Unit: Slow Viruses - CNRS URA 1930
Director: BRAHIC Michel
The Unit has two main research domains: neurovirology and AIDS. We study the pathogenesis of the disease of mouse due to Theiler's virus and of the disease of rat due to Bornavirus. As far as AIDS is concerned, we are using the attenuated Schwarz strain of measles virus as a vector to immunize concurrently against measles and HIV and we study thymocyte maturation in the course of HIV/SIV primo-infection.
Pathogenesis of the infection by Theiler's virus (Michel Brahic, Jean-François Bureau)
Theiler's virus, a mouse picornavirus, causes a persistent central nervous system (CNS) infection. The virus persists in glial cells of the white matter, in particular oligodendrocytes and infiltrating macrophages. Infection is accompanied by chronic inflammation and primary demyelination very reminiscent of active plaques of multiple sclerosis (MS).
Theiler's virus infects neurons first but persists in glial cells. We study virus trafficking between these different cells using tools of genetics and cell biology, in particular mixed, myelinating cultures of neurons and oligodendrocytes. In the C3H shiverer and rumpshaker mice, which bear mutations in two main myelin proteins, MBP and PLP, the virus infects neurons for 1-2 weeks but does not persist, suggesting that the oligodendrocyte and/or the myelin play a key role in persistence. In vitro, oligodendrocytes from wild type and mutant mice are equally permissive to the virus, showing that resistance is not due to a replication defect in the oligodendrocyte cell body. Resistance could be due to the characteristic alterations of myelin structure of the mutant mice or to changes in virus/macrophages interactions since MBP and PLP are also expressed in these cells, although at very low levels.
Susceptibility to Theiler's virus induced disease is multigenic and concerns mainly the control of viral load by the immune system during persistence. Tmevp3 is a susceptibility locus that we mapped on chromosome 10. It is located, together with interferon gamma and IL-Tif/IL-22, in a cluster of cytokine genes. No functional polymorphism has been found in the coding sequences of the cytokine genes suggesting that Tmevp3 controls gene expression. Tmevpg1, a gene that codes for a non-coding RNA expressed in T-lymphocytes is a candidate gene for the Tmevp3 locus. We are examining the role of the human homologue of Tmevp3 in the susceptibility to MS. Preliminary results suggest a small effect of the locus.
Neuronal pathology due to Bornavirus (Daniel Gonzalez-Dunia)
Borna disease virus (BDV), a non-segmented, negative single-stranded RNA virus, persistently infects the SNC of several species and causes behavioral diseases in mammals. There is good evidence that BDV infects humans and it has been suggested that it could be implicated in the etiology of certain psychiatric diseases.
BDV chiefly infects neurons, without causing any cytopathic effect and any inflammation when the animals are inoculated at birth. However, this persistent infection alters some neuronal functions. Our goal is to identify the mechanisms by which BDV interferes with neuroplasticity and synaptic function.
We studied the effect of BDV on primary cultures of rat hippocampal neurons, the main viral target cell. The neurons are very permissive to viral expression, although they survive and their morphology and the expression of structural markers remain unchanged. However, BDV causes a specific down-regulation of the expression of proteins implicated in synaptic plasticity, such as GAP-43, synapsin, VAMP-2 and synaptophysin. Furthermore, BDNF and NGF induced synaptogenesis is abrogated in infected neurons, due to a lack of phosphorylation of the ERK1/2 kinase. A recent study of synaptic activity in these neurons showed alterations in potentiation mechanisms.
We showed that 1-ß-D-arabinofuranosylcytosine (Ara-C) blocked BDV replication both in vitro and in vivo. Less toxic inhibitors were identified, including 2'-Fluoro-2'-déoxycytidine (2-FdC). 2-FdC is a promising, specific anti-BDV drug that could find uses in the field.
Bivalent vaccines derived from live attenuated measles vaccine (Frédéric Tangy)
Live attenuated strains of measles virus (MV) are very safe and potent vaccines that can be made into vectors to express foreign genes. We use this approach in an attempt to obtain a bivalent vaccine against measles and AIDS.
The Schwarz vaccine strain of MV has been cloned and made into a vector by adding 2 additional transcription units to the genome. Recombinant MV vaccines expressing various forms of the Gag, Pol and Nef SIV proteins and of the Env and Tat proteins of HIV have been obtained. They induce strong cellular and humoral responses against the SIV and HIV antigens in mice transgenic for the MV receptor.
The env gene of HIV98.6, from which the hypervariable V1, V2 and V3 regions were deleted, either singly or in various combinations, to increase immunogenicity was cloned into the MV vector. Anti-HIV T-cell responses and neutralizing antibodies were obtained in transgenic mice after a single injection. The sera neutralized 90-100% of the infectivity of primary HIV isolates at a 1/100 dilution. After 2 injections, the same levels of antibodies were found in mice which had been pre-immunized against MV. The same result was obtained in rhesus macaques pre-immunized 1 year earlier, suggesting that recombinant MV vaccines can be used in individuals with anti-measles immunity. A challenge experiment is ongoing with 18 macaques immunized with Schwarz MV vectors expressing the Gag, Env, Tat and Nef genes of SHIV89.6. Strong cellular responses have been obtained in all immunized animals and neutralizing antibodies in some of them.
Thymic function during the primo-infection by HIV and SIV (Remi Cheynier)
The thymus plays a central role in naïve T-cell populations homeostasis in the adult and in immune system restoration during HIV-1 infection, particularly after anti-viral therapy. We set up a new technique that makes it possible to quantify T-cells recently emigrated from the thymus. We showed that their number is considerably reduced during HIV primo-infection, especially in young patients, and is partially restored by anti-viral therapy. On the contrary, an increase in thymic output is observed in lymphopenic patients infected with HIV-2, a less pathogenic virus. This suggests that it might be possible to stimulate compensatory mechanisms to correct the T-cell depletion of HIV infected individuals.
We are studying the regulation of thymic homeostasis in SIV infected macaques to understand the mechanism of decreased T-cell output and the mechanism of restoration of this output that follows various treatments. These experiments should help us to identify molecules that could be manipulated to help restoring a naïve T-cell repertoire in HIV infected patients.
Axon myelination obtained in vitro. The neurons and their axons are stained green with an anti-neurofilament Smi31 antibody. The oligodendrocytes and the myelin are stained red with an anti-proteolipid protein (PLP) antibody. A number of axons have been surrounded by a myelin sheath.
Borna disease virus (BDV) blocks BDNF-induced synaptogenesis in primary rat hippocampal cultures. Control or BDV-infected neurons were treated with the neurotrophin BDNF, or left untreated. Synapses were visualized by double immunofluorescence using antibodies specific for VAMP-2, a presynaptic marker (in green) and PSD-95, a postsynaptic marker (in red). The BDNF-induced formation of synapses is clearly visible in control neuronal cultures (top panels). In contrast, it is blocked in BDV-infected neurons (bottom panels).
Measles virus (Schwarz vaccine strain) expressing HIV Env proteins.
Keywords: Theiler's virus, Borna disease virus, AIDS vaccine, Thymus, Pathogenesis