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Genetic and immunological studies in HIV+ European and African long term non progressors

Final Report Summary - GISHEAL (Genetic and immunological studies in HIV+ European and African long term non progressors)

The GISHEAL looked at long term HIV-infected patients that remain healthy without medication. As partial exception to the rule that treated HIV-1 infection leads to the development of the acquired immunodeficiency syndrome (AIDS) and death within 5-10 years, a minority (< 5 %) of infected individuals maintain exceptionally prolonged survival in good, healthy conditions without administration of antiretroviral therapy. These individuals have been viably defined, and are here referred to as 'HIV-1 long-term nonprogressors (LTNP)'. Since their first description in 1995, several cohorts of LTNP have been identified in the United States (US) and Europe. Given their paucity, national networks of clinical and research centres have been formed firstly in France (ALT cohort) and then in Italy (ELVIS cohort) to better study their particular features.
GISHEAL, however, represents the first collaborative European consortium formed with the specific goal of creating a common database containing all the relevant information of European (France, Italy, United Kingdom(UK)) and African (Ugandan) LTNP. This database will be instrumental for investigating the host genetic background and the related gene expression profiles as well as the adaptive and innate immunological responses of LTNP in comparison with control cohorts of infected individuals with chronic progressive (CP) disease. A genome-wide approach based on screening of > l00 000 single-nucleotide polymorphisms (SNPs) followed by proper validation steps is on the verge of describing novel genetic polymorphisms tightly associated with the LTNP condition in addition to validating those already described. The adaptive T lymphocyte (CD4, CD8) immune responses as well as the NK cell and gamma-delta T cell responses to HIV have been studied in LTNP and CP and they will be linked, when possible, to their genomic and post-genomic profiles. The integration of all the gathered genetic, post-genomic and immunological information is currently being analysed by appropriate statistical programs in order to identify natural correlates of non-progression that could be of relevance to the general population of HIV-infected individuals, and to vaccine design.

The project structure

The project has been structured in two distinct work packages (WPs) and by a central 'core' represented by the database implemented and updated under the direct responsibility of one of the partners as well as of the project coordinator.

The two WP principal investigators (PI) and their co-PIs have guaranteed that all the planned activities were developed smoothly and have provided a first line response to unexpected hurdles that, however, have been minimal and always solved without conflicts. At the same time, they have promptly communicated with the project coordinator and discussed variations from the planned development whenever required.
The general philosophy of GISHEAL has been inspired by two lines of work: on the one hand, each group has maintained its individual research on specific aspects of the LTNP condition. On the other hand, forces have been joined to maximise effort and to allow integration among scientists of different institutes and countries. This has allowed the construction of a common European database on HIV-1+ LTNP that has been also the basis for performing genomic studies.

All key investigators from Italy and the UK have met in Paris to perform the genomic analysis with the Illumina HapMap550 platform under the expert supervision of Ioannis Theodorou. On the immunological side, the teams of Patrice Debre and Federico Martini have exchanged protocols and results in order to characterise the innate cell-mediated immune response of LTNP versus control populations, particularly concerning the role of NK and of T cells. A Ugandan research assistant has received in depth training at the IC in London, UK and has exported then technology and immunological expertise to the associated MRC Centre in Entebbe, Uganda.

The close contact among the key scientists and physicians involved in GISHEAL has been instrumental to the overall efficiency of the consortium as it will be demonstrated soon in terms of scientific publications and related dissemination activities.

Main achievements of the second reporting period

The second year of GISHEAL has been characterised by the consolidation of the different activities started during the first year. In particular, the database has been consolidated in terms of documents submitted using the software programme 'Access' (included in the PC version of Microsoft Office) that has been used for data supplements after their initial collection either in Excel or other software or directly on paper documents; a network of dedicated personnel from the individuals centres has been implemented in order to channel the codified information to the coordinator and responsible for the database. After a critical review of this preliminary filing has been satisfactory with an overall acceptance rate of circa 80 % (more precisely: the data relative to 213 patients out of 270, 79 %, submitted were found eligible for inclusion in the database as of June 2008) the original centres have verified and resubmitted their files achieving a global 90 % of candidate LTNP fitting the defined inclusion criteria.

The second major activity of the second year has been the execution of a genome-wide association study (GWAS) on 144 validated Caucasian LTNP using the Ilumina HapMap550 platform. A population of 605 Caucasian HIV-1 seroconverters served as control for LTNP, different Single nucleotide polymorphisms (SNPs) in the Major histocompatibility complex (MHC) region were found strongly associated to the LTNP condition. In particular, these SNPs encompassed both Class I and Class HI genes. Six out of the l0 SNPs showing the highest statistical association with the LTNP condition were found in the HLA-B region, confirming previous studies on so-called 'elite controllers' (i.e. individuals who maintain their viremia levels undetectable in the absence of anti-retroviral therapy) indicating that Class I genes are indeed strongly correlated to the natural control of disease progression, Furthermore, we observed that circa 65 % of our LTNP naturally resist to HIV disease progression independently of HLA-B27 or B57. In this regard, quite strikingly, in our LTNP cohort 3 out of 32 SNPs with a Q-value < 0.05 were located in the MHC Class HI region supporting the concept that different MHC loci significantly contribute to long-term natural control of HIV disease progression in the absence of ART. Therefore, GISHEAL will provide novel evidence of a seminal role of MHC class III gene polymorphisms in determining the LTNP condition, at least in Caucasians.

Concerning the immunological activities, several parallel and cooperative efforts have been made in terms of exchange of SOPs for T cell functional assays, as well as in defining the panels of phenotypic markers and functional assays to be used for identification of immune response profiles of LTNP versus control populations. In particular, first line testing of cellular samples by Elispot has been accomplished while the role of CD4 plus and CD8 plus T cells directed against the dominant epitopes of HIV-l Gag and of the regulatory and accessory Nef and Tat proteins with specific regard to the magnitude, cell differentiation and functional characteristics were also defined. Furthermore, the correlates of protection against disease progression mediated by I-HV-l specific helper T-cells, with particular regard to their proliferation and cytokine secretion, were defined. Second line testing on fresh samples of CD4 and CD8 T cells by multi-parametric phenotypic analysis including CF SE staining and the production of IFN-gamma and IL-2 was performed by use of an eight-colour LSRII flow cytometer by various partners. Concerning innate immunity, a significant modulation of Natural killer receptors (NKR) expression on the surface of gamma-delta T cells has been found in LTNP in comparison to control patients with progressive disease and ongoing studies are verifying the role of these NKR expression on gamma-delta T cell effector function.

The exchange of information with prominent European scientists not belonging to the GISHEAL consortium, but sharing a common interest in LTNP and in correlates of natural protection from HTV disease progression, have been further consolidated. These scientists include Jose Alcami, Majadahonda, Spain, Hanneke Schuitemaker, Amsterdam, the Netherlands, Christine Rouzioux, Olivier Lambotte, Laurent Abel from Paris, France.

Many scientific papers and articles relevant to GISHEAL have been published by the consortium.