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  Director : Bruno HURTREL (bhurtrel@pasteur.fr)


  abstract

 

Our Unit investigate essentially the mechanisms involved in the early control of viral replication during the primo-infection phase, and their relationships to disease progression. Knowledge of these mechanisms should facilitate the identification of protective correlates of primary infection and this is clearly important for the development of an effective HIV vaccine.Report:



  report

cale

Despite substantial advances in HIV/AIDS research, a better understanding of the physiopathology of the infection is still urgently needed to define new strategies for vaccination and treatment. The aim of our team is to contribute to this field, in two main areas: 1) Mechanisms involved in the early control of viral replication during the primo-infection phase, and their relationships to disease progression. This work should improve understanding of the obstacles to vaccine development. 2) Physiopathological mechanisms responsible for HIV-induced encephalopathy. The aim is to provide insight into the development of dementia associated with HIV infection.

1 - early stages of infection and their relationship to disease progression

Following acute primary infection, the plasma viral load decreases and remains relatively stable at levels that correlate with disease outcomes, from rapid progression of AIDS to long-term survival. Although the significance for prognosis of the viral load threshold is broadly acknowledged, the regulatory events that occur during the initial stage of infection remain poorly understood.

Clinical studies have suggested that the severity of acute infection is associated with the rapidity of disease progression. However, the relationship between early viral burden and disease progression is not clear, as measurement of HIV RNA copy number in the blood at seroconversion does not always distinguish between rapid and slow progressors. Lymphoid organs play a central role in HIV pathogenesis. The early mechanisms of viral replication and dissemination and immune response within lymphoid tissues are unclear, as are those involved in the transition between primary and clinically latent stages. Knowledge of these mechanisms should facilitate the identification of protective correlates of primary infection and this is clearly important for the development of an effective HIV vaccine.

Major role played by apoptosis in acute SIV infection

To elucidate the acute immunopathological events responsible for the post-acute control of viral replication and disease outcome, an exhaustive follow up of viral load and immune response was performed in plasma, PBMC and LN during acute infection in rhesus macaques infected with the SIVmac251 strain. To characterize the dynamics of viral infection, we used a sensitive quantitative competitive (QC) reverse transcription (RT)-PCR based assay to quantify plasma viral load and for the LN, the standard in situ hybridization technique, in association with a highly quantitative technique to evaluate the frequency of infected lymphocyte CD4+ T cells and macrophages in whole LN by limiting dilution PCR. To determine precisely the role of apoptosis in primary infection, apoptotic cells were characterized and quantified in blood and LN sections, and the findings compared to LN changes and the ability of cells to proliferate in vivo as assessed by the expression of Ki67, a nuclear cell proliferation-associated antigen expressed at all stages of the cell cycle except G0. Our observations demonstrate that there is no significant relationship between disease evolution, measured at two and six months post inoculation., and viral load in LN at the peak of viremia during primary infection. However, high viral load in the LN was associated with suppression of the immune response to SIV (CTL and lymphoproliferative responses) and recall antigen (PPD). During this acute phase, the number of apoptotic cells in LN was correlated with the severity of immunosuppression and the course of the disease. In the nine animals studied, the number of apoptotic cells in the LN during primary infection was a significant predictor (p > 0.05) of disease progression. Moreover, extensive apoptosis associated with weak proliferation of cells in the same LN section was predictive of both immune anergy and very rapid disease progression. These results provide evidence that an early episode of immunosuppression due to apoptosis plays a major role on the rate of disease progression.

Early stages of SIV infection in macaques protected by SIVmac251?nef

This study was designed to assess the degree of protection provided by immunization with a live attenuated SIVmac251?nef virus. Numerous approaches have been tried, including the use of whole inactivated viruses, recombinant proteins, naked DNA and viral vectors alone or in combination. However, all attempts at vaccination to date have been largely unsuccessful at inducing long-term protection against heterologous viral challenge in macaques. Currently, the most successful vaccines in the macaque model are based on live attenuated SIV. We investigated the virological and immunological characteristics of five rhesus macaques immunized with a nef-deleted SIVmac251 clone (SIVmac251?nef virus) and challenged 15 months later with the pathogenic SIVmac251 isolate. Our results show that: i) monkeys were not totally protected against homologous virus challenge, but controlled such challenge very efficiently in the absence of a secondary immune response and, ii) challenge and vaccine viruses may persist in a replication-competent form for long periods after challenge, possibly resulting in recombination between the two viruses. These data show that vaccination with an attenuated live vaccine does not protect against superinfection but does protect against disease development.

2 - physiopathology of SIV encephalopathy

Involvement of the central nervous system (CNS) is frequent in acquired immune deficiency syndrome (AIDS): 30-60% of all patients with AIDS have neurological symptoms and these symptoms are a major cause of death and disability in the AIDS population. Although there have been a considerable number of reports dealing with HIV-associated nervous system syndromes, a number of questions concerning HIV neuropathogenesis remain unanswered. These questions can be addressed by studies in the SIV-macaque model. To investigate the neuropathological processes involved, a longitudinal analysis of CNS changes was performed using the SIV- macaque model. Five animals were studied during the early phase and thirteen during the asymptomatic and symptomatic phases. Histopathological analyses were performed on one fixed cerebral hemisphere. The other frozen hemisphere was studied by in situ hybridization, immunohistochemistry and RT-PCR. Viral load was quantified by in situ hybridization, CD4+ and CD8+ T cell infiltrates were analyzed by immunohistochemistry and mRNA cytokine expression (IL1b, IL2, IL6, TNFa, IFNg and TGF-b1) by semiquantitative RT-PCR. As reported for HIV-infected humans, the neuropathological analysis of SIV-infected animals revealed four different lesion profiles: minimal changes, early encephalitis, leukoencephalopathy and encephalitis. Early CNS infection was characterized by glial activation and increases in the levels of IL1b, TNFa and IL6 mRNA. During the asymptomatic and symptomatic phases, the increases in IL6 and IL1b mRNA levels correlated with gliosis and the development of myelin lesions. The absence of a significant relationship between neuropathological findings and numbers of SIV-replicating cells, especially in animals presenting minimal lesions and leukoencephalopathy, strongly suggests that cerebral lesions are caused by indirect mechanisms. The inflammatory cytokine pattern associated with severe lesions provides evidence that glial activation plays a key role in the SIV neuropathological process.

Keywords: Virology, AIDS, primary-infection, apoptosis, encephalopathy



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Guesdon, Sylviane,cayre@pasteur.fr Hurtrel Bruno, Researcher,bhurtrel@pasteur.fr

Estaquier Jérôme, Researcher,jestaqui@pasteur.fr

Ho Tsong Fang Raphaël, PhD student,rhotsong@pasteur.fr

Viollet, Laurence, PhD student,lviollet@pasteur.fr

Cumont Marie-Christine, Technician,mcumont@pasteur.fr

Monceaux Valérie, Technician,vmonceau@pasteur.fr


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