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  Impatvir


  Director : Rivière Yves (riviere@pasteur.fr)


  abstract

 

Cell mediated immune responses are a major determinant in the pathogenesis of viral infections. The functions of antigen specific T lymphocytes are required for recovery from viral infections, for clearance of virus or control of persistent infection. On the other hand, an antiviral immune response may also result in immunopathology when an inflammatory response destroys cells infected by a relatively non cytopathic virus. The major goal of our group is to study -in vitro and in vivo-the role of viral-specific cytotoxic responses (CTL) in two human viral infections, Human Immunodeficiency Virus type1 (HIV-1), and Hepatitis C virus (HCV).



  report

cale

A . Epitope mapping. Cross-clades reactivities F. Buseyne, coll with C. Rouzioux and S Blanche ,Necker Hospital. Cross-recognition of several HIV subtypes by specific CTL is frequently observed. However, it has not been reported if CTL cross-recognition of a defined epitope is a general property of all CTL specific for this epitope or if cross-reactivity will differ for each CTL population. We tested CTL activities directed to the same epitope in order to compare CTL cross-reactivity among patients. We have selected 7 HIV-infected children, which carry either HLA-B53 or HLA-B57 molecules, that present the QASQEVKNW CTL epitope from the HIV-p24gag protein. Their PBMC were tested against 10 variants of the epitope in chromium release assay. The five responders recognize 2 to 9 of the 10 sequences. Furthermore, each patient recognized a distinct set of sequences. Our data confirm that most CTL responses are broadly cross-specific, though variant specific-CTL response are also observed in some patients. For the first time, we show the high inter-patient variability in cross-recognition of mutant CTL epitopes. These inter-individuals variations in the CTL response to the same epitope should be taken into account for the design and the evaluation of vaccines (Buseyne et Coll , J. Virol., 2001).

B . Exogenous presentation of HIV particles to CD8+ lymphocytes, F.Buseyne, T. Paquet, coll. with O. Schwartz (Institut Pasteur) et J.P Abastado (IDM, Paris).In most cases, MHC-class-I epitopes are derived from endogenously synthetized proteins. An alternative pathway of peptide presentation derived from extracellular antigens has been shown in APC such as dendritic cells and macrophages.We show that epitopes from incoming HIV-1 virions are presented through this exogenous pathway in APCs, leading to CTL activation in the absence of viral neosynthesis. Exogenous presentation requires adequate virus-receptor interactions and fusion of viral and cellular membranes. This results are important in the understanding of CTL activation and have implications for anti-HIV vaccine design (Buseyne et Coll., Nature Medicine 2001).

C. Control of viral replication by CD8+ T lymphocytes, M. Février. CD8+ lymphocytes from HIV-infected patients are able to suppress in vitro replication of HIV in CD4+ infected T cells by a non-cytolytic mechanism, involving secreted CD8+ cell-antiviral factor(s) (CAF). We demonstrated that the ability to inhibit HIV replication by CAF secretion is not restricted to HIV specific CD8+ T lymphocytes, as an Epstein-Barr virus (EBV) specific CTL line is as efficient as HIV specific effector cells to suppress in vitro monotropic or lymphotropic HIV strain replication. Altogether, our data suggest that the non-cytolytic control of HIV replication by CD8+ T cells is not restricted to HIV-specific CTL induced during HIV infection and is distinct from a viral entry blocking (Le Borgne et al, 2000). CD8 T lymphocytes are divided in different subtypes according to the nature of their cytokine production: Tc 1 for gamma interferon, Tc2 for interleukine 4, and Tc0 for both gIFN and IL4. We have been able to show that the capacity of control of virus replication in our experimental system was not linked to a particular subtypes of CD8 T cells.

D. Frequency and phenotyping of HIV-specific CD8+ T cells in HIV-infected children using peptide/MHC-class I tetramers F. Buseyne, B.Corre, F. Porrot, N. Bellal, E. Bui . Coll : D.Scott-Algara, Institut Pasteur, S.Blanche, C.Rouzioux , Hopital Necker. HLA-A*02 tetramers complexed to HIV-Gag SLYNTVATL and HIV-Pol ILKEPVHGV peptides were used to characterize HLA class I-restricted CD8+ T cells in 41 HIV-infected children. The frequencies and the phenotype of specific circulating CD8+ T cells were determined in whole blood samples by cytometric analysis. Background staining of the 13 HLA-A*02 negative patients was below 0.01% of CD8+ T cells. Of the 28 HLA-A*02 positive patients, twenty-six stained at least once with the Gag tetramer (mean 0.87% of CD8+ T cells, range: 0.1-3.9%), and twenty-one with the Pol tetramer (mean: 0.59% of CD8+ T cells, range 0.1-5.5%).The tetramer-binding cells were CD27-/+, CD28-, CD45RA-, CD45RO+, HLA-DR+, CD69- T lymphocytes. Longitudinal follow-up of a patient following initiation of a new antiretroviral treatment showed a positive correlation between the frequency of tetramer-positive cells and viral load. In conclusion, HIV-specific CD8+ T cells can be easily detected in peripheral blood of HIV-infected children using HLA tetramers combined to HIV peptides. These cells are memory, activated, CD28-, CD8+ T lymphocytes (Scott-Algara et al., J. inf.Dis., 2001). Our present goal is to study the effects of treatment with a combination of reverse transcriptase and protease inhibitor agents on the recovery in CD8+ T-cell CTL functions (immune reconstitution).

E. Animal model: DNA based immunisation in macaque. [coll. with A.L Puaux et M.L. Michel (UREG, INSERM U163, Institut Pasteur), R.Legrand (CEA, Fontenay) et Anne Marie Aubertin, (INSERM, Strasbourg)]

DNA immunization can present antigenic proteins to the host for recognition by all arms of the immune system. In addition, it provides, the opportunity to delete any genes of the infectious organism which code for part of antigens that may have deleterious effects. We have tested the effectiveness of genetic immunization with an epitope presentation system carrying the V3 domain from HIV-1 gp 120. We showed that intramuscular and intradermal injections of DNA expression vectors encoding an HIV epitope fused to the surface protein of the hepatitis B virus (HBsAg) induced in mice and non-human primates specific and strong humoral and cytotoxic responses to antigenic determinants of both virus. Challenge experiments with a chimeric SHIV virus expressing HIV-1 env on a SIV backbone resulted in infection of all immunized animals. However in one of the V3-immunized animals, peripheral virus load was significantly lower. Interestingly, this animal was the one which had the highest anti-HIV CTL precursor frequency (LeBorgne et al, 2000, 2001). We have designed DNA constructs encoding for multiple dominant viral CTL epitopes of the SIV gag and nef proteins and for B epitopes within the HIV-1 viral envelope. The aim of our present study is to study the immunogenicity of these constructs in Rhesus macaques allowing experimental challenge with a chimeric SHIV virus in this later animal model.

F Detection of HCV-specific CTL in peripheral blood of patients co-infected with HIV and HCV. Genevieve Janvier, Françoise Porrot, coll with H.Fontaine, H.Zylberberg and S.Pol, Hopital Necker.The goal of this work was to study HCV-specific CD8 responses in infected patients using IFNgamma enzyme linked immunospot (ELISPOT) and chromium release assays after in vitro expansion. Compare to the HIV-specific responses the frequency of patients with positive HCV specific CD8+ effector cells in the peripheral blood was very low.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

JEWIARZ Danielle (djewiarz@pasteur.fr)

BUSEYNE Florence, IP (florence@pasteur.fr)

FEVRIER Michèle, CNRS (mfevrier@pasteur.fr)

RIVIERE Yves , IP (riviere@pasteur.fr)

BELLAL Nassima médecin stagiaire

FAVRE David , stagiaire Université Nantes (dfavre@pasteur.fr)

IGLESIAS Maria Candela, DEA (candela@pasteur.fr)

PAQUET Thierry thésard (tpaquet@pasteur.fr)

CORRE Béatrice, technicienne (bgs@pasteur.fr)

JANVIER Geneviève, technicienne (gjanvier@pasteur.fr)

PORROT Françoise, technicienne (fporrot@pasteur.fr)


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