Unit: Viral Immunopathology
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, N. Bellal,. Coll :D.Scott-Algara, Institut Pasteur, S.Blanche, C.Rouzioux , Hopital Necker; J LeChenadec, MJ Mayaux ,INSERM 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.
We have addressed the role of CTL in children who survived after 5 years of age. Memory HIV-specific CTL directed against Env, Gag, Nef and Pol proteins were measured in a group of 47 HAART naive HIV-infected children. Both Gag- and Pol-specific CTL were positively correlated with CD4+ T cell numbers. Gag, Nef, and Pol-specific CTL were inversely correlated with viral load The inverse correlation between viral load and Gag-specific CTL was independent of CD4+ T cell numbers. In conclusion, this study showed the beneficial role of HIV-specific CTL in children who survived after five years of age (Buseyne et al, J.Infec. Dis, 2002).
Alternative techniques based on the detection of specific cytokines secreted by CTL after specific activation (Elispot assay, intracellular staining) or by the surface expression of a specific T-cell receptor (tetramers) have been developed. An IFN-γ based Elispot assay was evaluated for the study of ex-vivo activated CTL. We tested synthetic peptides to stimulate HIV specific CD8+ T cells. We studied 20 HLA A02*01 positive HIV-infected children. The IFN-γproduction in response to stimulation with two HLA A02*01-restricted immunodominant CD8 epitopes (SL9 in Gag and IV9 in Pol), was tested using the Elispot assay. Comparison of results from tetramer and Elispot assays suggests that only a fraction of HIV-specific CD8+ T cells were functional. Most importantly, we found that the frequencies of IFN-γ producing CD8+ T cells were positively correlated with viral load, whereas frequencies of tetramer-binding CD8+ T cells were not. In addition, our results show that the ex vivo activated IFN-γ producing HIV-specific CD8+ T cell subset is dependant upon continuous antigenic stimulation (Buseyne et al., J Virol., 2002).
Most of the HIV-infected children are now receiving association of several antiretroviral drugs that are able to suppress HIV replication. The observational cohort is still ongoing, and our major goal is now to study the effect of treatment on the HIV-specific CD8+ T cells and other immune parameters
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 DCs 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. 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 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 (Février et al. J.Clin.Immunol., in press).
D. Animal model: DNA based immunisation in macaque. coll. with AL Puaux et ML. Michel (INSERMU163, et IP),R.Legrand (CEA, Fontenay) et AM Aubertin,(INSERM, Strasbourg).
DNA immunization can present antigenic proteins to the host for recognition by all arms of the immune system. 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.
E Role of HCV-specific CTL in peripheral blood of HCV-exposed non-infected individuals. G. Janvier coll with S.Pol, Hepatology, and JL. Bresso ,CIC, Hopital Necker.The goal of this work is to study the role of HCV-specific CD8 responses in HCV-exposed non-infected individuals using IFNgamma enzyme linked immunospot (ELISPOT) and intracellular staining after in vitro expansion.
Keywords: Antigen presentation, CTL, HIV-1, Infant, Immune reconstitution, HCV