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  Director : BRAHIC Michel (mbrahic@pasteur.fr)


  abstract

 

The Unit studies the pathogenesis of two persistent infections of the central nervous system (CNS): The demyelinating disease of mouse due to Theiler's virus, a model for multiple sclerosis, and the behavioral disease of rats infected at birth by Borna disease virus (BDV). The Unit has constructed a chimeric HTLV-I virus which will allow studying the pathogenesis of this virus in mice. Another important goal of the Unit is to use live attenuated measles vaccine as a vector to immunize against HIV.



  report

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

Theiler's virus is a picornavirus of mouse which infects neurons in the brain and spinal cord for about 2-3 weeks, before being eliminated by the immune system. However, in some genetically susceptible strains of mice, the virus is not eliminated and persists in glial cells of the white matter of spinal cord. This persistent infection is accompanied by chronic inflammation and primary demyelination resembling active plaques of multiple sclerosis.

Only viral strains which infect white matter glial cells may persist. The trafficking of the virus between neurons and glial cells and the role of myelin and oligodendrocytes in the establishment of persistent infection are studied by taking advantage of the resistance to persistent infection of the shiverer and rumpshaker mutant mice. These mice bear mutations in, respectively, Mbp and Plp, two genes coding for structural myelin proteins. Our studies call for pure primary oligodendrocyte and mixed oligodendrocyte/neuron cultures. In the latter, oligodendrocytes form myelin sheaths around axons in vitro. A technique which combines in situ hybridization with a fluorescent probe and immunofluorescent detection of antigens has been set up in the course of these studies. The technique allows quantitative studies on cell populations analyzed by FACS.

The group headed by Jean-Francois Bureau has been studying the genetic susceptibility to this disease for several years. The human homologues of some of the genes identified are studied as candidate gene for the susceptibility to multiple sclerosis.

In crosses between susceptible and resistant inbred mice, there is a correlation between high viral load in the CNS and clinical disease. However, high viral load is not sufficient for the appearance of clinical symptoms and a susceptibility locus for clinical disease has been mapped on chromosome 11.

Two loci for susceptibility to persistence infection, Tmevp2 and Tmevp3, have been mapped on chromosome 10, close to the gene for gamma interferon. Positional cloning has been initiated for Tmevp3. A candidate gene, Tmevpg1, and its human orthologue, TMEVPG1, have been characterized. They code for non-coding RNA expressed in CD4+ and CD8+ T-lymphocytes. They are turned off when these lymphocytes are activated, whereas the interferon gamma gene is turned on.

2- Neuronal dysfunction due to Bornavirus (Daniel Gonzalez-Dunia).

Borna disease virus (BDV), a single-stranded, non-segmented, negative RNA virus, causes persistent infections of the CNS with behavioral diseases. There is some evidence, both serologic and molecular, that BDV infects humans and that it could be implicated in some psychiatric diseases.

BDV infects neurons primarily and is non-cytopathic. In rats infected at birth, BDV persistent infection causes anomalies of the postnatal development of specific parts of CNS, in particular hippocampus and cerebellum. The animals do not show symptoms of meningitis or encephalitis; instead they show various behavioral disturbances. This system offers a unique opportunity to study structural and functional abnormalities due to a persistent viral infection, in the absence of inflammation. Our goal is to elucidate the mechanisms by which BDV alters the postnatal development of the CNS and neuroplasticity in general.

We showed that BDV infected PC12 cells are resistant to the neuronal differentiation induced by NGF. Resistance is linked to reduced expression of the NGF receptors, and also to alterations in the MEK/ERK NGF signaling pathway. We hypothesized that BDV interferes in vivo with the response of neurons to neurotrophins.

We studied the infection of primary cultures of hippocampal neurons by BDV. The virus does not alters the survival of the neurons, their morphology or the expression of neuron specific structural markers. Instead, the infection causes a specific down-regulation of the expression of proteins implicated in synaptic plasticity such as GAP-43, synapsin, VAMP-2 and synaptophysin. Furthermore, infected neurons do not respond normally to the neurotrophins BDNF and NT3, as shown by studying both the neurotrophin signaling pathway and the synaptogenesis normally induced by BDNF.

We showed that 1-ß-D-arabinofuranosylcytosine (Ara-C), an inhibitor of mitosis, was a powerful inhibitor of BDV, both in vitro and in vivo. This antiviral activity is specific for BDV. It is not linked to the effect of the drug on cellular DNA synthesis. New anti-BDV drugs, derived from Ara-C, but with reduced cellular toxicity are being investigated.

3- HTLV-I pathogenesis (Frédéric Tangy).

HTLV-I is the agent of tropical spastic paraparesis, a chronic demyelinating disease for which there is no good animal model. We constructed a chimeric HTLV-I in which the envelope protein is replaced by that of ecotropic Moloney murine leukemia virus. This chimeric virus lost its tropism for human cells but acquired a tropism for murine cells. Several inbred mouse lines were inoculated with this virus. The virus persisted in most of them, as integrated provirus, in CD4+ T-lymphocytes in lymphoid organs. Although the level of proviral expression was low, infected lymphocytes could spread the infection to naive ones.

4- A recombinant measles virus vaccine to immunize against HIV (Frédéric Tangy).

Live attenuated strains of measles virus are efficacious and safe vaccines which are widely used to immunize children. These vaccine can now be turned into viral vectors expressing foreign genes. A recombinant vaccine which could immunize at the same time against measles and against HIV would be a considerable asset in the fight against AIDS.

The Schwarz vaccine strain was cloned from a commercial batch of measles vaccine and made into a vector by inserting two additional transcription units. After two passages on chick embryo fibroblasts, the cell used to manufacture the vaccine, the sequence of the cloned measles vector was identical to that of the original vaccine. The immunogenicity of the cloned virus was tested in mice transgenic for the measles receptor (CD-46) and in macaques. Antibody titers and T cell responses were identical to those obtained with the same dose of the commercial vaccine.

A large series of recombinant measles viruses expressing various forms of the Gag, Pol and Nef proteins of SIV ant the Env and Tat proteins of HIV was constructed. Good humoral and cellular responses against these proteins were obtained by inoculating mice transgenic for the CD-46 receptor.

Several mutations were introduced in the env gene of HIV 89.6, a primary isolate, to increase the immunogenicity of the envelope protein, in particular the induction of neutralizing antibodies. The hyper-variable V1, V2 and V3 loops were deleted, singly or in various combinations. In some of the constructions, the V3 loop was replaced by the so-called "Katinger" neutralizing epitope. The constructions were obtained for the gp140 as well as the gp160 form of the Env protein. They were introduced in a measles vector and tested for their immunogenicity after a single low dose injection in mice transgenic for the CD-46 receptor. When the V3 loop was deleted, the neutralizing titers obtained were similar to those of human reference neutralizing sera or of a mixture of the 2F5 and 2G12 neutralizing monoclonal antibodies.

Recombinant measles/HIV-env viruses were inoculated to CD-46 transgenic mice which had been previously immunized against measles virus alone. After two injections of recombinant viruses, the anti-HIV neutralizing titers were identical to those obtained with naive animals. These results indicate that it might be possible to use recombinant measles vaccines in adults as well as in children.

Keywords: Theiler's virus, Borna disease virus, HTLV-I, AIDS vaccine, Pathogenesis



  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

MOLLET Lucile

TANGY Frédéric

BAJRAMOVIC Jeffrey, Postdoc

DELEBECQUE Frédéric, PhD student

HANS Aymeric, PhD student

KARACHTCHOUK Galina, Postdoc

KHIAR, Hind, PhD student

LORIN, Clarisse, PhD student

MARTINAT Cécile, PhD student

MENA Ignacio, Postdoc

ROUSSARIE Jean-Pierre, PhD student

SARTORIUS Leah, Fulbright fellow

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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