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

XIV International AIDS Conference

Abstracts WePeA5732-WePeA5735 11

Cell purification: PBMC underwent sequential steps of purification with magnetic
beads giving rise to CD14+ and CD8-depleted subsets. Proviral HIV DNA was
measured by two combined TaqMan Real time PCR assays. The first accurately
quantitates all clades of the HIV-1 M group; the second normalizes the cellular
genome content of each sample.
Results: In most patients the CD8-depleted fraction harbored the highest viral
load, followed by PBMC. However, a consistent viral load was measured in all
patients also in circulating CD14+ monocytes. One patient who at tO had a concomitant hepatitis B infection presented a viral load in the CD14+ fraction higher
than in the CD8- and the PBMC. Further, viral load was detected also in the purified fractions of a patient infected with a clade G HIV-1 virus. The HIV copy number/106 cells ranged from 200 to approximately 20000 at tO. Upon treatment a
strong decrease was observed especially at t2 in the CD8-depleted cells; in most
cases the PBMC and the CD14+ mirrored their kinetics, with two exceptions.
Conclusions: After an effective antiretroviral treatment the proviral DNA showed
a biphasic decay in all three cellular fractions. Although the CD8-depleted cells
constitute the favourite target of HIV, the circulating CD14+ fraction represents
since the earliest phases of HIV infection a relevant viral reservoir.
Presenting author: Priscilla Biswas, Scientific Institute S. Raffaele, Immunology
and Infectious Diseases Dept., Via Stamira d' Ancona 20, 20127 Milan, Italy, Tel.:
++39 02 2643 7930, Fax: ++39 02 2643 7989, E-mail: Priscilla.Biswas@ hsr.it
WePeA5732 Unintegrated 2LTR circular DNA is a possible
marker of HIV-1 cryptic replication
O.E. Varnier1, J.L. McDermott', F. Bertolotti', I. Martini', I. Levreril,
G. Murdaca2, A. Campelli2, F. Puppo2. 'Inst. Microbiology, School of Medicine,
University of Genova, Genova, Italy; 2DIMI, School of Medicine, University
Genova, Italy
Issues: Measurement of plasma RNA levels is considered the best prognostic
marker for predicting clinical outcome in HIV-1 infection and is also a marker of
therapy efficacy. Viral load is measured every 3 to 6 months in treated subjects,
yet following HAART, it is drastically reduced to undetectable levels. However, latent reservoirs of integrated proviral DNA in resting CD4+T lymphocytes provides
a long-lived source of replication-competent virus and small numbers of cells continue to be infected ex novo giving rise to unintegrated DNA. If therapy is interrupted or reduced, viral rebound may occur and resistant viruses may account for
therapy failure.
Description: Total cellular DNA obtained from 171 peripheral blood mononuclear
cell (PBMC) samples collected from 22 aviremic subjects. A 120-bp region of the
U3-U5 junction between the two LTRs of unintegrated 2LTR DNA was amplified
and detected using a colorimetric microplate protocol. For some samples 2LTR
test sensitivity was increased by the use of an episomal DNA sample extracted
from a second PBMC aliquot. HIV-1 pol gene sequence analysis was performed
for DNA and RNA samples.
Lessons learned: The analysis of sequential PBMC samples revealed the presence of 2LTR DNA in 117 (68.4%)of the tested samples, demonstrating active
cryptic HIV replication in these subjects. For 33 samples 2LTR detection was only
possible in the episomal DNA aliquot where a sensitivity of 1 infected cell/106
PBMC was achieved. Drug resistance was obtained in episomal samples and the
profiles were similar to those seen in total DNA and plasma RNA for the same
subject.
Recommendations: When undetectable plasma RNA levels are sustained, 2LTR
DNA becomes the only available virological marker. Its presence in about 70% of
the samples from aviremic subjects indicates ongoing cryptic replication, which
might anticipate therapy failure. DNA sequencing analysis might detect resistant
viruses responsible for viral rebound.
Presenting author: oliviero varnier, institute of microbiology, school of medicine,
university of genova, largo r. benzi 10, 16132 genova, Italy, Tel.: [phone]
7649, Fax: [phone], E-mail: [email protected]
WePeA5733 Caspase-8 is a useful disease activity marker in
HIV infected individuals
B. Kumar', A. Wanchu', A. Bhatnagar2. 'Department of Dermatology,
Venereology and Lepro/ogy Postgradaute Institute of Medical Education and
Research, Chandigarh, Department of Dermatology Venereology and
Leprology Pgimer, Chandigarh, PIN - 160 012, India; 2postgradaute Institute of
Medical Education and Research, Chandigarh, Chandigarh, India
Background: Apoptosis is an important cause of destruction of CD4 + T lymphocytes among patients with HIV infection. Transduction of apoptotic signals is
mediated, among others by Fas ligand. It signals through an intracellular transduction pathway via an adapter molecule called Fas-associated death domain
(FADD), which converts interleukin-1 b-converting enzyme (Caspase-8). The latter is an upstream member of the family of Caspase proteins. We carried out this
study to determine if measurement of level of Caspase-8 could indicate disease

progression and hence, act as a surrogate marker for the same.
Methods: This study was carried out in 19 HIV infected patients who were seen
at the Postgraduate Institute of Medical Education and Research, Chandigarh,
India were studied. Their mean age was 28.6+6.5 years. There were 11 males.
CD4 counts were measured on a flowcytometer. Caspase-8 levels were measured using an in-house single-step ELISA and results were expressed as OD
value.

Results: Mean CD4 counts of the patients were 235+111.6 per cumm. The
mean OD value for Caspase-8 was 0.927+0.25. There was a negative correlation between CD4 counts and the OD value for Caspase-8 (r value -0.897, p
value<0.01).
Conclusion: This study shows a negative association between CD4 counts and
an apoptotic marker, Caspase-8. Wherever quantitation of the latter is possible,
it might be a cheaper option to use and may be a surrogate marker to assess
disease activity.
Presenting author: Bhushan Kumar, Department of Dermatology, Venereology
and Leprology, Pgimer, Chandigarh, PIN - 160 012, India, Tel.: [phone], EXT 522, 310, Fax: [phone], 745 078, E-mail: [email protected]
WePeA5734 T cell turnover does not normalize after 24 weeks
of HAART and remains elevated in patients with
failure of CD4 recovery despite effective viral
suppression
K. Anthony', I. Seretil, J. Orenstein2, R. DerSimonian3, B. Herpin', J. Metcalf',
H.C. Lane', M. Polis1. 1'NIAID, NIH, NIHINIAID, 10 Center Drive, Bldg. 10, Room
1 1B05, Bethesda, MD, United States; 2George Washington University Medical
Center, Washington, DC, United States; 3DAIDS, Rockville, MD, United States
Background: CD4 lymphopenia is a cardinal manifestation of HIV infection. Increased T cell turnover has been proposed as an important mechanism of CD4
depletion. To study the effect of changes in T cell turnover induced by HAART on
CD4 recovery, blood and lymph nodes (LN) were evaluated in patients before and
after HAART initiation.
Methods: PBMCs from 32 ARV-naive HIV+ patients were collected at week 0 and
24 of HAART for immunophenotypic and intracellular Ki67 (marker of recent proliferation/turnover) staining. LN biopsies done at week 0 and 24 in 13/32 patients
were stained for Ki67. Student's t-test, Wilcoxon signed rank test, and Pearson
correlation were used for statistical analysis.
Results: At week 24 CD4 count increased from a mean of 341 cells/ul at baseline to 481 cells/ul, CD8 count decreased from a mean of 865 to 731 cells/ul, and
viral load (VL) decreased from a median of 215175 to <50 copies/mI. 27/32 patients achieved VL<50. The %CD4 cells expressing Ki67 decreased from 13.6%
at baseline to 8.5% at week 24 (p=0.0007; 13 HIV- controls: 2.6%) and the %CD8
cells expressing Ki67 decreased from 15.3% to 6.1% (p<0.0001; HIV-: 2.7%). In
12/13 LN biopsies the number of Ki67+ cortical/paracortical cells decreased at
week 24 compared to baseline. At week 24 there was a strong correlation between the %Ki67+CD4 cells and CD4 count (R=-0.722, p<0.0001). Of note in
patients with VL<50 at week 24, those with CD4<200 had a higher %Ki67+CD4
cells than those with CD4>200 (13.2% vs. 6.2%, p=0.04).
Conclusion: T cell turnover is increased in HIV infection and decreases significantly after HAART initiation in blood and LN. T cell turnover in blood does not normalize after 24 weeks of HAART despite VL suppression and remains negatively
associated with CD4 count. These data suggest that increased T cell turnover in
both blood and LN is an important contributor to CD4 depletion in HIV infection
and plays a central role in the failure of CD4 recovery after HAART initiation.
Presenting author: Kara Anthony, NIH/NIAID, 10 Center Drive, Bldg. 10, Room
11B05, Bethesda, MD, United States, Tel.: [phone], Fax: [phone], E-mail: [email protected]
WePeA5735 Longitudinal analysis of proliferating T cells and
thymic output in HIV-associated T cell
homeostasis failure
P.K. Chattopadhyay', K. Chadwick', J.P Phair2, R. Detels3, C.R. Rinaldo4,
M.K. Hellerstein5, J.B. Margolickl. 'Johns Hopkins Bloomberg School of Public
Health/Johns Hopkins University, Baltimore, MD, United States; 2Northwestern
University Medical School, Chicago, United States; 3Schools of Public Health
and Medicine/University of California Los Angeles, Los Angeles, United States;
SGraduate School of Public Health/University of Pittsburgh, Pittsburgh, United
States; 5Department of Nutritional Sciences and Toxicology/University of
California Berkeley, Berkeley, United States
Background: T cell homeostasis (TCH) failure, which usually precedes AIDS, is
characterized by declining CD4+ and CD8+ T cell counts, but whether it results
from increased T cell destruction, insufficient T cell proliferation, or decreased
thymic output is unknown.
Methods: Using longitudinally stored peripheral blood mononuclear cells (PBMC)
from HIV+ individuals with TCH failure, we measured T cell proliferation (Ki67 expression) in naive and memory T cells and thymic output (T cell receptor excision
circles, TREC) in PBMC. For each subject, at least 3 time points before and after
TCH failure were analyzed. Cross-sectionally, we compared Ki67 expression to
the fractional rate of T cell replacement (by deuterated glucose incorporation) in
14 other HIV+ individuals (4 with early HIV, 7 with intact TCH, 3 with failed TCH).

Results: After TCH failure, the absolute number of Ki67+ cells declined among
total CD4+ (mean =32 vs 15/uL, p=0.001), naive (CD45RA+) CD4+ (3 vs 1/uL,
p=0.002), and memory CD4+ cells (24 vs 12/uL, p=0.003). Numbers of Ki67+
CD8+ cells did not change significantly. TREC levels were detectable while TCH
was intact (median=2764 TREC/106 PBMC), but were generally undetectable
within 12 months after TCH failure (mean<100 TREC/106 PBMC; p=0.03). In
subjects studied cross-sectionally, Ki67 expression by naive and memory T cells