Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

4 Abstracts 11106-11112

12th World AIDS Conference

10*/11106 Co-receptors and cellular targets for HIV-1 in the
human female genital tract
Florian Hladik1, G. Lentz2, R.E. Akrioge2, H. Kelly2, Alef McElroy1,
Julie McElrath2. 1Fred Hutchinson Cancer Research Center, 1124 Columbia
Street, #M785, Seattle; 2University of Washington, Seattle, WA, USA
Objectives: To identify the cells first attacked by HIV-1 in the human genital
tract and to elucidate mechanisms for selection of viral variants during sexual
transmission of HIV-1.
Methods: We established an isolation procedure whereby human vaginal or
cervical mononuclear cells selectively emigrate from mucosal explants over 36 h.
The isolated unstimulated cell populations were analyzed for HIV-1 co-receptor
expression and the infectivity of M-tropic and T-tropic HIV-1 strains.
Results: 5 major cell populations were identified by flow cytometry using phenotypic markers: CD3+ T cells, CD83+ dendritic cells (DC), clusters of CD83+ DCs
and CD3+ T cells, CD14+ macrophages, and clusters of CD14' macrophages and
CD3+ T cells. Examining surface expression by flow cytometry in >10 individuals,
CCR5 was present on >80% of mucosal T cells and the majority of macrophages,
but weak or absent on DCs. These findings were corroborated by mRNA expression in sorted mucosal mononuclear cells (MMC) subpopulations. By contrast,
CXCR4 expression was abundant on all MMC. Infection with both CCR5-tropic
and CXCR4-tropic HIV-1 isolates was highly productive when T cells were co-cultured with DCs, but not when cultured alone. However, fully reverse transcribed
viral DNA, as determined by nested LTR/gag PCR, was clearly present in both
single T cells and T cell/DC clusters.
Selection of viral strains does not occur by predominance of CCR5 over
CXCR4 expression on MMC. Expression of CCR5 on the majority of T cells
and macrophages suggests that these cells are the early targets for M-tropic
HIV-1 in the human genital mucosa. The block of the viral live cycle following
reverse transcription in unstimulated cervicovaginal T cells can be very effectively
overcome by contact with local DCs.
57*/11107 Bicyclams are selective antagonists of the
chemokine receptor CXCR4 and potent inhibitors of
HIV
Dominique Schols1, J.A. Este2, S. Struyf1, J. van Damme1, G.J. Bridger3,
G.W. Henson3, E. de Clercg1. 'Rega Institute, Minderbroedersstraat 10, B-3000,
Lueven, Belgium; Institut de la Recerca de la Sida, Caixa, Baldalona, Spain;
3Anormed, Langley, BC, Canada
Bicyclams are a class of antiviral compounds which are highly potent and selective
inhibitors of the replication of HIV-1 and HIV-2 strains. The prototype compound,
AMD3100, 1,1'-[1,4-phenylenebis-(methylene)]-bis-1,4,8,11 -tetraazacyclotetradecane, has an IC50so of 1-10 nM, which is a least 100,000 fold lower than the
cytotoxic concentration. When the bicyclams were evaluated against macrophagetropic HIV strains, they proved inactive. Because of the specific and potent inhibitory effect of AMD3100 on T cell line-tropic viruses, but not macrophage-tropic
viruses, it was verified whether bicyclams interact with the CXC-chemokine receptor CXCR4, the main coreceptor used by T cell line-tropic viruses. Bicyclams dosedependently inhibited the binding of a specific CXCR4 mAb to T cells, as monitored
by flow cytometry. In addition, bicyclams blocked the Ca2+ flux and chemotactic
responses induced by stromal cell-derived factor la//I (SDF-lu/I), the natural
ligand for CXCR4. The anti-HIV potency of the bicyclams closely correlated with
their potency in inhibiting the binding of anti-CXCR4 mAb and inhibiting the Ca2+
flux induced by SDF-la/p. The bicyclams had no effect on the Ca2+ flux induced
by the chemokines MIP-la, MIP-1, RANTES, MCP-3, IL-8 and GCP-2 (which
are ligands for CCR-1, CCR-2, CCR-3, CCR-4, CCR-5, CXCR-1 or CXCR-2).
The bicyclams are the first low-molecular-weight anti-HIV agents to be found
to specifically interact with CXCR4, the main coreceptor for T cell line-tropic HIV
strains. AMD3100 strongly binds to CXCR4 and through this interaction potently
inhibits viral replication. AMD3100 holds great promise as a candidate anti-HIV
drug and clinical phase I/11 trials with the compound have been started.
1|11108 Frequency of the A32 deletion allele of CCR5 coreceptor
gene in HIV infected and normal population of Central
Europe
Bela Kemeny1'2, K. Nagy2, A. Horuath2. 1Maria U. 41 1085 Budapest; 2Natl Inst
of Derm-Vener Budapest, Hungary
Objectives: CCR5 is a chemokine receptor and also serves as co-receptor for
non-syncitia inducing, macrophage-tropic strains of HIV. It had been shown that a
32 base-pair deletion in the gene results in a severely truncated protein and leads
to a partial resistance in HIV infection. As the majority of the sexually transmitted
HIV-1 isolates are macrophage-tropic, we studied the polymorphism of the CCR5
gene in our HIV infected patients as well as in normal population.
Methods: The following groups were included in our study: long-term non
progressor (LTNP) HIV-infected individuals, HIV-infected persons with usual progression of infection, their sexual contacts, and healthy individuals. The analysis

was done by PCR of DNA isolated from PBMCs of the patients.
Results: Out of 117 determinations, heterozygote mutations of the CCR5 gene
were demonstrated in 40% of the LTNPs, in 16% of HIV progressors, in 17% of
the HIV negative contacts and 21% of healthy individuals. Homozygous form of
A32 deletion has been found in two cases (1.9%), none of them were infected by
HIV. Allele frequency of the deletion is 0.120.

Conclusions: The high prevalence of A32 deletion of CCR5 gene among
LTNPs supports findings, that mutation in co-receptor gene may provide a certain
protection against HIV infection and/or contribute to the delayed progression of
AIDS.
| 11110 Isolation of MIP-l c-resistant macrophage-tropic HIV-1
selected by passage in cell culture
Yosuke Maeda~2, S.M. Matsushita2, M.B.F. Foda2, K.N. Nanke2, H.M. Maeda2
S.H. Harada2. 12-2-1 Honjo Kumamoto; 2Kumamoto University Kumamoto,
Japan
Background: It has been shown that chemokine receptors, CXCR4 and CCR5,
serve as the major coreceptors for T- and macrophage-tropic human immunodeficiency virus (HIV-1) isolate, respectively, and that natural ligands for CCR5,
including MIP-lao, MIP-1/~, and RANTES, block macrophage-tropic but not
T-tropic HIV-1 infection. It has been also shown that the disease progression in
HIV-1-infected individuals is associated with the change in the coreceptor usage
from CCR5 to CXCR4, and with a loss of sensitivity to these CC-chemokines.
Recent data showed that the change of coreceptor was associated with changes
in the third variable (V3) region of env gene. However, little is known which region
in HIV-1 is responsible for the reduced sensitivity to the CC-chemokines in vivo.
Methods: In order to elucidate its molecular mechanism, a variant of
macrophage-tropic HIV with reduced sensitivity to MIP-Il has been selected
by passage of virus in CCR5-expressed MOLT-4#8 cells in the presence of increasing amount of MIP-la. The sensitivities of these variants were determined
using CCR5-expressed CD4-HeLa/LTR-/-gal (MAGI/CCR5) cells.
Results: The variant from a macrophage-tropic infectious clone, JR-FL, had
moderately decreased sensitivity (fourfold) to MIP-1a compared with wild type
JR-FL. This resistant variant had also decreased sensitivity to MIP-1/I and
RANTES, but did not change the cellular tropism, suggesting that this variant is
still using CCR5 for its coreceptor. Analyses of the env sequences of the isolate
had V-- M substitution at codon 166 in the second variable (V2) region, whereas
no remarkable changes were observed in the V3 region.
Conclusions: These results indicated that the CC-chemokines-resistant mutant could emerge from macrophage-tropic HIV-1 without any changes in the V3
region in vitro.
11111 Study of co-receptors used by NSI HIV-1 strains that infect
CD4+ T-cell lines, Molt4 and SupT1
Nathalie Dejucq, Graham Simmonds, S. Hibbitts, P. Clapham. Institute of Cancer
Reseach, London, UK
Background: Non-syncytium-inducing (NSI) CCR5-using strains usually infect
macrophages but not T cell lines. A variant of JR-CSF (called C3) selected
for replication in the CD4+ T-cell lines Molt4 and SupT1 differed by a single
amino-acid change in the VI loop of gp 120 (Boyd et al., 1993). The aim of this
study was therefore to assess the co-receptor use by C3 and other Molt4/SupT1
tropic primary HIV-1 strains.
Methods: To check co-receptor-use by C3 and other HIV-1 strains, we used
the human U87 and the feline CCC cell lines transfected with CD4 and the following chemokine receptors: CCR1, CCR2b, CCR3, CXCR4, CCR5, CCR8, Bob,
Bonzo, GPR1 or V28. The expression of known chemokine receptors was assessed on Molt4 and SupT1 using RT-PCR, flow cytometry analysis and confocal
microscopy. Inhibition of infection by different chemokines was assessed. Infection
of A32/A32 CCR5 PBMC by C3 and other Molt4/SupT1 NSI strains was studied.
Results: C3 and three other NSI strains that infect Molt4/SupT1 used CCR5
only and did not infect U87 or CCC cells transfected with any other co-receptors.
Molt4 and SupT1 were shown to express mRNAs coding for CXCR4, CCR5,
CCR3 and Bonzo. CCR5 and CCR3 were however barely undetectable on the
cell surface. Inhibition experiments using AOP RANTES, a strong inhibitor of viral
entry for strains using either CCR3 or CCR5, blocked infection of both Molt4
and SupT1. Eotaxin, a CCR3 ligand, had no effect on the viral replication, while
MIP1/l, a specific ligand for CCR5, totally inhibited the infection. No infection of
A32/A32 CCR5 PBMC by C3 or other Molt4/SupT1 tropic strains was observed,
even after long term culture.
Conclusions: Our results indicate that some HIV-1 strains with an expanded
tropism for Molt4 and SupT1 have no change in co-receptor use and use CCR5 for
infection of these cell lines. The implications of these results for HIV-1 co-receptor
use and cell tropism in vivo will be discussed.
S11112 Tropism, characterization, and co-receptor usage of an
HIV-1 isolate derived from HIV +ve person homozygous
for CCR5A32
Hassan Naif', M. Alali', S. Li', R. French2, G. Stewart3, A. Cunningham'.
'Centre for Virus Research Westmead Hospital sydney 2145; 2Sydney Children
Hospital Sydney NSW; 3lImmunology Dept Sydney NSW, Australia

Objectives: To investigate the mechanism of infection of an HIV+ve person homozygous for CCR5A32 and identification of co-receptors other than CCR5 that
are used by isolated viruses.
Methods: The patient was repeatidly positive for HIV antibody by ELISA and
western blot. Homozygosity for CCR5A32 mutation was detected by PCR and
confirmed by DNA sequencing. Virus growth in MT2 cell line, macrophages and
lymphocytes was measured by CPE, p24 antigen ELISA and DNA-PCR. The