Unit: Slow Viruses - CNRS URA 1930

Director: BRAHIC Michel

The Unit has two main interests : neurovirology and AIDS. We study the infection of mouse by Theiler's virus, one of the best models for multiple sclerosis. We develop a candidate AIDS vaccine, as well as other human viral vaccines, using measles vaccine as a vector. We study the regulation of thymic function during the acute infection by HIV and SIV. We develop a high throughput analysis of the global interactions between viral proteins and host proteins.

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 it persists in white matter glial cells, in particular in oligodendrocytes, the myelin making cells, and in 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 virus does not persist in C3H shiverer and rumpshaker mice which bear mutations in the Mbp and Plp myelin genes respectively. We are using these mice to test the role of myelin and of oligodendrocytes in persistence. We showed that their resistance to persistent infection was not immune-mediated and was not due to reduced permissiveness of the cell body of oligodendrocytes and macrophages to the virus. These results suggests that resistance could be due to the myelin abnormalities of mutant mice. We are testing this hypothesis by inoculating the virus in the vitreous body of the eye and following its migration in the optic nerve. In wild type mice, the virus infects retinal neurons and is found a few days later in myelin and oligodendrocytes in the ipsilateral optic nerve (Figure 1). The contralateral optic nerve is not infected, ruling out hematogenous spread. In C3H shiverer mice, the infection of oligodendrocytes is considerably reduced. We are studying the role of axon/myelin contacts in viral spread.

The susceptibility of mice to Theiler's virus persistent infection varies considerably between mouse strains and is multigenic. The differences of susceptibility are linked mainly to the control of the infection by the specific immune responses. We mapped a susceptibility locus, Tmevp3, at the telomeric end of chromosome 10. We reduced the interval corresponding to Tmevp3 to approximately 500 Mb. It contains 4 genes : Mdm1, Il-22, Ifng (interferon gamma) and Tmevpg1, a " new " gene coding for a non-coding RNA (Figure 2). Tmevpg1 has an ortholog in the human genome and could be implicated in the regulation of the expression of interferon gamma. Several polymorphisms between susceptible and resistant mice have been found in the regulatory sequences of Il-22, Ifng and Tmevpg1. Results obtained ex vivo with mouse lymphocytes show that the expression of interferon gamma and of Il-22 varies according to these polymorphisms. Interestingly, the human homolog of Tmevp3 has been implicated in susceptibility to several auto-immune diseases, including 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 the Env gene of HIV 89.6 with single or combinations of deletions of the V1, V2 and V3 hypervariable loops. These vectors induced T-cell responses and neutralizing antibodies after a single injection to mice transgenic for the measles virus receptor. 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 and a reduction of viral load during primo-infection were observed after a rectal challenge with SHIV89.6.

A Schwarz measles vector expressing HIV antigens is being developed in collaboration with GlaxoSmithKline Bilogicals. The expression of the transgene and its immunogenicity have been tested in transgenic mice. A phase I trial in healthy volunteers without antibodies to measles is planned to confirm the lack of toxic effects and to study the possibility of vaccine shedding by the volunteers. A phase I/II trial will test the induction of HIV-specific CD4+ and CD8+ T-cells in healthy volunteers with pre-existing immunity to measles.

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 challenge given 8 days after immunization. This candidate vaccine is being tested in monkeys. A tetravalent dengue vaccine is being tested.

3- T lymphocyte homeostasis and viral infections (Rémi Cheynier)

The homeostasis of naïve T-cells requires a fine tuning of their production, proliferation, survival and death. The thymus, including in the adult, is the organ that ensures the production of new T-cells with a large range of antigenic specificities. During infections, the diversity of the repertoire must be maintained in spite of the major changes due to the specific immune responses against the pathogen. We study these complex regulations during the acute infection of macaque monkeys by measles virus and by SIV. We showed that measles virus causes, early in infection, an increase in the number of peripheral naïve T-cells due both to increased thymus output and to peripheral proliferation. SIV, on the other hand, causes an increase of peripheral proliferation but no increase of thymic output. Therefore, it is likely that viruses responsible for AIDS cause an early reduction of the T-cell repertoire diversity which may hamper the specific immune responses to infectious agents in general. We are currently studying the mechanism behind the dimished thymic output during SIV infection and the consequences of this reduction on the naïve repertoire. We are testing the possibility of restoring thymic function in these animals by a treatment with recombinant IL-7.

4- High-throughput mapping of virus-host protein-protein interactions (Pierre-Olivier Vidalain)

In collaboration with Y. Jacob (Institut Pasteur, Paris), C. Rabourdin-Conbe et V. Lotteau (IFR-128 Lyon-BioSciences) we nitiated I-MAP, a large-scale mapping project of virus-host protein interactions using high-throughput technologies. The cornerstones of the project are a collection of viral open reading frames (ORF) cloned in a versatile recombination-based system (Gateway and a high-throughput yeast two-hybrid screening platform.

These tools are currently used to identify cellular targets of paramyxoviruses including measles virus, and the recently isolated Nipah virus, Hendra virus and human metapneumovirus (hMPV). All paramyxoviridae enter the organism through the respiratory tract, but their ability to invade other organs and their virulence varies. Whereas respiratory syncytial virus and hMPV are usually responsible for inflammatory lesions restricted to the respiratory tract, measles virus, Nipah virus and Hendra virus can spread to other tissues including the central nervous system. Comparing, at the protein level, the interactions of these viruses with the host will uncover molecular steps that are specific for their respective pathogenesis. Preliminary results obtained with a pathogenic and a vaccine strain of measles virus demonstrate the usefullness of the approach to identify interactions that determine virulence. Because they are phylogenetically related, paramyxoviridae are likely to share common strategies to hijack antiviral immune responses. Such strategies, based on conserved virus-host protein-protein interactions, represent potential targets for generic antiviral drugs.

In addition to the program on paramyxoviridae, we collaborate with several groups from the Pasteur Institute to extend this strategy to other virus families and eventually obtain a comprehensive map of human viruses/host interactions.

FIGURES :

Figure 1: An infected oligodendrocyte in the optic nerve of a mouse, 4 days after intra-ocular inoculation with Theiler's virus. Red: anti-viral antibody. Green: anti CNPase (an oligodendrocyte marker) antibody. Blue: Nuclei stained with DAPI.

Figure 2: Genetic map of the Tmevp3 susceptibility locus.

Figure 3: Measles virus (Schwarz vaccine strain) expressing HIV Env proteins.

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


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