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).

A. CTL and Pediatric infection : Not all tetramer binding CD8⁺ T cells can produce cytokines and chemokines involved in the effector functions of virus-specific CD8⁺ T lymphocytes in HIV-1 infected children. F. Buseyne, E.Montchatre, N. Bellal,, coll with :D.Scott-Algara, Institut Pasteur; S.Blanche, C.Rouzioux ,CHU Necker-Enfants Malades; J LeChenadec, J Warszawski ,INSERM CHU Bicêtre.

In human immunodeficiency virus type-1 (HIV-1) pediatric infection, the presence of cytotoxic T lymphocytes (CTL) is associated with a slow progression to AIDS. However, in spite of their high frequency, CTLs are unable to eradicate virus, and a selective reduction of HIV-1 specific CTL frequency has been observed in the advanced stages of HIV-1 infection .These findings could be due to virus replication along with its antigenic variability or to the functional inability of CD8⁺T cells to lyse the virus-infected target cells.The secretion of cytokines by CTLs may be critical in the control of viral infection. We used the combination of cell surface and intracellular staining to study the functionality of tetramer binding CD8⁺ T cells recognizing two HIV-1 immunodominant epitopes, in peripheral blood mononuclear cells from HIV-1-infected children. A fraction of tetramer positive CD8⁺ T cells produce cytokines (IFN-γ, TNF-α) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlation between the plasma viral load and the percentage of CD8⁺ Tetramer Gag⁺ T cells secreting IFN-γ_This is the first report in the context of pediatric HIV-1 infection showing that only a fraction of HIV-1-specific CD8⁺ T cells have the capacity to produce cytokines and chemokines implicated in their antiviral functions (Scott-Algara et al in press).

B. Exogenous presentation of HIV particles to CD8+ lymphocytes: role of non-replicative HIV particules in the induction of primary HIV-specific CD8+ T lymphocytes. F.Buseyne, C.Retiere, M.Nowakowski coll. with A. Morris,O.Schwartz (Institut Pasteur) et J.P Abastado (IDM, Paris)

We have shown that viral particules entering dendritic cells (DC) are processed and presented by MHC-I molecules to HIV-specific CD8+ T lymphocytes, in absence of neosynthesis of viral proteins (Buseyne et al. 2001).The first aim of the research work is to investigate if exogenous HIV particules pulsed onto DCs are able to induce the differentiation of naïve CD8+ T lymphocytes into HIV-specific effectors. Then, we plan to compare the effectors induced by either exogenous HIV particules or endogenous HIV antigens deriving from the productive infection of the DC, in term of frequency and epitope specificity.

C. Control of viral replication by CD8+ T lymphocytes, M. Février, coll with A. Talarmin, Institut Pasteur de Guyane.

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). 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 interleukin 4, and Tc0 for both γIFN 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 (Février et al. J.Clin.Immunol., 2004).

D. Animal model: DNA based immunisation in macaque. Collaborative studies. with AL Puaux et ML. Michel (INSERMU163, et Institut Pasteur) ,R.Legrand (CEA, Fontenay) et AM Aubertin,(INSERM, Strasbourg).

Recent efforts to design an HIV-1 vaccine candidate have focused on means of eliciting antiviral T-cell responses. We tried to improve the immunogenicity of DNA vaccines by designing hybrid DNA constructs encoding hepatitis B surface antigen fused to antigenic domains of simian/human immunodeficiency virus (SHIV 89.6P). Immunisation with hybrid DNA induced both effector and long-lasting precursor T cells. Following boosting with a recombinant modified vaccinia Ankara (rMVA) producing full-length SIV and HIV antigens, it appeared that priming with hybrid DNA had increased virus-specific T-cell responses in terms of both the number of virus-specific IFN-γ-secreting T cells and virus-specific lymphoproliferation. After intrarectal challenge with SHIV 89.6P, immunised animals demonstrated early control of SHIV 89.6P replication and stable CD4+ T cell counts (Puaux et al, 2004).

E Role of HCV-specific T cell responses in HCV-exposed uninfected infected individuals. G. Janvier coll with S.Pol, Hepatology, JL. Bresson,CIC, ML Chaix, Virology, CHU Necker-Enfants-Malades.

Hepatitis C virus (HCV) specific cytotoxic T lymphocytes have been detected in the peripheral blood and the liver of patients with chronic HCV-infection. In most cases, these activities have been evidenced after multiple cycles of virus-specific stimulation in vitro. A beneficial role of HCV-specific T cell responses has been proposed during acute HCV infection: a vigorous T cell response has been evidenced in subjects with spontaneous recovery of infection. In addition, in experimentally infected chimpanzees, intrahepatic CD8 T-cell responses directed to multiple regions of HCV were correlated with the recovery of HCV infection. Our goal was to study the role of virus-specific T cell responses in exposed uninfected individuals (stable partners of HCV-chronically infected subjects) which may explain the protection from infection despite the absence of detection of HCV specific antibodies, and to identifiy virus-specific T-cell epitopes involved in protection. Characterization of HCV-core specific responses was performed from fresh and in vitro stimulated PBMC from 20 exposed uninfected individuals and their 20 HCV-infected spouses and 20 unexposed uninfected volunteers. The specificity of virus-specific T cell responses was studied by γ-interferon Elispot assays using a panel of fifteen-mer peptides overlapping by 10 residues and covering the entire core sequence. When memory Tcell were expanded after in vitro stimulation, the frequency of responders was higher in chronic carrier and in exposed uninfected compared to the non exposed uninfected control group. Both the N-terminal and the central regions were the main targets for the chronic HCV carrier and or exposed uninfected groups while in the control group the target of the T cell responses was the central region.The results suggest a protective role of viral specific T cell activities in exposed uninfected spouses of HCV-infected patients. The identification of HCV epitopes in a such protection might be of importance for vaccine development.

Keywords: HCV, CTL, HIV, Infant, Immune reconstitution, Antigen presentation