Unit: Viral Immunopathology - URA CNRS 1930
Director: Rivière Yves
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. F Buseyne, E Montchatre, Coll D Scott-Algara, I.P ; S Blanche, M Burgard, C Rouzioux , CHU Necker; J LeChenadec, J Warszawski ,Inserm CHU Bicêtre.
The pediatric HIV infection has several characteristics: i- in the mother-to-child transmission, the time of infection is well defined, mainly a few weeks before and at the delivery; ii- at the time of infection, the immune system of the child is partially immature, and has not been exposed to the pathogens responsible for opportunistic infections which develop in immunosuppressed subjects; iii- finally, in the absence of anti-retroviral treatment, the evolution of the disease in the children is bimodal, as one-fifth of the children develop rapid evolution leading to AIDS before one year of age and the remaining children evolve more slowly to the disease, like most infected adults.
A significant part of the children are included in protocols of anti-retroviral therapy (ART) with or without anti-protease. The efficiency of the antiviral treatments is evaluated by the measurement of the viral load (plasmatic, cellular viremia and integrated DNA) in the laboratory of the Pr. Rouzioux. In the child, because of the higher viral replication rate and of a more active thymopoiesis, the impact of ART on the immune system is different from that observed in the adult. We have studied the reconstitution of the immune responses in infected children and receiving ART. Our goal is to quantify the different subpopulations of CD4+ and CD8+ T- lymphocytes as well as their functions, and in particular the virus-specific cytototic T lymphocytes, in relation to the modifications of the viral load induced by the treatments. The effects of ART were studied during neonatal period and in children after several years of infection. The activities of the virus-specific CD8+ T lymphocytes are evaluated by various methods including Elispot (Buseyne et al, J Immunol.,Methods 2005), and Intra Cellular Staining of cytokines coupled to tetramers surface staining (Scott Algara et al, J Clin.Immunol., 2005). Our goal is to evaluate the level of reconstitution of the immune response according to age and of the degree of immunosuppression at the time of treatment initiation. Taking into account the potential side effects of continuous ART, this information is significant for the establishment of the optimal time to initiate the therapy. We showed among patients receiving a first combined line of multitherapy that the level of the memory cytotoxic activities T at the beginning of treatment is conversely correlated with the plasmatic viremia after 12 months of treatment independently of the plasmatic viral load level at the beginning of treatment (Buseyne et al. AIDS Res Hum Retro 2005). In addition, the measure of the production of IFNg by virus-pecific CD8+ T lymphocytes by Elispot showed an increase in the responses with age in treated and untreated children. In the ART-treated children the reduction of the viremia was associated to a reduction of the virus-specific T CD8+ responses (Buseyne and al, J.I.D 2005). In a previous study, the use of tetramers of the HLA-A2 molecule complexed with two immunodominant peptides [p17(Gag) and RT (pol)] had enabled us to show that the large majority of PBMC of HLA-A2 children -PBMC bind the two tetramers. We have extended this observation and showed that only a fraction of tetramer positive CD8+ T cells produce cytokines (IFN-g, TNF-a) or chemokines (CCL4, CCL5) after ex vivo stimulation with the cognate peptide. There was a negative correlationbetween the plasma viral load and the percentage of CD8+ Tetramer Gag+ T cells secreting IFN-g (Scott-Algara et al., J Clin.Immunol, 2005).
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 (I.P) 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. 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. Animal model: DNA based immunisation in macaque. Collaborative studies with AL Puaux et ML. Michel (Inserm U163 et I.P), 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) (Marsac et al. 2005). 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). We investigated the fate of vaccine-induced T cells after the SHIV challenge. Vaccine-induced CD8+ T cells were recalled during acute infection, whereas none of the vaccine-induced SHIV-specific CD4+ T cells were recalled (0/7 epitopes). Moreover, most of the CD4+ T-cell responses (5/7 epitopes) became undetectable in peripheral blood or lymph nodes even after in vitro peptide stimulation. We persistently detected CD4+ T-cell responses specific for control recall antigens in infected animals. These results show that SHIV 89.6P challenge results in functional impairment of vaccine-induced SHIV-specific CD4+ T cells and suggest that attention should be paid to CD4+ T cell responses when designing vaccines (Puaux et al., 2005).
E Role of HCV-specific T cell responses in HCV- infected individuals. G Janvier, S Ferris coll with S Pol ,H Fontaine, Hepatology, JL. Bresson,CIC, ML Chaix, Virology, CHU Necker-Enfants-Malades.
The cellular immune response to hepatitis C virus (HCV) plays a critical role in determining the clearance or persistence of HCV. Moreover, in chronic HCV infection, these responses that are insufficient to eradicate virus completely may cause liver injury. The memory T cells responses specific to the core protein were measured by interferon-g Elispot assay after in vitro stimulation of peripheral blood mononuclear lymphocytes from chronically infected subjects. Ten out of the 22 patients studied (45%) present a core-specific response with a preferential recognition of the N-terminal and central parts. There was no relationship between T cell responses and the parameters of disease evolution as determined by ALT (serum alanine transaminase levels), and histologic hepatic damage (Metavir score A and F), but there was a positive relationship between the presence of a core-specific T cell responses and the viraemia (Janvier et al., 2005).
Keywords: HCV, CTL, HIV, Infant, Immune reconstitution, Antigen presentation