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

XIV International AIDS Conference

Abstracts WePeA5722-WePeA5725 9

notyping and ultrasensitive fluorescence in situ hybridisation (UFISH) using FACS
analysis. In individual patients, intracellular p24 was determined additionally.
Results: HIV RNA was detected in all patients in both CD4+ and CD8+ lymphocytes. However, HIV RNA was mainly confined to CD4+ T lymphocytes in the
majority of patients. During HAART, a significant and permanent suppression of
HIV RNA was noted in both CD4+ and CD8+ lymphocytes in 8/25 patients, correlating with a rapid reduction of plasma viral load to < 50 copies/ml. 17/25 patients
showed persistence of viral RNA in CD4+ and CD8+ lymphocytes, often resulting
in a small population of cells carrying peak levels of HIV RNA. Although a significant drop in plasma viral load was noted in all patients, 12 of these patients had
subsequently blips or an increase of plasma viral load again.
Conclusions: HIV RNA was detected (using UFISH and/or intracellular p24) in
CD4+ and CD8+ lymphocytes in all patients studied. A decrease in both CD4 -and CD8 associated HIV RNA during HAART was consistenty associated with
permanent suppression of plasma viral load. In contrast, blips and rebounds of
plasma RNA were detected in 12 of 25 patients with persistent cellular associated HIV RNA. We conclude that, in addition to CD4+ T cells, CD8+ T lymphocytes represent a circulating reservoir of HIV RNA and respond to HAART in a
substantial number of patients. Further studies are warranted to evaluate whether
persistence of HIV RNA in CD4+ or CD8+ lymphocytes may correlate with late
treatment failure.
Presenting author: Andrea Rubbert, Med Klinik I, Joseph-Stelzmann Str 9,
50924 Cologne, Germany, Germany, Tel.: [phone], Fax: [phone]
6459, E-mail: [email protected]

We PeA5722

Evaluation of Persistence and Residual
Replication of HIV-1 in patients on HAART with
suppressed plasma viremia by analysis of proviral
HIV-1 DNA, HIV-1 DNA preintegration circles and
immune activation in the peripheral blood of
patients on HAART with suppressed plasma
viremia

T. Harrer', J. Hauber 2, P. Ldw 1, M. Schmitt 1, E. Schwingel2, J.R. Kalden ',
I. Hauber 2. 'Department of Medicine Ill, University of Erlangen, Erlangen,
Germany; 2Institute of Molecular and Clinical Virology, University of Erlangen,
Erlangen, Germany
Background: Although HAART can suppress HIV-1 plasma viremia below the
limit of detection of the available assays, there are concerns that low level HIV
replication could lead to emergence of drug resistance. Thus, there is a need for
sensitive assays to measure HIV-1 replication beyond plasma RNA assays. To
assess persistence and residual replication of HIV-1 in PBMC we measured in
patients on HAART with suppressed plasma viremia HIV-1 DNA preintegration
circles, proviral HIV-1 DNA and the CD4/CD8 ratio as a marker for HIV-1 induced
immune activation.
Methods: We analyzed by PCR both HIV-1 proviral DNA and 1-LTR- and/or 2 -LTR-containing HIV-1 DNA circles in PBMC. These DNA circels indicate recent
de novo infection of cells. PCR findings were correlated to absolute numbers of
CD4+ and CD8+ T-cells and to the CD4/CD8-ratio. 84 HIV-1 infected patients on
3 to 6 drug combination regimens were included in this study All patients showed
an HIV plasma viremia of below 50 copies/ml as measured by the b-DNA assay.
Results: We could detect proviral HIV-1 DNA in PBMC from 78 (92.9%) of the
84 patients. Viral DNA circles (PID) could be found in PBMC at any time point in
51/84 (60.7%) and at the last evaluation still in 31/84 (37%) of the patients. 45.1%
of patients showed CD4-counts below 500 (49.1% of PID - and 37.9% of PID +
patients) and 67.5% of patients a CD4/CD8-ration of <1.0. Elevated CD8-cells
above 700/1I, which are indicative of continuous antigenic stimulation of the immune system by HIV, could be observed in 46.3% of the patients (47% of PID -
and 48% of PID + patients).
Conclusions: Despite potent HAART therapy proviral HIV-1 DNA persists in
PBMC of >90% of patients. The high proportion of patient with either positive
HIV-1 DNA circles or an elevation of CD8 cells demonstrates, that in many patients the current drug regiments are not able to block completely viral replication
in the peripheral blood.
Presenting author: Thomas Harrer, Department of Medicine Ill, University of
Erlangen, Krankenhausstrasse 12, 91054 Erlangen, Germany, Tel.: [phone], Fax: [phone], E-mail: [email protected]
I WePeA5723 Protease diversity changes in HIV-1 infected
individuals receiving highly active antiretroviral
therapy
M.A. Martinez, M. Parera, B. Clotet. Fundacio irsiCaixa, Hospital Universitari
Germans Trias i Pujol, Badalona, Spain
We have previously studied the reservoir of HIV-1 present in PBMCs of 10 infected patients on highly active anti-retroviral therapy (HAART) by measuring over
time levels of unintegrated and integrated cell-associated viral DNA. In addition,
we have also investigated the HIV-1 integrated provirus sequence variation in
the PBMCs from four of these patients. Concordant with the decline of virus in
plasma, a significant decrease in total HIV-1 DNA was observed after 48 weeks
of therapy However, no statistically significant reduction was detected when copy
numbers of integrated HIV-1 DNA were compared. Moreover, 2 years of suc
cessfully HAART did not significantly reduce the genetic repertoire of the integrated LTR sequence population. Here, we present the HIV-1 protease provirus
sequence variation in the PBMCs from six of the previously analyzed patients.
Proviral fragments of the protease-coding region taken at baseline and at 108
weeks of HAART were PCR amplified from PBMCs and 10 to 12 individual clones
were sequenced for each time point. Phylograms for each patient revealed an intermingling of sequences from the two time points. Visual inspection of these phylograms showed no significant decreases in the genetic diversity of the proviral
protease quasispecies after two years of plasma viremia suppression. This trend
was quantitatively evaluated by calculating the intrasample genetic distances. The
six patients studied showed no statistically significant decrease in DNA sequence
variation after 2 years of successfully HAART. These results showed that the
current antiretroviral therapies are ineffective at reducing the HIV-1 latent genetic
repertoire. Thus, treatment suppressive protocols that include structured interruption of therapy have to take into account that the virus population that will emerge
may contain their entire repertoire of variants present before the beginning of the
therapy.
Presenting author: Miguel Angel Martinez, Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, Carretera del Canyet s/n, 08916 Badalona, Spain,
Tel.: [phone], Fax: [phone], E-mail: [email protected]
WePeA5724 Detecting the residual replication level to
potentiate HAART regimen for a salvage therapy
K. Yoshimura, T Kimura, F Wang, J. Kim, A. Koito, S. Matsushita. Center for
AIDS Research, Kumamoto University Division of Clinical Retrovirology and
Infections diseases, Center for AIDS Research, Kumamoto University, 2-2-1
Honjo, Kumamoto 860-0811, JAPAN, Japan
Background: Ongoing HIV-1 residual replication during HAART remains a major
obstacle in our therapeutic effort, especially for structured treatment interruptions
(STI). To better understand the level of residual replication in vivo, we examined
both of proviral DNA levels and turnover of T lymphocytes in patients with detectable or undetectable plasma viral load on HAART.
Methods: Proviral DNA levels in PBMCs from HIV-1+ patients were measured
using a novel hypersensitive nested PCR in the LTR region, 1st PCR was used
by common PCR method and 2nd PCR was performed by a quantitative real-time
PCR fluorogenic assay. We also investigated CD4+ and CD8+ T cell turnover in
both healthy and HIV-1+ patients by measuring the nuclear antigen Ki-67 with
four-color flow cytometry analysis.
Results: We measured the HIV DNA level in PBMCs of 199 samples from 57 patients. Only 11 subjects had undetectable proviral DNA level (<3 copies/reaction),
although plasma HIV viral loads remained at <50 copies/ml in more than 80%
subjects. In these subjects we selected three patients who were two primary and
one chronically HIV-1 infected for detected prospective analysis. CD4+Ki67+ and
CD8+Ki67+ subset decreased as a drop of proviral DNA level even after <50
copies/mi plasma RNA in the all subjects. Significant decline of the proviral DNA
level was observed when the regimen of HAART was changed to more potent
combination.
Conclusions: Our study suggest that the measurement of both proviral DNA
level and T cell turnover is suitable candidate for detecting the residual replication
in patients, especially at the time of concerning whether the regimen of HAART
should be changed or not, because there is no adequate indices to compare potency of the antiviral regimen.
Presenting author: kazuhisa yoshimura, Division of Clinical Retrovirology and Infections diseases, Center for AIDS Research, Kumamoto University, 2-2-1 Honjo,
Kumamoto 860-0811, JAPAN, Japan, Tel.: [phone], Fax: [phone], E-mail: ykazu @ kaiju.medic.kumamoto-u.ac.jp
WePeA5725 Towards therapy for the reservoir of human
immunodeficiency virus type 1 in resting T cells
D.M.M. Marqolis', H.J. Johnson2, L.L.Y. Ylisastigui2. 'Univ Texas
Southwestern/Dallas VAMC, Dallas, TX, United States; 2Univ. Texas
Southwestern, Dallas, United States
Background: Due to persistent HIV-1 infection within resting CD4+ T cells, eradication of HIV infection is not yet possible. The host factors YY1 and LSF cooperatively recruit histone deacetylase 1 (HDAC1) to the HIV-1 long terminal repeat
(LTR) and inhibit transcription and virus production. Therapies that target these
factors may disrupt latency of integrated HIV.
Methods: Lymphopheresis of HIV+ donors, tissue culture, in vitro phosphorylation, chromatin immunoprecipitation
Results: The HDAC inhibitor trichostatin A or the HIV activator Tat decrease
HDAC1 occupancy and increase acetylation and expression of the LTR. Conversely, YY1 blocks Tat activation, increasing HDAC1 occupancy and decreasing
nuc 1 acetylation. Both a YY1 mutant lacking its HDAC1 interaction domain and a
dominant negative mutant of LSF capable of blocking repression cause increased

acetylation and decreased occupancy of HDAC1 at the LTR.
Conclusions: LSF, YY1, and HDAC1 may maintain hypo-acetylation of the LTR,
and hence its quiescence in resting infected cells. These findings suggest targeted disruption of latency in the resting CD4+ reservoir of HIV infection without
global activation of uninfected T cells may be possible. In combination with potent antiretrovirals, such an approach may allow the clearance of HIV infection.
(Supported by amfAR, RO1-AI-45297, UO1-AI-46376).