Unit: Molecular Retrovirology
Director: WAIN-HOBSON Simon
The lab is working on five projects in the areas of HIV immunopathology, vaccination and mutation.
1. Mechanism of APOBEC3 retroviral G->A hypermutation
Rodolphe SUSPENE, Myrtille RENARD, Vincent PETIT, 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 HIV, Foamy viruses, 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 %. We have also analyzed the impact of murine APOBEC on the friend Leukemia virus replication. 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, Vincent PETIT and 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. 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
In a recent study published in the Journal of Clinical Investigation in 2005 (115; 348-358), we have demonstrated that the HIV-1 promoter clade-specific polymorphism impacts on viral replication in different tissue environments target of the infection. To perform this study, 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 minimal 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 STR-E. At weeks 10, 22 and 31 post-infection, relative chimeras distribution in the peripheral lymph nodes has been analysed. In this body compartment, the predominance of STR-B chimera was verified in accordance with data from rPBMC. At sacrifice, the distribution of the different STR chimeras could be determined in many lymphoid and non-lymphoid tissues. Hence, 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.
The aim of a successive study (in press in AIDS 2006) was to determine the main source of STR-C production during primo-infection. Hence, two new rhesus macaques have been intravenously co-infected with the three STR chimeras and sacrificed 18 days post-infection. The follow up analysis confirmed data obtained at primo-infection in the previous study: the STR-C dominates in the serum till 18 day post-infection while the STR-B dominates in the rPBMC compartment. The analysis of the gut-associated lymphoid tissue (GALT) and faeces allowed identifying this compartment as the main source of STR-C production at primo-infection. Data obtained supported the hypothesis that the clade C HIV-1 promoter is particularly well adapted for viral replication in the GALT, an environment enriched in IL-7. This cytokine is under evaluation to be used in anti-HIV-1 immunotherapy because of its benefit effects on T cell homeostasis and its potentialities in purging viral reservoirs. Supplementary studies on IL-7 impact on activation of subtype specific HIV-1 replication are on going in the URM. All our results have issued in a model on the specificity of HIV-1 subtypes in the temporal and spatial dynamics of viral colonisation of the different compartments target of infection (in press in Current Opinion in HIV-1 and AIDS 2006).
5. Optimisation of an HIV-1 polyepitope and characterisation of the induced cellular response in the HHD mice transgenic model. Development of a new anti-HIV-1 vaccine strategy throught transgenic plants.
Marie-MICHEL, Denise GUETARD and Monica SALA-SCHAEFFER
The main aim of the present project (submitted for publication to Journal of Virology 2006) is to quantitatively and qualitatively increase the response of cytotoxic CD8+ T lymphocytes (CTL) against HIV-1 in infected individuals. The CTL are the cornerstone of the immune response against HIV-1. Nevertheless, CTL are not able to prevent the evolution of the infection towards disease, suggesting that their lyse activity is deficient. To increase CTL response in infected individuals, 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 human worldwide population. We have designed different anti-HIV-1 polyepitopes optimised in their nucleotide sequences, intracellular processing and presentation to CTL. These polyepitopes have been designed to be associated to virus like particles (VLPs) formed by the HBV surface antigen (HBsAg). The efficiency of one of these polyepitopes to induce a strong anti-HIV-1 CTL response has been evaluated in the HHD and HHD/DRB1 mice animal models. We could demonstrate that by optimising polyepitopes, it is possible to obtain higher VLPs secretion in culture and an in vivo augmentation of the global activation state of HIV-1 specific T lymphocytes.
Based on these results, in collaboration with the University of Milan (Italy) and the INRA of Versailles, we are developing a first generation of transgenic plant expressing the optimised HIV-1 polyepitope tested in vivo in the mice models for its immunogenicity. Actually, recombinant vaccine products are available in many transgenic organisms, as bacteria, yeast and mammalian cell lines (i.e. the HBV vaccine). Biotechnology applied to plants has recently improved and allowed using these organisms as vector for obtaining edible immunogenic derivatives in fruits, plant extract and/or purified proteins which can be orally delivered. Oral administration allow the induction of an immune response at the mucosal level 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, split between the lamina propria and the epithelium. These cells are far more represented in this tissue that in the peripheral blood and the lymphoid organs. Hence, being the GALT the preferential site of HIV-1 infection and replication at primary infection, it is fundamental to target this tissue in the development of preventive and therapeutic vaccines. We hope to achieve this objective by oral administration of transgenic plants expressing recombinant VLPs bearing HIV-1 polyepitopes.
Keywords: SIV, HIV, Vaccine, multiple infections, APOBEC3, cytidine deamination