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

12th World AIDS Conference

Abstracts 41184-41188 779

the binding of 12G5 antibody. AMD3465, together with the previously reported bicyclam, are among the first nonpeptidic, low molecular-weight compounds shown
to specifically block HIV replication through interaction with the CXCR4 receptor.
Conclusion: AMD3465, a novel N-benzyl cyclam, is a potent anti-HIV agent
targeted at the chemokine receptor CXCR4 which is used as coreceptor for
T-tropic, syncytium-inducing strains of HIV. AMD3465 should be further studied
for its potential use as therapeutic agent against HIV infection.
41184 Accumulation of gag-pol polyprotein intermediates in
HIV-infected cells treated with protease inhibitors
Renae Speck, C. Flexner, X-F. Yu. 1Johns Hopkins University, Osler 524, 600
North Wolfe Street Baltimore MD 21 287 - 6180, USA
Objectives: To compare the concentration-dependent effects of HIV-1 protease
inhibitors on gag and gag-pol polyprotein processing in chronically infected T-cell
lines.
Methods: Chronically infected T-cell lines (H9/HIV-11liB, H9/HIV-1MN,
CEM/HIV-1MN) were suspended at 1X105 cells per ml in RPMI 1640 media
containing 10% fetal bovine serum. Mock infected and chronically infected T-cell
lines with no protease inhibitor were used as controls. An HIV-1 115, proteasedefective, H9 human T-lymphoid cell line was used as a negative control. Cells
were incubated with 0.05, 0.5, 5, 50, and 500 times the published ECso of indinavir, nelfinavir, ritonavir, saquinavir, and SC52151. After 24 hours of incubation,
the cells were harvested and resuspended in fresh media containing inhibitor.
Cells and supernatant were collected after an additional 48 hours and processed
by SDS polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose and probed with either human anti-HIV-1 serum or murine HIV-1 reverse
transcriptase (p51/p66) specific monoclonal antibodies.
Results: HIV-1 protease inhibitor treatment caused accumulation of gag and
gag/pol polyprotein intermediates in chronically infected cells. Many of the observed intermediates were not present in control chronically infected T-cell lines.
The specific processing intermediates seen in cell extracts and virus particles
changed as a function of drug concentration. All HIV-1 protease inhibitors produced surprisingly similar, concentration-dependent inhibition of gag and gag-pol
processing in H9/HIV-1111B. Complete inhibition of accumulation of gag-pol processing intermediates was not achieved. Based on comparison of the effects of
one inhibitor, saquinavir, on gag-pol processing in different cell lines, it appears
that this phenomenon is not cell type nor virus type specific.
Conclusion: Comparison of cells treated with the same relative concentration
of different protease inhibitors showed virtually identical patterns of inhibition of
gag and gag-pol processing. At drug concentrations as high as 500 times the
EC50 (well above the levels achieved in vivo), gag-pol precursor processing was
not completely inhibited. This may have relevance to the substantial overlap of
resistance pathways seen with these drugs.
411851 Effects of HAART on HIV viral load and CD4 T cells in the
mucosa-associated lymphoid tissue (MALT) and blood
Wolfgang Schmidt', M. Schaefer2, O. Faculer2, U. Bauer3, N. Mueller-Lantsch2,
E.O. Riecken3, R. Ullrich3. 1Medical Clinic UK Benjamin Franklin
Hindenburgdamm 30 12200 Berlin; 2Department Virology University of Saarland,
Homburg; 3University Clinic Benjamin Franklin Germany, Berlin, Germany
Objective: To determine the effect of HAART on HIV-1 RNA, proviral DNA and
CD4 T-lymphocytes in the MALT.
Methods: We investigated rectal biopsies and blood samples from HIV-infected
patients before and after initiation of HAART. Viral RNA and CD4-T-lymphocytes
were determined in both compartments by RT-PCR and threecolor flow cytometry,
resp. HIV-DNA was extracted from blood and biopsies and measured by qcPCR
using highly conserved nef primers.
Results: Preliminary analysis of samples from five patients before and 4 weeks
after onset of HAART revealed that two ART-naive patients had a decline in viral burden in peripheral blood (-0.93 log, -0.42 log; resp.) and rectal mucosa
( 0.42 log, 2.05 log) as well as a rise of CD4 T-cells (+122, +159 cells/il blood
and +160, +257 cells/mg biopsy) in both compartments. In the other patients,
discordant results were observed. One pretreated patient had an increase in viral
burden (+0.92 log) and only minor changes of CD4 T-cells (+15///1) in the blood,
and a decrease in mucosal CD4 T-cells ( 52/,/g) with only minor changes in
viral burden in the mucosa (-0.39 log). Another pretreated patient had a rise
in CD4 T-cells in the blood (+81//Il) without changes in HIV-1 RNA in the blood
and an increase of viral burden (+1.90 log) with a decrease of CD4 T-cells in
the mucosa (-20/mg). One seroconverter had a reduction of viral burden (-1.20
log) with an increase of CD4 T-cells (+291/pl) in the blood but an increase of
RNA in the mucosa (+0.57 log) associated with a decline in CD4 T-cells in the
mucosa (-20/mg). In the responding patients, viral RNA decreased with a median
half-time of 7.3 days and 4.6 days, and CD4 T-cells increased with a median
half-time of 20 days and 5.5 days in blood and mucosa, resp. Even in patients
without detectable HIV-RNA, HIV provirus was still found.
Conclusion: Early effects of HAART may differ between mucosa and blood.

Significant differences were also observed between patients, which may be due
to pretreatment or stage of disease. Follow-up data from our patients and quantitative analysis of proviral DNA will be presented. HIV replication kinetics and
CD4 T-cell turnover seem to be more rapid in the mucosal compartment than in
the blood.

S41186 Anti-HIV and immunomodulatory effects of interferon--r,
a new type I interferon
Marc Martin', N. Dereuddre-Bosquet2, P. Clayette2, P. Fretier2, G. Gras2,
J. Martal3, D. Dormont2. 1CEA Service de Neurovirologie DSV-DRM, BP6 92265
Fontenay AUX, Roses; 2CEA DSV/DRM/SNV, Fontenay/Roses; 3Jouy en Josas,
France
Interferon (IFN)-a has been reported to be effective in the treatment of patients
with human immunodeficiency virus (HIV)-related Kaposi's sarcoma. Nevertheless, its use was limited by severe side effects. On the contrary, few in vitro and in
vivo toxicity has been described up to now for IFN-r, a new type I IFN. Moreover,
because IFN-T has immunosuppressive effects towards CD4+ T lymphocytes and
capacity to increase interleukin (IL)-10 production and decrease tumor necrosis
factor (TNF)-u synthesis, we investigated its anti-HIV activity, its mode of action
and the possible implication of its immunomodulatory effects in its antiviral activity.
Antiretroviral activity of IFN-r was evaluated in primary cultures of human
peripheral blood mononuclear cells (PBMC), lymphocytes (PBL) and monocytederived macrophages (MDM), infected either with reference strains (HIV-1-LAI,
HIV-1/Ba-L) or primary isolates. Moreover, in order to evaluate the role of interferon (IFN)-r immunomodulatory activity in its antiviral effects, the expression
of pro- or anti-inflammatory cytokines such as tumor necrosis factor (TNF)-u,
interleukin (IL)-1/, IL-6 and IL-10 was evaluated in IFN-r-treated MDM cultures.
Cytokines were measured in cell culture supernatants by ELISA, and cytokine
mRNA expression by RT-PCR.
In these experiments, four main results were obtained. 1) A high antiretroviral
effect of IFN-r was evidenced, in the absence of cytotoxicity, against reference
and primary isolates. 2) No significant modulations of IL-1/ and TNF-u, and
their specific mRNA were detected when MDM were treated with IFN-r. 3) Weak
modulations of IL-10 mRNA expression and IL-10 protein secretion were only
observed in unstimulated uninfected IFN-r-treated MDM. 4) IL-6 and its mRNA
were modulated in response to MDM treatment by IFN-r. Strong relationships
were elsewhere described between other IFN type 1 and IL-6, and this participation of IL-6 to the antiviral activity of IFN-r in MDM may be supported by the
lower antiviral activity of this IFN when IFN-r-induced IL-6 production is blocked
by a neutralizing anti-IL-6 monoclonal antibody.
In conclusion, our results show that IFN-r could be considered as part in future
anti-HIV therapeutic strategies, as far as it combines antiviral and immunomodulatory effects susceptible to decrease the viral load and to modulate cytokine
network.
41187 Targeting anti-HIV agents to nucleocapsid protein zinc
knuckles without affecting cellular zinc finger proteins
William Rice, J.A. Turpin, G.M. Janini, A. Maynard, D.G. Covell, M. Huang.
National Cancer Institute - FCRDC, PO Box B, Bldg. 431 T-B, Frederick, MD,
USA
Objectives: HIV-1 nucleocapsid p7 (NCp7) protein Zn knuckles have emerged as
prime targets for antiviral therapy due to the conserved nature of their CysCysHisCys (CCHC) motif. Numerous chemotypes have been identified that modify the
nucleophilic sulfur atoms of the Zn-coordinating Cys residues of the Zn knuckles, and we sought to determine if these agents selectively target retroviral Zn
knuckles over other types of Zn fingers.
Design and Methods: Effects of agents on Zn ejection from and modification
of NCp7 structure and nucleic binding properties were biochemically evaluated.
These findings were compared to the effects of agents on the cellular CCHC
Zn finger protein poly (ADP-ribose) polymerase (PARP) and on the nucleic acid
binding properties of the cellular CCHH-containing Spl and the CCCC-containing
GATA-1 transcription factors. Computational studies employed interatomic surface molecular modeling, density function theory calculations and Fukui function
measurements, and were used to probe the selectivity of agents for the HIV-1
NCp7 Zn knuckles.
Results: Three chemotypes promoted Zn ejection from NCp7, modified NCp7
structure, and caused loss of nucleic acid binding by NCp7. These effects on
NCp7 occurred at concentrations of agents that did not affect PARP activity, the
nucleic acid binding properties of Spl or GATA-1, or the in vitro transcriptional
capacity of HeLa nuclear extracts. Computational efforts identified docked configurations of agents to NCp7 Zn knuckles having binding energies and molecular
orbital overlap that provide for favorable reactivities, suggested these agents
target a saddle-shaped binding domain on the surface of the knuckles that can
yield productive chemical reactivities, and suggested that the favored site for
electrophilic attack of NCp7 is the S49 atom in the carboxy-terminal knuckle.
Conclusions: Both experimental and computational approaches provided
strong evidence that antiviral agents can be developed that will selectively target
the HIV-1 NCp7 Zn knuckles without affecting the functions of cellular Zn finger
proteins.
S41188 Anti-HIV-1 activity of combinations and covalent
conjugates of negatively charged albumins and AZT

Mirjam E. Kuipers', P.J. Swart', M. Witvrouw2, J.A. Este2, E. De Clercq2,
D.K.F. Meijer1. 1 UCF Section of Pharmacokinetics Ant. Deusinglaan 1 9713 Av
Groningen, The Netherlands; 2Rega Institute for Medical Research Leuven,
Belgium
Background: Human serum albumins, that have been charge- modified by
succinylation or aconitylation at the terminal -NH2 functions of the lysines, are