Unit: Molecular Retrovirology

Director: WAIN-HOBSON Simon

The lab is working on four projects in the areas of HIV immunopathlogy, vaccination and mutation.

1. Mechanism of APOBEC3 retroviral G->A hypermutation Rodolphe SUSPENE, Michel HENRY, Denise GUETARD, Jean-Pierre VARTANIAN

The HIV-1 Vif protein prevents incorporation of two host cell cytidine deaminases into the virion, notably APOBEC3F and 3G which are part of a seven gene locus on chromosome 22. In the absence of the vif gene, HIV genomes are peppered by endless C->U mutations. To explore the generality of these findings in retrovirology we analyzed the potential impact of these deaminases on HBV and HTLV-1 replication. Although four APOBEC3 cytidine deaminases were able to deaminate nascent viral DNA, they did so at low frequencies, less than a few %. Detection of such relatively rare events was possible via a variant PCR protocol developed in the lab that exploits the fact that G->A hypermutants denature at a few degrees below that of the parental sequence. Simply put PCR is performed using a reduced denaturation temperature. Hypermutated genomes at frequencies as low as 10-6 could be amplified. Present work is aimed at understanding the domain structure and packaging of APOBEC3 molecules.

2. Stoichiometry of HIV infection /Quantitating HIV DNA from reverse transcription to integration in vivo Rodolphe SUSPENE, Michel HENRY, Denise GUETARD et Jean-Pierre VARTANIAN

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. 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.

Building on the observation that in vivo cells harbour multiple proviruses, the question we ask here is how many viruses infect a cell in vivo? It is known that proviruses are accompanied by unintegrated forms in the nucleus. By extensive sequencing of cloned PCR material from microdissected nuclei from human splenocytes, the number of discrete sequences asymptote to a finite number. The ratio of unintegrated/integrated DNA is ~ 10-20. Using infected PBMCs, as opposed to established T cell lines, and the proteasome inhibitor epoxomycin we have shown by TaqMan quantitation of nascent DNA formation that the proteasome degrades up to 75% of incoming virions. Combining the two studies the fraction of HIV RNA that gets converted into a provirus could be as low as 1:100. Hence, as a splenocyte may harbour 3-4 proviruses in vivo, this means that the cell was originally infected by ~300-400 virions. The high ratio and wide range in the number of virions making it to the provirus indicates a substantial stochastic component to the infection process.

3. A novel means to attenuate SIV by exchanging the viral promoter. Nicole 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. 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 and 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 <1000 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 (Blancou et al., J. Virol;, Feb 2004).

A new approach based on long-term viral antigen production by genital mucosal epithelium is investigated. SIVmac239 LTR promoter was exchanged in SIV full length genome for the involucrin gene promoter. The involucrin promoter has been extensively studied and was shown to specifically express in the terminally differentiated epithelial cells localized in the upper part of epithelia. High tittered of VSV pseudo-typed recombinant virus will be prepared. When intradermally or intravaginally administered to the animal, this virus should integrate into epithelial basal stem cell and viral antigen should express in the daughter cells engaged into the differentiation process. Transduced stem cells should escape from immune control while antigens producing cells should boost the immune response by cross presentation to Langherans resident cells.

Molecular constructs have been performed and vaccinal stocks characterization is underway.

4. Optimisation of an HIV-1 polyepitope and characterisation of the cellular response in the HHD mice transgenic model. Development of an anti-HIV-1 vaccine strategy throught transgenic plants. Marie-MICHEL, Mireille CENTLIVRE and Monica SALA-SCHAEFFER

The main aim of the present project is to quantitatively and qualitatively increase the response of cytotoxic CD8+ T lymphocytes (CTL) against HIV-1 in infected individuals. CTL are the cornerstone of the immune response against HIV-1. To induce CTL responses, many studies have focused on the design of an HIV-1 polyepitope bearing epitopes restricted to class I HLA alleles, in particular HLA-A2.1, an allele present in 40% of humans. We have designed different anti-HIV-1 polyepitopes and optimized their expression using HBsAg as a carrier. These polyepitopes to induce strong anti-HIV-1 CTL responses is evaluated in the HHD mouse model. In collaboration with the University of Milan, a first generation of transgenic plants expressing an HIV-1 polyepitope has been established. Oral administration of an immunogen would allow the induction of mucosal immune responses and in particular at the lymphoid tissue associated to the intestinal mucosa (the GALT: gut associated lymphoid tissue). The GALT bears the 80% of the total activated memory CD4+ T lymphocytes of the body. These cells are far more represented in this tissue that in the peripheral blood and the lymphoid organs. As the GALT the preferential site of HIV-1 replication during primary infection, it is important to target this tissue in the development of any form of a vaccine.

5. The HIV-1 promoter plays a major role in the dynamics of colonisation of different target tissue compartments. Mireille CENTLIVRE, Marie MICHEL and Monica SALA-SCHAEFFER

We have recently demonstrated that the HIV-1 promoter clade-specific polymorphism impacts on viral replication in different tissue environments target of the infection (Journal of Clinical Investigation in press, 2005). A SIV chimera was engineered where the SIV genome presents non-overlapping Nef and LTR elements (STR). In the STR clone, the SIV homologous region has been replaced by the core/enhancer portion of the HIV-1 B, C or E promoters. These chimeras have been in vitro characterised throughout replication and competition kinetics experiments. Then, two rhesus macaques have been co-infected by the three chimeras. The classical follow-up parameters for SIV infection have been determined over eight months. Data showed that in the rPBMC and in the peripheral lymph nodes, the predominant provirus was the STR viruses bearing the clade B HIV-1 promoters. By contrast, the viruses found in the plasma were mainly the STR-C and -E. At weeks 10, 22 and 31 post-infection, relative chimeras distribution in the peripheral lymph nodes has been analysed. In this compartment, the predominance of STR-B chimera was verified in accordance with data from rPBMC. Sequence analysis on samples from lymph nodes, rPBMC and serum of weeks 10, 22 et 31 post-infection revealed no mutations in the reengineered promoter region showing that the SIV-STR configuration is genetically stable in vivo. At sacrifice, it was possible to show that although the STR-B was the predominant viral form in all analysed compartments, the STR-C and to a later extent the STR-E persist in all organs. Two new rhesus macaques have been intravenously co-infected with the three STR chimeras and sacrificed 18 days post-infection. Follow up confirmed data obtained at primo-infection in the previous study: the STR-C dominates in the serum up to 18 day post-infection while the STR-B dominates in the rPBMC compartment. The analysis of different tissue compartments is on going and it will allow us to identify the main source of STR-C production at primo-infection.

Keywords: SIV, HIV, vacine, multiple infections, APOBEC3, cytidine deamination

Activity Reports 2004 - Institut Pasteur

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