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  Director : WAIN-HOBSON Simon (simon@pasteur.fr)


  abstract

 

Work is directed in two directions. The first seeks to generate novel attenuated SIV strains that can strongly protect against challenge by pathogenic strains and has been pursued with some success. The second line of research is showing that in vivo cells are infected by hundreds of viruses of which only a minute fraction ever survive to become integrated proviruses.



  report

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Multiply HIV-1 infected cells in vivoRodolphe SUSPENE, Michel HENRY, Jean-Pierre VARTANIAN

In vivo cells are generally multiply infected (Jung et al 2002, Nature 418, 414). By studying splenocytes from two HIV-1 infected individuals, it was possible to show by fluorescent in situ hybridization (FISH) that, on average, the proviral copy number per cell was 3-4 with a range of 1-8. Greater than 75% of infected cells harboured two or more proviruses. By laser microdissecting individual FISH+ nuclei, followed by PCR, cloning and sequencing, it was possible to show that a single cell harboured a genetically diverse collection of genomes with up to 29% amino acid variation in the V1V2 hypervariable regions of the envelope protein. Whenever multiple infection occurs in retrovirology, recombination invariably follows. Hence the work may not only provide insights into the infection process in vivo but also highlight the tempo of recombination and its impact on HIV evolution. Many important questions remain : 1) How many viruses infect a cell in vivo? 2) Might integration sites be clustered in the genome ? 3) Why is there so much sequence variation in one cell? Preliminary results indicate that in vivo a cell is infected by >100 virions, of which only a minute fraction generate proviruses. Finally the nature and complexity of HIV on cell surfaces may help us to understand the genetic complexity of HIV and generation of huge numbers of recombinants.

A novel means to attenuate SIV by exchanging the viral promoterNicole CHENCINER, Philippe BLANCOU, Denise GUETARD

Among the many HIV/SIV immunogens, only some attenuated live viral vaccines have afforded strong protection against intravenous challenge with a pathogenic SIV isolate. They have invariably been obtained by deleting gene segments. To date there has been a general inverse correlation between the degree of attenuation and protection. The exchange of the SIV promoter by other viral or cellular promoters may confer novel properties to the chimera, notably attenuation. Alternatively if viral expression is shifted away from the crucially important CD4+ T lymphocytes to other cells, such as macrophages and dentritic cells, this may preserve sufficient help to allow the immune system to contain infection.

We have worked extensively on one particular chimera where the core SIV promoter just 3' of nef nd 5' of TAR has been replaced by the powerful immediate early promoter of human cytomegalovirus (CMV-IE). The chimera (SIVmegalo) grows to very low titres in vivo — median titres for 15 animals were <400 copies/ml. This represents >1000 fold reduction in peak viremia compared to the parental virus. When challenged by the pathogenic virus SIVmac251, viremia was contained by >1000 compared to naive controls. In view of this, novel promoter chimeras are being tested in pilot studies.

We hope to greatly improve the safety of SIVmegalo in protecting against SIVmac251 by the intravenous, vaginal and intrarectal routes. We would like to know where SIVmegalo is replicating, why it is so attenuated and learn as much as possible about the nature of the immune responses it induces. The long term course of SIVmegalo infection will become clear. We should have an answer to the physiological relevance of HIV core promoter polymorphism, as some insight into whether shifting tropism into macrophages and dentritic cells spares CD4+T lymphocytes.

Construction of SIV chimeras. Compartmentalisation of viral replication throughout specific promoters usage.Mireille CENTLIVRE, Marie MICHEL and Monica SALA-SCHAEFFER

The aim of this project is to analyse the influence of different promoters on viral transcription, replication, cellular tropism and pathogenesis of immunodeficiency viruses. To develop this study in vivo, SIV chimeras have been constructed where the SIV genome presents non-overlapping Nef and LTR elements (STR). In the STR clone, the SIV homologous region has been replaced by either the enhancer/core portion of the HIV-1 B, C and E subtypes promoters, or the human promoters for the IL-2, IL-4 and INF-g genes. These chimeras allow the phenotypic analysis of subtype-specific enhancer/core promoter HIV-1 regions. Moreover, compartmentalisation of viral replication in Th1 (IL-2 and INF-g promoters) and Th2 (IL-4 promoter) cells will clarify the impact on pathogenesis of viral replication into a specific cellular subset.

Contribution of different cell compartments to persistence and pathogenesis of SIV in vivoPeter Sommer

The molecular mechanisms leading to post-integration latency of HIV/SIV as well as the contribution of different cellular compartmenats to viral persistence and AIDS pathogenesis remain poorly understood. These important aspects of HIV/SIV infection rely on viral gene expression and replication, which are regulated by promoter elements located within the viral LTR. The function of these elements depends on the presence of cellular transcription factors. Our laboratory has concentrated on the construction of chimeric SIVs with promoters modified by the insertion of constitutive and tissue-specific regulatory elements. This novel approach will allow to dissect the cell tropism of SIV in vivo and to address experimentally the impact of different cellular compartments on viral persistence and pathogenesis in the macaque model.

Use of the SIV genetic variability in the study of the in vivo immunopathology in vivo, and stimulation in silico.Céline RENOUX-ELBE

The present study tries to analyse the role played by the activation of the immune system on the dynamics of viral replication. To do that, rhesus macaques immunised with different antigens were infected with SIVmac251. At regular time points, booster injections were realised in order to favour the infection of CD4+ T cells responding to those antigens. Then, delayed hypersensibility tests were made to create antigen-specific inflammatory sites. By comparing SIV quasispecies at these sites to those in peripheral blood during booster injections, it should allow to describe lineages of SIV within T cells specific for particular antigens. These results will permit to draw up a model of virus replication taking into account antigenic stimulation of the immune system.

Keywords: SIV, HIV, Vaccine, multi-infection



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  WAIN-HOBSON Simon, Institut Pasteur, Professor,simon@pasteur.fr

CHAHINE Michèle, Institut Pasteur, Secretary,mchahine@pasteur.fr

CHENCINER Nicole, phD, Institut Pasteur,nchencin@pasteur.fr

SALA-SCHAEFFER Monica, pHd, Institut Pasteur,joo@pasteur.fr

VARTANIAN Jean-Pierre, phD, Institut Pasteur,jpvart@pasteur.fr

CENTLIVRE Mireille, graduate student, University Paris VI,mcentliv@pasteur.fr

MICHEL Marie, graduate student, University Paris XI Orsaybiomarie@pasteur.fr

RENOUX-ELBE Céline, graduate student, University Paris VIIrenoux@pasteur.fr

SOMMER Peter, Post-Doc,psommer@pasteur.fr

SUSPENE Rodolphe, graduate student, University Paris VI suspene@pasteur.fr

GUETARD, Denise, Engineer, Institut Pasteur,dguetard@pasteur.fr

HENRY Michel, Technician, Institut Pasteur ;michael@pasteur.fr


Activity Reports 2002 - Institut Pasteur
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