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  Director : BRAHIC Michel (mbrahic@pasteur.fr)


  abstract

 

The Slow Viruses Unit studies the pathogenesis of two model neurological diseases : the demyelinating disease of mouse due to Theiler's virus, a model for multiple sclerosis, and the behavioral disturbances of rats caused by Borna disease virus. The Unit constructed a chimeric MoMuLV/HTLV-I virus that infects mice and could provide a model for HTLV-I infection. A major goal of the Unit is to develop an anti-HIV vaccine based on live attenuated measles virus vectors.



  report

cale

1-Pathogenesis of the infection of mouse by Theiler's virus (Jean-Francois Bureau, Michel Brahic).

Theiler's virus, a picornavirus of mouse, infects neurons of brain and spinal cord for approximately 2 weeks. This is followed by a persistent infection of glial cells in the white matter of spinal cord with chronic inflammation and primary demyelination. These lesions resemble active plaques of multiple sclerosis closely. All mouse strains are susceptible to the early phase of infection. However, persistence and demyelination occurs only in genetically susceptible strains.

For several years, the group headed by Jean-Francois Bureau has been characterizing genes making mice susceptible to persistence of the infection and/or to demyelination. The human equivalents of some of these genes are studied as candidate susceptibility genes for multiple sclerosis.

A correlation between viral load and clinical signs has been documented in a cross between susceptible and resistant inbred mice. A causal relationship between the two phenotypes has been demonstrated by showing that a line of congenic mice selected for its low viral load does not present with clinical signs.

The Tmevp1 locus, located in the H-2D region of the MHC, has a major effect on susceptibility. All the properties of this locus are those of a Class I gene. However, SJL/J mice are very susceptible to persistent infection whereas B10.S mice are resistant, although both bear the same H-2s haplotype. Experiments using lethal irradiation followed by bone marrow reconstitution showed that hematopoietic cells, most likely immune cells, were responsible for this difference. Using these strains, we mapped two susceptibility loci, Tmevp2 and Tmevp3 on chromosome 10, near the interferon gamma gene. The interferon gamma gene having been excluded as a candidate gene, the Tmevp3 region was sequenced and a candidate gene, expressed in lymphocytes, is being characterized. Interestingly, the human homologue of this gene, in the 12q15 region, could be a susceptibility gene for multiple sclerosis.

The virus persists mainly in CNS macrophages in which virus replication is restricted. Virus replication was studied in primary cultures of bone marrow macrophages from SJL/J mice. The cells express large amounts of viral antigens at the beginning of the infection. Thereafter, viral replication becomes restricted, in great part due to interferon type I, and the cells survive the infection. A technique of purification of inflammatory CNS cells followed by double-labeling and FACS analysis has been set up. It allowed comparing the infection of macrophages in SJL/J and B10.S mice.

2- Pathogenesis of the infection of new born rats by Borna disease virus (Daniel Gonzale-Dunia).

Borna disease virus (BDV), a non segmented negative strand RNA virus with a wide host range, is responsible for persistent CNS infections with behavioral abnormalities. Sero-epidemiological and molecular data suggest that BDV infects humans and could be implicated in the etiology of certain psychiatric diseases.

Infection of newborn Lewis rats leads to a persistent, non-inflammatory CNS infection associated with postnatal development abnormalities in hippocampus and cerebellum, and behavioral disturbances. The main focus of our studies is to elucidate the mechanisms whereby BDV alters neuroplasticity, postnatal brain development and causes cognitive impairment.

BDV is a non-cytolytic virus and the mechanisms underlying its neurotoxicity are not well understood. We hypothesized that BDV interferes with the response to neurotrophins. We showed that, indeed, infected PC12 cells are resistant to NGF-induced differentiation. This resistance is linked to a downregulation of the expression of the NGF receptor and to defects in the MEK/ERK signaling cascade triggered by NGF.

We extended these observations to primary cultures of rat hippocampal neurons. These neurons are permissive to BDV replication and spread, although the infection is non-cytopathic and does not cause overt morphological changes to the cells. However, we found that infection selectively blocked the expression of molecules involved in neuroplasticity, such as GAP-43, synapsin, VAMP-2 and synaptophysin. Moreover, BDV-infected neurons responded only weakly to the neurotrophins BDNF and NT3, both in term of ERK signaling cascade activation and of BDNF-induced synaptic remodeling.

We observed that the mitotic inhibitors 1-ß-D-arabinofuranosylcytosine (Ara-C) was a very potent inhibitor of BDV RNA and protein synthesis. The expression of viral proteins was almost exclusively restricted to the nucleus in Ara-C-treated cells. This nuclear sequestration prevented cell-to-cell spread of BDV almost completely. The inhibitory effect of Ara-C appears to be specific for BDV.

3- Pathogenesis of HTLV-I infection (Frédéric Tangy).

HTLV-I is responsible for a chronic demyelinating neurological disease named tropical spastic paraparesis. The pathogenesis of this disease is poorly understood, in part because of the lack of a good animal model. We constructed a chimeric HTLV-I virus in which the envelope protein has been replaced by that of ecotropic Moloney murine leukemia virus. This virus has acquired tropism for murine cells and lost its tropism for human cells. It replicates, albeit at low levels, in murine cells and persistently infects several inbred mouse strains.

4- Anti-retroviral vaccines (Frédéric Tangy).

Live attenuated measles vaccine strains are widely used in human medicine. They can now be used as vectors for expressing foreign genes. Recombinant measles viruses expressing various genes from HTLV-I, SIV and HIV have been constructed. These viruses express their respective transgene at high level, are stable and grow normally in tissue culture. They induce good anti-HTLV-I Gag, Env and Tax antibody responses in saimiri macaques even if the animals have been preimmunized with standard anti-measles vaccine.

Several recombinant measles vaccine viruses expressing different forms of the Gag, Pol and Nef proteins of SIV and of the Env and Tat proteins of HIV have been constructed. Mice transgenic for the measles virus receptor and the HLA A2.1 molecule have been obtained. The kinetics and levels of anti-measles immune responses were similar in these mice and in rhesus macaques. Good antibody and cellular responses against the SIV and HIV proteins were obtained after inoculation of the mice with recombinant measles viruses. Mutant HIV Env proteins have been constructed in order to produce antibodies that neutralize primary isolates of HIV. Measles virus expressing these mutated Env proteins induced high antibody titers in mice.

A new measles vector based on an approved commercial vaccine has been cloned and produced in Vero cells and primary chick embryo fibroblasts. The immune responses induced in rhesus macaques by the original Edmonston based measles vector and this new vector have been compared. The SIV and HIV genes will be introduced in the new vector for immunization and challenge experiments in macaques.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

GAU, Mireille

BRAHIC, Michel

BUREAU, Jean-François

GONZALEZ-DUNIA, Daniel

JAROUSSE, Nadine

MOLLET, Lucile

TANGY, Frédéric

AUBAGNAC, Stéphanie, PhD student

BAJRAMOVIC, Jeffrey, Postdoc

DELEBECQUE, Frédéric, PhD student

HANS, Aymeric, PhD student

LORIN, Clarisse, PhD student

MARTINAT, Cécile, PhD student

MENA, Ignacio, Postdoc

VIGNEAU, Soline, PhD student

VOLMER, Romain, PhD student

COMBREDET, Chantal, Technician

LEVI-ACOBAS, Fabienne, Technician

LEVILLAYER, Florence, Technician

NAJBURG-LABROUSSE, Valérie, Technician

SYAN, Sylvie, Technician


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