the
cavity to the inner domain of the protein. We further extended our panel and
demonstrated that critical residues within the stem of the V3 loop of gpl20
are necessary for BMS-806's antiviral effects. Specifically, mutation of residues
at positions 305, 307, 309, and 317 from Lysine, Isoleucine, Isoleucine, and
Phenylalanine, respectively, to Alanine result in escape from antiviral effects of
BMS-806, without any reduction in overall replication compared to wild-type
viruses. Moreover, these same mutations resulted in a significant decrease
of BMS-806 binding compared to the wild-type as determined by [3H]BMS806 direct binding assays. These data suggest a preferred site for BMS-806
binding within V3 loop of gp120. In addition, the results indicate a high
degree of correlation between the amino acid changes in gpl20 that affect
BMS-806 binding and those that allow virus escape from inhibition, suggesting
drug binding site on the gp120 monomer and on the functional trimer must
coincide.
Conclusions: Identification of new residues important for BMS-806 binding
provides significant new insights into BMS-806 envelope interaction. Together
with our previous studies, these results point to a conformation within gp120
that is critical for BMS-806 binding, providing useful information for rational
drug design.
MOPEO002
Envelope determinants of highly efficient
macrophage-tropism and neutralization sensitivity
conferred by a brain derived HIV-1 envelope
M.J. Duenas-Decamp, P.J. Peters, P.R. Clapham. University of Massachusetts
Medical School, Worcester, United States
Background: HIV-1 binds CD4 and CCR5/CXCR4 to infect cells. We previously
described R5 envelopes amplified from uncultured patient tissues that
conferred distinct tropisms. R5 envelopes from brain were highly macrophagetropic, while lymph node (LN) envelopes infected macrophages inefficiently.
Macrophage-tropism correlated use of low CD4 levels for infection. Envelope
determinants and in vivo selective pressures that confer macrophage-tropism
are poorly understood. Highly macrophage-tropic brain envelopes may have
adapted for replication in resident macrophages and microglia. However, low
concentrations of neutralizing antibodies (nabs) in brain tissue protected
by the blood brain barrier may also play a role. Moreover, brain envelopes
from two individuals were sensitive to neutralization by the CD4 binding site
(CD4bs) mab, b12 while LN envelopes were resistant. Here, we map envelope
determinants conferring macrophage-tropism and b12 sensitivity.
Methods: NA420 envelope B33 (brain) is highly macrophage-tropic and b12
sensitive, while LN40 (LN) is non-macrophage-tropic and b12 resistant. B33
N283T was constructed. N283 is associated with enhanced macrophagetropism, is predominant in brain but rare in LN. Chimeric B33 envelopes
were constructed containing non-overlapping LN40 envelope regions, C2-C3
and C3-C5. Mutant envelopes were expressed on reporter viruses to analyze
phenotypes.
Results: N283T reduced macrophage infection and infection via low levels of
CD4, but did not affect b12 sensitivity. LN40 C2-C3 abrogated macrophage
and low CD4 infection. LN40 C3-C5 conferred a minor reduction in macrophage
infection but entirely modulated b12 sensitivity.
Conclusions: Macrophage-tropism of B33 brain envelope was mainly
determined by C2-C3 env region. N283 and C3-C5 env region contributed to

lesser extents. B12 sensitivity mapped to C3-C5. Thus, envelope determinants
of macrophage-tropism are mainly distinct from those conferring neutralization
sensitivity. Our results do not support a role for CD4bs nabs as a selective force
for evolution of macrophage-tropic variants in brain.

MOPEO003
Identification of new antiviral compounds targeting
HIV-1 entry
S. Janvierl, F. Hamdan2, M. Bouvier2, N. Heveker'. 'University of Montreal,
Sainte-Justine Hospital Research Center, Biochemistry, Montreal, Canada,
2University of Montreal, Biochemistry, Montreal, Canada
Background: HIV-1 entry into target cells is currently the most promising
validated new antiviral target. Several molecules have been developed in order
to block viral entry by targeting the HIV-1 co-receptors CCR5 or CXCR4 some
of which are currently in clinical phase III testing. Until now, only one molecule
targeting HIV-1 fusion, enfuvirtide (T20), has been approved for treatment.
Methods: We identified 4 new compounds, by High Throughput Screening,
that can inhibit the activation of CCR5. We named them A, Aa (a derivative
of A), B and C. The BRET screening assay technique detects the effect of
those compounds on the CCR5-13-arrestin2 interaction following activation of
the receptor by RANTES thereby identifying receptor antagonists. We also
demonstrated that a molecule from green tea extract, EGCG, and two of its
derivatives, TD and TM, possess similar activity. Our objective was to test
the antiviral activity of those 7 molecules on HIV-1 entry via the co-receptor
CCR5.
To test the selectivity of the compounds for CCR5, we used stable cell lines
expressing either CCR5 (R5.3) or CXCR4 (X4.15) as well as p-galactosidase
under the HIV-1 LTR promoter. Dual tropic HIV-1 (89.6) viral particles were
used to test the inhibitory effect of the 7 molecules on the two reporter cell
lines.
Results: We demonstrate that EGCG, TD, TM and compounds Aa, B and C, in
contrast to compound A, show little or no co-receptor specificity in inhibiting
HIV-1. That means that those compounds could either non-selectively inhibit
HIV-1 entry, or other steps of viral replication. In contrast, compound A
demonstrates a specific inhibitory potential against CCR5 mediated HIV-1 entry
with an IC50 of 4,95 M ~ 1,55.
Conclusions: This compound could serve as a new lead structure for the
development of new molecules with higher CCR5 affinity.
MOPEOOO4
Sialoglycoprotein (Sgp) pattern and
sialyltransferase (ST) activity of HIV-1 in acutely
infected cells under inhibition of glycosylation
R. Gavazova', S. Ivanovl, D. Ivanovl, S. Raleva2, L. Froloshka2, P. Genova3,
D. Dundarova3, R. Argirova2. 'Institute of Experimental Pathology and
Parasitology - Bulgarian Academy of Sciences, Dept. of Biochemistry, Sofia,
Bulgaria, 2National Centre of Infectious and Parasitic Diseases, Dept. of
Virology, Lab. of Retroviruses, Sofia, Bulgaria, 3National Centre of Infectious
and Parasitic Diseases, Dept. of Virology, Lab. of Cell cultures, Sofia, Bulgaria
Background: Sialoglycoproteins (Sgps) are directly involved as epitopes in the
recognition process permitting access to receptors. Formation of Sgps is type
specific and catalyzed by specific sialyltransferases (ST). Earlier we created a
laboratory model to study HIV-1 epitope structures. High and virus-specific
rate of sialylation of HIV-1LAI in acutely and chronically infected cells was
shown. To further understand changes of epitope structures conferring viral
escape in HIV-infected individuals we studied first the Sgp profile of HIV-1LAI
in MT-2 infected cells treated with tunicamycin (Tu) - a well known inhibitor of
glycosylation as well as corresponding ST activies.
Methods: A study of N-acetyl-[C14]-mannosamine (14C-NAcMan)
incorporation in cytosols of HIV-1LAI acutely infected and uninfected MT2 cells treated with Tu (0,5pg/ml) was carried out. Fractions derived after
isoelectrofocusing (IEF) were measured for 14C-incorporation, protein content
and reverse transcriptase (RT) activity (Cavidi, Sweden). ST-activity was
detected by Serafini-Cessi F. method.
Results: Higher incorporation of 14C-NAcMan for MT-2/HIV + Tu compared
to MT-2 + Tu was observed. MT-2 IEF profile compared to that of MT-2 + Tu
showed a shift of the latter to the basic zone and only two common pI peaks.
On the contrary, a number of common pI peaks in MT-2/HIV and those treated
by Tu were seen. ST activity was reduced by 330/ for MT-2 + Tu compared
to MT-2 v. 20,6 %/ for MT-2/HIV + Tu compared to MT-2/HIV (inhibition of
glycosylation 27,8 and 14,20/ resp.).
Conclusions: Higher inhibition of glycosylation by Tu in MT-2 cells compared to
MT-2/HIV probably confers the higher inhibition of sialylation and the changes
in Sgp profiles of Tu treated cells. The differing effect of equal concentrations of
Tu on glycosylation in MT-2 and MT-2/HIV under experimental conditions might
at least partially explain already described viral escape in HIV-infected persons
by changes in glycosylation.
MvOPEOOO5
Polymorphism of CCR5 and CCR2 genes associated
with HIV-1 resistance in Mongolia
Y. Amarjargal', N. Munkhtuvshin', T. Lkhagvasuren', C. Baigalmaa',

K.-W. Kim', A. Tumanov6, E. Kazennova6, M. Bobkova6. 'Institute of Public
Health, Ulan Bator, Mongolia, 'National Institute of Medicine, Ulan Bator,
Mongolia, 'Health Science University of Mongolia, Ulan Bator, Mongolia,
4National Center for Communicable Diseases, Ulan Bator, Mongolia, sHealth
Research Institute of Korea, Seoul, Korea, Republic of, 6DI Ivanovsky
Institute of Virology, Moscow, Russian Federation
Background: The CCR5 and CCR2 chemokine receptor gene polymorphism
has been shown to influence susceptibility to HIV-1 infection and disease

XVI INTERNATIONAL AIDS CONFERENCE * 13-18 AUGUST 2006 * TORONTO CANADA * ABSTRACT BOOK VOLUME 1