Abstract Book Vol. 2 [International Conference on AIDS (14th: 2002: Barcelona, Spain)]

10 Abstracts WePeA5726-WePeA5731

XIV International AIDS Conference

Presenting author: david margolis, 5323 harry hines blvd, mailcode 9113, dallas,
texas 75390, United States, Tel.: [phone], Fax: [phone], E-mail:
[email protected]
WePeA5726 IEstablishment of quantitative assay for cellular
HIV-1 mRNA by real-time PCR
H. Nagai', K. Wada, Y Tawada2, T. Morishita3, M. Utsumi, Y. Nishiyama4,
T. Kaneda1. i'Department of Clinical Research, Nagoya National Hospital,
Department of Clinical Research, Nagoya National Hospital, 4-1-1 Sannomaru
Naka-ku Nagoya 460-0001 Aichi, Japan; 2Department of Clinical Laboratory
Nagoya National Hospital, Nagoya, Japan; 3Department of Microbiology Aichi
Prefectural Institute of Public Health, Nagoya, Japan; 4Laboratory of Virology,
Research Institute for Disease Mechanism and Control, Nagoya University
School of Medicine, Nagoya, Japan
Background: A method to quantify cellular HIV-1 mRNA, multiple-spliced-mRNA
(MS-mRNA) and unspliced-mRNA (US-mRNA), was established. Using this
quantitative assay method, we monitored the transcriptional activity of HIV-1
provirus in CD4+ T lymphocytes from HIV-1 infected patients.
Methods: Total RNA was extracted from CD4+ T lymphocytes, and was then reverse transcribed using OligodT12.18 primers. The synthesized cDNA was used for
the quantitative assay. Quantification of MS-mRNA and US-mRNA was achieved
with a Roche LightCycler using two sets of primers and a Taqman probe. Twentyone patients receiving HAART and 6 naive patients were enrolled in this study.
Results: The minimum level of detectable mRNA was 5 copies(c)/assay for
both MS- and US-mRNA. Average levels of MS-mRNA in HAART-receiving patients with a plasma viral load (VL)<50c/ml and with VL>50c/ml were 0.6c/cell
(n=12) and 1.7c/cell (n=9), respectively; that in naive patients was 0.6c/cell (n=6).
Average levels of US-mRNA in HAART-receiving patients with VL<50c/ml and
with VL>50c/ml were 0.03c/cell (n=12) and 0.05c/cell (n=9), respectively; that
in naive patients was 0.12c/cell (n=6). The levels of MS-mRNA and US-mRNA
in Molt4 which the HIV-1 IIIB strain persistently infects were 1.5~0.2c/cell and
173.9+35.9c/cell, respectively.
Conclusion: Even in successful cases of HAART, the transcription of HIV-1
provirus was not completely inhibited. Therefore, the suppression of plasma VL in
these patients may be due to the inhibition not only of the HIV-1 provirus transcription, but also of other cell mechanisms, such as translation, post-transcriptional
regulations, and/or viral assembly
Presenting author: Hiromi Nagai, Department of Clinical Research, Nagoya National Hospital, 4-1-1 Sannomaru Naka-ku Nagoya 460-0001 Aichi, Japan, Tel.:
[phone] (ext. 2770), Fax: [phone], E-mail: hnaga@livedoor.
com
WePeA5727 Evolution of CD4 and CKR usage by SIV during in
vitro xenoinfection
S. lVenqar, J.G. Finny, H. Zhang, C. Chancey, D.H. Schwartz. Dept Molec Micro
& Immunol, Bloomberg School of Public Health, Johns Hopkins University Dept
of Molec Micro & Immunol, Bloomberg School of Public Health, Johns Hopkins
University, 615 N. Wolfe Street, Baltimore, MD, 21205, United States
Background: All HIV-1, 2, and SIV strains are CD4 dependent in cells of the
host species from which they were isolated, and it is widely stated that all SIV
strains use CCR5 rather than CXCR4. CD4 and/or CKR variation among primate
species could be barriers to lentivirus xeno-transmission. We examined the CD4
and chemokine receptor (CKR) usage of two SIV strains during adaptation to
human (hu) PBMCs.
Methods: Lymphocyte-tropic SIVmac239, and the neurovirulent macrophagetropic clone SIV/17E-Fr (constructed by replacing SIVmac239 env with env from
a serially brain passaged SIVmac239) were grown in macaque PBMCs prior
to being maintained in CEMx174 cells. SIV stocks from this cell line were assayed for CD4 and CKR dependent growth on macaque or huPBMCs by p27
EIA. Supernatants were fed with fresh allogeneic huPBMCs weekly. CKR binding
chemokines and mAbs were used at saturating concentrations in blocking experiments.
Results: Both stocks of SIV showed CD4 dependence and CCR5 usage when
grown on macaque PBMCs, as evidenced by blocking infection with anti-CD4
mAb (Leu 3A), CD4-IgG, a-CCR5 mAb (2D7) or 1 chemokines. Surprisingly, parallel experiments in huPBMCs showed no blocking of infection by Leu3A or CD4 -IgG. In some cases enhancement was seen. By the 5th passage in huPBMCs,
Leu3A caused partial blocking. By the 15th passage in huPBMCs, both SIV populations showed strong CD4 dependence, with complete blocking by Leu3A. Also,
by the 15th passage, both SIV strains had switched to CXCR4 usage, as evidenced by blocking effects of o-CXCR4 mAb (1 2G5), but no effects of 2D7 or
beta chemokines.
Conclusions: Interspecies SIV transmission may be facilitated by initial CD4 independence. But an advantage for CD4 dependent infection is rapidly apparent
for SIV in huPBMCs. A switch from huCCR5 to huCXCR4 usage by SIV infect
ing huPBMCs was unanticipated, and expands our appreciation of SIV's potential
CKR usage.
Presenting author: Sujatha lyengar, Dept of Molec Micro & Immunol, Bloomberg
School of Public Health, Johns Hopkins University, 615 N. Wolfe Street, Baltimore,
MD, 21205, United States, Tel.: [phone], Fax: [phone], E-mail:
[email protected]

WePeA5728I Reduced fitness of HIV-1 resistant to CXCR4
antagonists
M. Armand-Ugon1, M. Quinones-Mateu2, A. Gutierrez', J. Barretinal,
J. Blanco', D. Schols3, E. De Clercq3, B. Clotet', J.A. Este'. 'Fundacio
IrsiCaixa, Fundaci6 irsiCaixa, Hospital Trias i Pujol, C Canyet s/n, 08916
Badalona, Spain; 2Lerner Research Institute, Cleveland, United States; 3 Rega
Institute, Leuven, Belgium
HIV-1 strains with a syncytium-inducing phenotype that use CXCR4 (X4 strains)
have been associated with faster disease progression and AIDS. Antiviral agents
designed to block CXCR4 may prevent the emergence of X4 HIV strains but resistant strains that maintain the X4 phenotype can be raised by sequential passage in cell cultures. We have demonstrated that a laboratory adapted strain
(NL4-3) and a cloned clinical isolate (Cl-1) of HIV-1 cultured in the presence of
the CXCR4 antagonist AMD3100 became resistant to the compound without a
change in coreceptor use. These strains became resistant through an evolutionary change in divergence with respect to the wild type virus. Conversely, a clinical isolate made resistant to AMD3100, switched coreceptor use from X4 to R5
through a change in diversity from the original virus population. When dual infection competition/heteroduplex tracking assays were performed, all AMD3100 -resistant strains, regardless of coreceptor use showed a significantly diminished
fitness compared to the wild-type virus. Single virus infections, at a similar multiplicity of infection, also indicated that the wild-type strains posses better replicative ability than their corresponding resistant strains. Thus, viral resistance development to a CXCR4 antagonist, such as AMD3100-resistance is associated with
reduced viral fitness.
Presenting author: Jose A Este, Fundaci6 irsiCaixa, Hospital Trias i Pujol, C
Canyet s/n, 08916 Badalona, Spain, Tel.: [phone], Fax: [phone],
E-mail: [email protected]
WePeA5730 HIV-1 protease mutations as potential cause for
reduced viral fitness in therapy-naive individuals
Y. Maerki1, G.R. Kaufmann1, T. Klimkait2, M. Battegay3. 'University Hospital,
Med Poliklinik, Petersgraben 4, CH-4038 Basel, Switzerland; 2Institute of
Medical Microbiology, University of Basel, Basel, Switzerland; 3 University
Hospital, Basel, Switzerland
Background: A low plasma HIV-1 viral load <104 copies/mL is observed in a
small proportion of untreated HIV-1-infected individuals and is associated with a
superior prognosis of untreated HIV-1 infection and better response to antiretroviral therapy. Potential explanations include a poorer viral fitness or a stronger
HIV-1 specific immune response.
Patients and methods: Polymorphisms in the protease gene were compared in a
group of 10 untreated HIV-1 infected patients with low viral load (<104 copies/mL;
LOW) and a group of 5 untreated individuals with high viral load (>105 copies/mL;
HIGH). In addition, viral fitness was evaluated using cloned viral constructs harboring the patient's protease gene and the backbone of the laboratory strain
pNL4-3. Virus production was determined in culture supernatant by a quantitative PCR technique. Patients were matched for CD4 count.
Results: Both groups showed a median number of 3 protease mutations
(p=0.77). The most frequently observed alterations included L63P/T/S (n=8) and
193L (n=5). In the viral fitness assay, the HIGH and LOW-groups showed similar
median peak viral production (6.3 vs 6.0 log10 copies/2.5 uL; p=0.44), which was
reached at 11 days in both groups. Conversely, a comparison of HIV-1 strains
with low and high viral fitness (cutoff: peak production of 3 x 106 copies/mL)
demonstrated that strains with low viral fitness had a higher prevalence of mutation L63P/T/S (p=0.04).
Conclusion: Protease mutations and the in vitro fitness of viral recombinants do
not explain the magnitude of steady-state viremia in untreated HIV-1 infected individuals. It suggests that the immune response or mutations outside the protease
gene, affecting viral fitness may play a more important role. Nevertheless, mutations at codon 63 were associated with reduced viral fitness and may potentially
affect the response to antiretroviral therapy.
Presenting author: Manuel Battegay, Med Poliklinik, Petersgraben 4, CH-4038
Basel, Switzerland, Tel.: [phone], E-mail: [email protected]
WePeA5731 Longitudinal study of viral load in purified cell
subsets from patients with Primary HIV Infection
(PHI)
P. Biswas', M. Malnati2, A. Galli3, B. Capiluppi2, S. Piergiovanni2, G. Scarlatti2,
A. Lazzarin2, P. Lusso2, G. Tambussi2. 'San RaffaeleoScientific nstiute,
Scientific Institute S. Raffaele, Immunology and Infectious Diseases Dept., Via
Stamira d' Ancona 2 20127 Milan, Italy; 2San Raffaele Scientific Institute,
Milan, Italy; 3San Raffaelel Scientific Institute, Milan, Italy
Background: Plasma viral load presents a biphasic decay with rapid elimination

of free virus in the first weeks followed by a slower second phase decay of plasma
viremia. Aim of our study is to evaluate the kinetics of HIV-DNA in sequentially purified cell subsets, including peripheral blood mononuclear cells (PBMC), CD14+
monocytes, CD4+ enriched lymphocytes from PHI patients.
Methods: Patients: 15 individuals who attended our Clinic for an acute retroviral syndrome. Time points evaluated: tO, prior to the beginning of an antiretroviral therapy, followed by two (t2), six (t6) and twelve (t12) months post-therapy.