Unit: Slow Viruses - CNRS URA 1930

Director: BRAHIC Michel

The Unit has two main domains of research: neurovirology and AIDS. We study the infection of mouse by Theiler's virus, one of the best models for multiple sclerosis. With regards to AIDS, we study the effect of HIV infection on thymus function and the possibility of restoring this function using IL-7. We develop a candidate AIDS vaccine based on a vector derived from the measles Schwarz vaccine. We showed that this vector could protect mice completely against a lethal challenge by West Nile virus.

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

Theiler's virus, a picornavirus, causes a persistent infection of the central nervous system of mice. The virus infects neurons first, then, if the mice are genetically susceptible, the virus persists in white matter glial cells, in particular in oligodendrocytes, the myelin making cells, and in infiltrating macrophages. The infection is focal and is accompanied by chronic inflammatory lesions with primary demyelination, which resemble active plaques of multiple sclerosis very closely.

The C3H shiverer and rumpshaker mutant mice, which bear mutations in the Mbp and Plp myelin genes respectively, are resistant to persistent infection. This phenotype suggests that oligodendrocytes and/or myelin play an essential role in persistence. Mbp and Plp are also expressed at low levels in macrophages; therefore resistance could also be due to alterations of the virus/macrophage interactions. However we showed that macrophages, as well as oligodendrocytes, from wild type and mutant C3H mice are equally susceptible to Theiler's virus infection in vitro. Therefore, we formulated the hypothesis that resistance has to do with the structural abnormalities of myelin caused by the mutations. We are testing this hypothesis using mixed myelinating neuron/oligodendrocyte cultures.

Susceptibility of the mice to persistence of the infection and to demyelination is multigenic. The differences of susceptibility are linked mainly to the control of the specific immune responses to the virus. We identified a susceptibility locus, Tmevp3, at the telomeric end of chromosome 10. It belongs to a cluster of cytokine genes, including the genes for interferon gamma and for IL-Tif/IL-22. We did not find any polymorphisms between the susceptible and resistant mice in the coding regions of these genes, suggesting that the effect of Tmevp3 is due to differences in the regulation of gene expression. The results that we obtained with lymphocytes ex vivo are in favor of this hypothesis. Interestingly, the human homolog of Tmevp3 has been implicated in susceptibility to multiple sclerosis.

2- Bivalent vaccines derived from the Schwarz measles vaccine (Frédéric Tangy).

Live attenuated strains of measles virus are very efficacious and safe vaccines that can be made into vectors expressing foreign genes. We are using such vectors to design bivalent pediatric vaccines against measles and HIV that could be of great interest, particularly in developing countries.

We cloned the genome of the Schwarz vaccine strain and made it into a vector by inserting two new transcriptional units. We produced a series of recombinant vaccines expressing various forms of the HIV and SIV Gag, Pol, Env and Nef proteins. They induce strong anti-HIV or SIV humoral and cellular responses in mice transgenic for the measles virus receptor.

The regions coding for the V1, V2 and V3 hypervariable loops of the HIV 89.6 env gene were deleted singly or in various combinations and the deleted genes were introduced in the measles vector. These vectors induced T-cell responses and neutralizing antibodies after a single injection to transgenic mice. A 1/100 dilution of these sera neutralized 90-100% of the infectivity of HIV primary isolates. These titers are higher than those of human reference neutralizing sera. Mice pre-immunized against measles virus responded with similar titers of neutralizing antibodies if injected twice with the vectors. The same results were obtained with macaques, which had been pre-immunized against measles virus one year before immunization with the recombinant vector. These results suggest that recombinant measles vaccines could be used in individuals with pre-existing measles immunity.

An immunization/challenge experiment has been performed on 12 macaques using a mixture of vectors expressing the Gag, Env, Tat, and Nef proteins of SHIV89.6. Strong cellular responses were obtained in all monkeys and neutralizing antibodies were obtained in some of them. Intense anamnestic responses were observed in vaccinated animals following a rectal challenge with SHIV89.6. Four out of six immunized monkeys had reduced viral loads during primo-infection compared to the non-immunized controls. The effect of immunization on long-term pathology is being followed.

We are constructing recombinant measles vaccines expressing flavivirus proteins (Dengue virus, West Nile virus, Yellow fever virus, Japanese encephalitis virus). We showed that a single injection of Schwarz vector expressing the secreted form of West Nile virus Env protein induced sterilizing immunity against a lethal West Nile virus challenge given 8 days after immunization. Protection was long lasting. This candidate vaccine is being tested in monkeys.

3- Thymic function during primo-infection by HIV/SIV (Rémi Cheynier).

Thymus plays a major role in homeostasis of naïve T lymphocyte populations and in immune reconstruction, in particular in HIV infected individuals undergoing tri-therapy. Thymic T-cell output can be followed by measuring the amount of a DNA by-product of the rearrangement of the T-cell receptor genes. However, in circulating T-cells, the frequency of these by-product molecules depends also on T-cell peripheral proliferation. We designed a new assay which measured recent thymus emigrant T-cells independently of peripheral proliferation. With this assay we showed that thymopoiesis was strongly reduced during HIV primo-infection, in particular in young patients, and that anti-viral treatments could restore thymopoiesis to some extent.

In collaboration with the team of Nicole Israel (Biology of Retroviruses Unit), we showed that giving interleukin 7 (IL-7) to macaques during SIV primo-infection together with anti-viral drugs, increased the population of peripheral naïve T-cells significantly. This effect was due in part to peripheral proliferation but mainly to an increase of thymic output. These results give us hope that IL-7 treatment may have a similar effect in HIV infected patients.

Photos :

Photo 1

C3H wild type mice were infected intracerebrally with the DA strain of Theiler's virus and sacrificed 11 days later. The mice were perfused with paraformaldehyde, spinal cords were dissected out, snap frozen and 10 µm sections were cut. A: The section was post-fixed in acetone for 10 minutes, then stained for Theiler's virus antigen (green), for the CD11b macrophage marker (red), and counterstained with DAPI (blue). B: The section was post-fixed in ethanol for 5 minutes, then stained for Theiler's virus (red), and for the oligodendrocyte specific enzyme CNPase (green). The image was acquired with a x63 oil lens and the ApoTome microscope.

Photo 2

An oligodendrocyte and its processes myelinating the proximal axon. The oligodendrocyte is stained with an anti-PLP antibody (red) and the axon with an anti-Smi-31 antibody (green). Nuclei are stained with DAPI (blue).

Photo 3

Measles virus (Schwarz vaccine strain) expressing HIV Env proteins.

Keywords: Theiler's virus, AIDS vaccine, Thymus, Pathogenesis


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