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

4 Abstracts WeOrA1l261-WeOrA1l346

XIV International AIDS Conference

Results: We find that cultured PBMC subjected to a spreading infection display
G2 cell cycle arrest that is greatly augmented by the presence of Vpr. In the absence of Vpr, these cell cycle changes are markedly diminished and vary from
donor to donor. Importantly, a significant proportion of the activated T cells circulating in the peripheral blood of HIV-infected patients are arrested or paused at
the G2/M phase of the cell cycle. However, activated but uninfected T cells from
these same patients display a normal cell cycle distribution
Conclusion: These data thus provide the first evidence that HIV alters cell cycle
progression in vivo, likely in a Vpr-dependent fashion. This arrest may play a role
in not only virus production, but also in the accelerated T cell turnover observed
in HIV-infected individuals.
Presenting author: Warner Greene, 365 Vermont St., San Francisco, CA, 94123,
United States, Tel.: [phone], Fax: [phone], E-mail: [email protected]
WeOrA1l261 Maturation of HIV virions
A.G. Bukrinskaya, M. Stevenson. University of Massachusetts Medical School,
373 Plantation Street, Suite 319, Worcester, Massachusetts, United States
Background: HIV-1 particle assembly is directed by the GAG proteins which are
initially synthesized as a polyprotein p55 which is cleaved by virus-encoded protease to produce mature GAG proteins. Dramatic morphological changes occur
inside the released virions which lead to the appearance of a cone-shaped capsid. Although important information has been obtained about virus assembly, it is
not defined how the described processes correlate with virion infectivity. We studied kinetics of virus maturation with regard to RT activity, protein composition,
density in sucrose gradients and infectivity in MAGI cells.
Methods: Supernatants from HIV-1 transfected 293 T cells were taken every 4
hours after transfection. The virus was pelleted by centrifugation and purified by
centrifugation in 20-60% sucrose gradients. RT activity of purified virus was measured and the infectivity of the virus normalized to equal RT was determined in
MAGI cells. Viral proteins were analyzed by Western blotting.
Results: RT activity of the virus increased progressively from 12 to 36 hours after
cell transfection and then gradually decreased. The virus density in sucrose gradients increased from 1.14 g/ml in the samples taken from 12 to 16 hours after
transfection to 1.15 at 20hours and at 24 hours to 1.16 g/ml. The virus collected
before 20 hours was poorly infectious while virus normalize to the same RT collected at later intervals was highly infectious in MAGI cells. However, the marked
change in infectivity did not reflect similar differences in the level of processed
and precursor virion proteins.
Conclusion: Our results demonstrate a significant change in virus infectivity that
does not show an obvious correlation with the processing of precursor proteins.
This suggests a step in virus assembly independent to precursor maturation that
impacts the infectivity of the virion.
Presenting author: Alissa Bukrinskaya, 373 Plantation Street, Suite 319,
Worcester, Massachusetts, United States, Tel.: [phone], Fax: [phone], E-mail: [email protected]
WeOrA13441 cDNA expression array analysis of cell cycle
regulation genes in CD4 and CD8 T lymphocytes
from HIV-1 infected patients
M. Nobile, E. Aschwanden, M. Spasojevic, G.RP. Rizzardi, M. Khonkarly,
G. Pantaleo. Laboratory of AIDS Immunopathogenesis, Division of Immunology
and Allergy, Hopital Beaumont, 29 av. Beaumont, 1011 Lausanne, Switzerland
Background: Several mechanisms have been proposed to explain the functional
and the quantitative defects of CD4 T cells with HIV-1 infection. At the present,
limited information is available on the pattern of expression of genes involved in
cell activation and in cell cycle. The cDNA expression array represents a comprehensive tool as it allows the simultaneous screening of large number of genes.
Methods: Premade cDNA expression arrays spotted with 111 genes all related
to cell cycle was used to screen CD4 and CD8 T cells in donors (n=8), in HIV-1
infected patients (CD4, n=8; CD8, n=10), and in vitro stimulated cells with antiCD3 and anti-CD28 (n=3) during 60 hours. Expression was detected by radio
labelling of cDNA by reverse transcription and exposure 6 hours of arrays to Instantlmager. Data analysis was done with GeneCluster and TreeView softwares
(Stanford Uni.).
Results: An increased expression of the kinases, cyclins and CDKs gene families was observed in both CD4 and CD8 cells from healthy donors after in vitro
stimulation consistently with cell activation and cycling activity. A similar pattern
of gene expression was observed in CD8 T lymphocytes of HIV-1 infected subjects thus demonstrating increased activation and cycling activity in CD8 T cells.
CD4 T cells had increased expression of cell activation genes (e.e. kinase genes)
but lack expression of CDK2, a gene crucial for advancing cell cycle from the G1
to the S phase. Both CD4 and CD8 T lymphocytes of infected subjects express
higher level of p53 tumor suppressor gene. The expression of mdm2 gene, a re
pressor of p53 whose increased expression is associated with apoptosis, was
increased only in CD8 T cells.
Conclusions: These data confirm an activation state of CD4 and CD8 T cells in
HIV-infected patients. However, the immune activation is associated with active
cycling only in CD8 T cells while the pattern of gene expression observed in CD4
cells is rather in favour of a selective cell cycle arrest.

Presenting author: Massimo Nobile, Hopital Beaumont, 29 av. Beaumont, 1011
Lausanne, Switzerland, Tel.: [phone], Fax: [phone], E-mail: [email protected]
WeOrAl345 Immune activation and CD4 depletion are directly
linked both in HIV1 and HIV2 pathogenesis
A.E. Sousa1, J. Carneiro2, Z. Grossman3, R. Victorino1. 1Faculty of Medicine of
Lisbon, Clinical Immunology Unit, Faculty of Medicine of Lisbon, Av. Prof. Egas
Moniz, 1640 Lisbon, Portugal; 2Gulbenkian Institute of Science, Oeiras,
Portugal; 3University of Tel-Aviv, Tel-Aviv, Israel
Background: The causal relationships among CD4 cell depletion, HIV replication
and immune activation are not well understood. HIV-2 infection, "nature's experiment" with inherently attenuated HIV disease, provides additional insights into
this issue.
Methods and Results: We stratified and paired groups of HIV-1 and HIV-2 infected patients whose levels of CD4 depletion fell in the same range and found
that the two infections exhibited a similar imbalance in the naive/memory-effector
population ratios with comparable up-regulation of CD4 and CD8 cell-activation
markers (HLADR, CD38, CD69, Fas molecule). Moreover, there was a similar
increase in the frequency of cycling CD4 T cells (Ki67+), which was in strong correlation with the expression of activation markers, and a similar level of anergy,
as assessed by the in-vitro lymphoproliferative responses to CD3 stimulation and
to a panel of microbial antigens.
Conclusions: These findings are surprising considering that HIV2-infected patients at a given stage of CD4 depletion bear plasma viral loads two orders of
magnitude lower than HIV1 viremia, and are known to have slower rates of CD4
T-cell decline and a better clinical prognosis. Our data are consistent with a direct
causal relation between immune activation and CD4 cell depletion in HIV disease
and only an indirect relation of these parameters to the virus replication rate.
Presenting author: Rui Victorino, Clinical Immunology Unit, Faculty of Medicine
of Lisbon, Av. Prof. Egas Moniz, 1640 Lisbon, Portugal, Tel.: [phone], Fax:
[phone], E-mail: [email protected]
WeOrA1l346 Reversible anergy of HIV-specific CD4+ T-cells
detected by class II tetramers
B. Yassine Diabl, G. Breton', S. Younes', N. Bernard2, K. MacDonald3,
M. Legault4, J.P. Routy4, R.P. Sekaly1. 1Universitd de Montrdal, Universitd de
Montreal, Departement de Microbiologie et dImmunologie, 2900, bvd
Edouard-Montpetit, Montreal, Quebec, H3T 1J4, Canada; 2McGil/ Univ. Med.
Ctr, Montreal, Canada; 3Univ of Toronto, Ontario, Canada; 4 McGill Univ. Med.
Ctr., Montreal, Canada
Background: HIV-1 infection is marked by a gradual decline in the number of circulating CD4+ lymphocytes in general and a specific failure to develop functional
HIV-1-specific T helper cells in the majority of chronically infected individuals. The
major goal of this work was to characterize the presence of HIV-specific CD4+ T
cells during primary infection (PI) and to identify their fate during the natural history of HIV infection.
Methods: To directly assess the HIV-specific CD4+ T cell response in HIV+ individuals, we generated soluble tetrameric HLA-DR molecules from 5 alleles covalently linked to 8 immunogenic and conserved HIV peptides. We then performed
a prospective longitudinal study over 3 years on 20 PI patients using HLA-DR
tetramer staining and cytokine flow cytometry or CFSE-staining following HIVspecific peptide stimulation in vitro.
Results: Analysis of CFSE-staining on patients treated with HAART shows
clearly that proliferative response to several epitopes were present in early
treated patients (5 out 7 patients) entered in the study with a low viral
load (500<VL<5407 copies/ml) and CD4 cell counts in the normal range
(408<CD4<940/ml, m = 600). In most cases (80%), these patients maintain this
response overtime. Furthermore, the specific tetramers can stain the CSFE low
population.
In contrast, patients treated late (n= 11, 15313<VL<3500533 copies/mi and
126<CD4<512, m = 300), no peptide induced proliferative or cytokine response
to HIV epitopes was seen although these cells are recognised by specific
tetramers in some cases. These patients do recognize recall antigens as detected by the same assays. These anergic cells can be proliferate following SEA
or tetramer stimulation.
Conclusions: Our results show that CD4+ T helper response against multiple
HIV antigens is generated during the PI stage but these cells disappear rapidly.
Anergic state of CD4 cells may be in part responsible for to the lack of peptidespecific proliferative responses.
Presenting author: Bader Yassine Diab, Universite de Montreal, Departement
de Microbiologie et d'lmmunologie, 2900, bvd Edouard-Montpetit, Montreal, Quebec, H3T 1J4, Canada, Tel.: [phone] #5125 (office), #5114 (Lab), Fax:
[phone], E-mail: yassineb @ poste.umontreal.ca