Cell surface protein signatures have been successful to discriminate hematopoietic progenitor populations allowing major advances in understanding blood cell production, to define pathways in hematologic malignancies and to foster new therapeutic approaches. Limited knowledge on the phenotype of cells that participate in heart formation impairs our understanding of progenitors of the cardiac cell lineages and their eventual persistence in the adult organ. As a consequence, therapies to restore heart function after injury have been unsuccessful. A number of membrane proteins have been identified on cardiomyocytes; on cardiac fibroblasts; and on endothelial cells, however a multi-parametric analysis of the phenotype of the different cardiac cell compartments along development is still missing. We combined multi-parametric flow cytometry with transcriptional characterization, based on well-known gene expression patterns, to describe major cardiac cell-subsets. The expression of CD24, CD54, Sca-1 and CD90 allowed defining cardiac populations in the non-hematopoietic and non-endothelial cell fraction by flow cytometry. Transcriptional profiling of the sorted populations enabled the identification of cardiomyocytes, in the CD24+ population, while differential expression of CD54, Sca-1 and CD90 defined four cardiac stromal compartments. The identified subsets exhibited specific distributions in three analyzed regions (atria, auriculo-ventricular junction and ventricles). We have thus identified a panel of surface markers, some of which novel in the cardiac context, that allowed assigning surface signatures to different cellular fractions by their unique transcriptional profiles. This work is the foundation for comprehensive studies on the role of different cell fractions by their unique transcriptional profiles.

Stem cells are defined by their is their capacity for self-renewal and differentiation. Some adult tissues maintain a reservoir of stem cells, that generally reside within specialized microenvironments, known as stem cell niches, that regulate their behaviour. Skeletal muscle stem (satellite) cells are quiescent in homeostatic conditions in adults, and they are activated after muscle injury, when they re-enter the cell cycle, proliferate and differentiate into myoblasts, which will then fuse to form new muscle fibers. Satellite cells express the paired/homeodomain gene Pax7, which plays a critical role in satellite cell maintenance postnatally. Numerous experiments have shown that the skeletal muscle stem cell population is heterogeneous, therefore like many other stem cell systems, characterising the stem cell states is a major objective. In our laboratory, a reversible dormant cell state was identified, correspondent to a Pax7Hi quiescent subpopulation (top 10% of the Pax7-nGFP+ cells isolated from the transgenic mouse model Tg:Pax7-nGFP) with a lower metabolic activity and longer lag for the first cell division compared to Pax7Lo cells [1]. Muscle stem cells that survive for extended periods post-mortem are also dormant, suggesting that this property, in addition to anoxia [2] contributes to their viability. Therefore, different physiological states are associated with distinct cell states of muscle stem cells. Metabolism could play a critical role in dictating whether a cell remains quiescent, proliferates or differentiates. Stem cell metabolic plasticity in homeostasis and differentiation, as well as during cell reprogramming, is well described in different cell systems. However, unanswered questions remain regarding the metabolic regulation of satellite cell biology and skeletal muscle regeneration. In this project, we will investigate the behaviour of muscle stem cells in distinct physiological states, especially post-mortem and aging.

We search for the expressed transcript in mature normal sperm as compared to the infertil one.

Recherche de cible pour des miRNAs chez l'Humain.

The post-translational modification by SUMO is an essential regulatory mechanism of protein function that is involved in most challenges faced by eukaryotic cells. Gene expression is particularly regulated by sumoylation as many SUMO substrates are transcription factors and chromatin-associated proteins, including histones. The emerging paradigm for the proposed work is that sumoylation controls multiple aspects of chromatin structure and function in response to external cues. According to this view, sumoylation is expected to impact both global and specific transcriptional programs thereby affecting constitutive and inducible expression of both coding and non coding genes. Recently, we found SUMO as an integral and instructive component of chromatin in cell growth and senescence, thus establishing sumoylation as a new and paradigmatic chromatin modification. This work now paves the way for detailed understanding of the contribution of SUMO as a multifaceted modifier of chromatin.

La PF Transcriptome et Epigenome développe des projets de séquençage à haut débit (collaboration et service) avec des équipes du Campus. Ceux-ci couvrent l'ensemble des thématiques du campus ainsi qu'une large gamme d'organismes (des virus aux mammifères). La plate-forme exerce des activités de biologie humide (construction des librairies et séquençage) et de biologie sèche (analyse bioinformatiques et statistiques). La personne mise a disposition interagira étroitement avec les autres bioinformaticiens du pôle BioMics et du Hub. Ses activités concerneront notamment: - La participation à la conception et à la mise en place des projets avec les équipes demandeuses, la prise en charge des analyses et le reporting aux utilisateurs - La mise en place d'un workflow d'analyse bioinformatique des données de transcriptome /épigénome en étroite collaboration avec le C3BI, la DSI et les autres bioinformaticiens du pole. Ce workflow permettra le contrôle qualité des données, leur prétraitement, le mapping des séquences sur les génomes/transcriptomes de réference, et le comptage des reads pour les différents éléments de l'annotation - L'adaptation du workflow d'analyse aux questions biologiques et aux organismes étudiés dans le cadre des activités de la PF - L'activité de veille technologique et bibliographique (test et validation de nouveaux outils d'analyse, updates d'outils existants...) - La mise en place et le développement d'outils d'analyse adaptés aux futurs projets de la PF: single cell RNAseq, métatranscriptome, ChIPseq, analyse des isoformes de splicing.. Ceci se fera notamment via la réalisation d'analyses dédiées avec certains utilisateurs. Les outils mis en place et validés dans ce cadre seront ensuite utilisés pour l'ensemble des projets. - L'activité de communication et de formation (participation aux réunions du consortium France Génomique,formation permanente à l' Institut Pasteur… - la participation a d autres projets du Pole BioMics (selon disponibilité) Bernd Jagla, qui était le bioinformaticien de la plateforme a rejoint le Hub au 1er janvier 2016. Rachel Legendre est mise a disposition depuis le 2 novembre 2015 et remplace Bernd Jagla. Je souhaite que Rachel Legendre soit mise à disposition de la plateforme pour une durée d'au moins 2 ans.

Dans le contexte du LabEx Milieu Intérieur (MI) dirigé par M. Albert et L. Quintana-Murci, 300 lignées de fibroblastes cutanés ont été préparées à partir de donneurs sains stratifiés selon l’âge et le sexe. Le projet « Fibroblastes » du Labex MI mené à la plateforme d’Imagerie dynamique contribue à l’étude de la variabilité de la réponse immune de donneurs sains en analysant la réponse immune innée des fibroblastes.L’étude du transcriptome est devenue un moyen important pour caractériser les phénomènes biologiques. Le but de ce projet est de mettre à contribution des données déjà publiées d’expression de gènes obtenues par hybridation sur microarrays dans des situations expérimentales pertinentes (questions posées,cellules étudiées) pour définir les gènes d’intérêt pour l’étude de la variabilité des cellules et des réponses dans les 300 lignées de fibroblastes du LabEx MI.

L'extraction des protéines humaines qui interagir avec des protéines virales par le séquençage des barcodes associés avec les protéines humaines et virales. Analyses structurales des protéines virales et humain pour trouver les sites d’accrochage.

Analyses fonctionnel des protéines humains de type ubiquitin qui interagir avec les virales protéines de la grippe

The Center for Human Immunology (CIH) supports researchers involved in translational research projects by providing access to 16 different cutting edge technologies. Currently, the CIH hosts over 60 scientific projects coming from 8 departments of the Institut Pastuer and 5 external teams. In order to respond to the growing needs of these projects in the area of single cell analysis, the CIH has introduced a significant number of single-cell/single-molecule technologies over the past 2-3 years. These new technologies, such as the Personal Genome Machine (PGM) and Ion Proton sequencers, iSCAN microarray scanner, Nanostring technology for transcriptomics profiling and real-time PCR machine BioMark, give rise to large datasets with high dimensionality. Such trend, in terms of data complexity, is also true for flow cytometry technologies (currently reaching over 20 parameters per cell). The exploration of this data is generally beyond the scope of scientists involved in translational research projects. In order to maximize the research outcomes obtained from the analysis of these rich datasets, and to ensure that the full potential of our technologies can be served to the users of the CIH, we would require a proximity bioinformatics support. A CIH-embedded bioinformatician would: 1) design and implement standard analysis pipelines for each of the data-rich technologies of the CIH; 2) provide regular ‘bioinformatics clinics’ to allow scientists the possibility to customize standard pipelines to their specific needs; 3) run trainings on the ‘R software’ platform and other data analysis tools (such as Qlucore) of interest for the CIH users. The objective would be to empower the users to run exploratory analysis by themselves, and to teach good practices in terms of data management and data analysis.    

Protein sequencing by MS: Automation of analysis

Analysis of the immune response to yellow fever 17D vaccination

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, T-cell response and type I interferon (IFN-I) are important for an effective response to LASV infection. Among dendritic cells (DC), myeloid DC can induce T-cell activation and plasmacytoid DC are specialized in IFN-I response. DC are also the first target of LASV during the infection of a new host, and studies on in vitro differentiated DC suggest a role of DC in T-cell response to LASV infection. Therefore, plasmacytoid and myeloid DC could be important for an efficient response to LASV infection.

The objective of this study is to evaluate how the different programming of distinct CD4+ T cell subsets affected their susceptibility to HIV infection and the survival of infected cells. Dr. Saez-Cirion's Team evaluates how the metabolic state of CD4⁺ T cells and the regulation of cellular factors shape the distribution of the HIV reservoir in distinct CD4⁺ T cell subset. This project uses new technologies and reagents to analyze cells from uninfected donors, and from HIV patients in acute infection, chronic infection and HIV remission.

  Shigella is an enteroinvasive bacterium that induces bacillary dysentery. It invades human intestinal epithelial cells causing the inflammatory destruction of the colonic epithelium. Our previous work reported that Shigella inhibits the secretion of its host cell (Mounier et al Cell Host Microbes 2012) via several virulent proteins injected by the bacterium such as IpaJ and VirA. These two effectors may have redundant action. Thus, to address the effect of Shigella on the secretome of its host cell and in particular define the precise action of IpaJ and VirA, we conducted a mass spectrometry (MS) analysis on apical (A) and basal (B) secretion of polarized infected human intestinal cells using several strains: wild type (WT), avirulent strain (mxiD) and mutated strains either on virA, ipaJ or both (virAipaJ) and compared to non-infected cells (NI). We have in total 6 infection conditions and we collected apical and basal secretion medium (total 12 MS data). Most of the conditions were done in triplicate. We have now these quantitative MS data (excel file) that gave between 300 (basal secretion) to 1000 proteins (apical secretion) secreted differently among the different conditions. We need your collaboration to analyze our data to: Check whether our protein candidates are in databases of secreted factors Obtain protein clusters of our hits among the 6 conditions used (12 datasets)

Recent progress in sequencing and bioinformatics methods have given access to ancient DNA. This project aims at understanding the differential evolutionary pressure on the key proteins implicated in the nervous system.

Label free quantification of proteins after the infection with M. tuberculosis. Macrophages isolated from seven patients were used in this study. Four conditions were compared.

The mechanisms governing ageing, which is a multifactorial process, have not been resolved and constitute a fundamental open question in cell and organismal biology. Exceptionally, in rare genetic diseases like the Cockayne syndrome (CS), ageing is dramatically accelerated. We have recently identified a novel pathway that is altered in cells from CS patients and is not affected in a linked disease (UVSS) that is not associated with precocious ageing. This is a unique case in the literature of diseases due to mutations in the same proteins but only one resulting in precocious ageing. The defect linked to the progeroid phenotype consists in oxidative stress-dependent overexpression of a little known proteases, HTRA3, which degrades the key mitochondrial DNA polymerase and in turn affects mitochondrial function. We succeeded in rescuing the defective phenotype in patient cells using two independent strategies. We aim to understand the mechanism by which oxidative stress induces overexpression of HTRA3 and we have indications that this event may be linked to the methylation of target genes. Genome wide methylation will be analysed in these rare patient cells.

Skeletal muscle stem cells constitute a population of cells with heterogeneous properties. Interestingly, muscle stem cells have a remarkable capacity to regenerate muscle fibres after regeneration. We are performing a molecular analysis of these stem cells.

It has been found in the past that the peptidoglycan (PGN) degradation fragments DAP-containing muramyl tripeptide (M-triDAP) and muramyl di-peptide (MDP) stimulate innate immune receptors Nod1 and Nod2. However, it remains to be clarified how the fragments reach Nod1 and Nod2, since these receptors are intracellular. The aim of the project is thus to investigate novel factors in peptidoglycan signalling, using gene trap mutagenesis in human near-haploid cells to randomly knock out genes and do a genome wide screen to establish a library. There is a pressing need to find novel therapeutic strategies, and it has been shown in the past that the above described technique of finding genes works well for this (see Carrette et al., 2011). Results will be validated in relevant mouse models and epithelial cell lines using CRISPR-Cas.

As a part of BIRDY program (http://www.charliproject.org/), this project aims to study the microbiome composition and its dynamics in early infancy in Madagascar. It will provide genomic knowledge on antibiotic resistant strains and resistance genes circulating in this island. It will identify factors influencing the outgrowth of pathogenic bacteria, including ESBL-PE and decipher the role of the gut microbiome. The developed comprehensive modeling framework will be easily adaptable to other epidemiological questions related to the impact of any human intervention on the gut microbiome and of other environments such as skin, nose or urogenital tract. This project will contribute to the transfer of expertise in genomics and bioinformatics to the IP Madagascar partners.

One of the best models for the study of bacteria-host interactions is Listeria monocytogenes. One interesting facet of this bacterium is its ability to modify host chromatin. Recently, we have shown that Listeria causes a drastic deacetylation of histone H3 on lysine 18 (H3K18dc). Interestingly, to impose this modification, Listeria highjacks previously undescribed host machinery: the host protein sirtuin 2 (SIRT2) is relocalized to the nucleus where it causes genes repression during infection. SIRT2 is a NAD-dependent deacetylase that has been implicated in the regulation of complex processes such as aging and cancer. This protein had been mainly studied in the cytoplasm, and although SIRT2 had been show to shuttle between the cytoplasm and the nucleus, the mechanism and its biological role remained unknown.        In order to further characterize the role of SIRT2 during infection and identify all the genes at which it is recruited, we are performing ChIP-seq analysis. These studies are expected to bring new insights into the function and regulation of SIRT2 by characterizing this new phosphorylation site.

Measles virus protein C interplay with cellular apoptotic pathways; applications for cancer treatment.

The aim of the project is to search for the effects of hepatic inflammation on microbiota.

Over the last years, innate lymphoid cells (ILC) have been increasingly investigated. Despite the absence of antigen specific receptors, they belong to the lymphoid lineage and represent important sentinels for tissue homeostasis and inflammation. They contribute to numerous homeostatic and pathophysiological situations via specific cytokine production. ILC are currently divided into three groups based on the expression of specific transcription factors and secretion of cytokines. We focus this study on fetal ILC3 development. We have observed that contrary to lymphocytes, ILC can migrate toward lymphoid organs, tissues and mucosal sites as lymphoid precusors and terminate their developmental program in situ. In the fetal spleen, we observe different stages of ILC3 with precursors that are already RORgt+ but could still give rise to other ILC fate. Hence, these splenic ILC3 precursors were sorted and analyzed by microarrays. The identification of gene expression differences was used to design a single cell transcriptomic assay. The single cell transcriptomic assay is based on this specific selection of primers for transcription factors and cytokine receptors. We evaluate their differential expression in single cells at different stages of their plasticity. The aim is to decipher the progression from an ILC precursor stage to an another in one cell. We are also using the new polaris technology to detect and evaluate at different early timepoints the sequence of molecular events for changing ILC cell fate. In this case, we chose to use the sc RNAseq technology. The single cell transcriptomic will be analyzed and bioinformatic programs will be applied in order to organize the sequential molecular events and to build a hierarchical developmental model in case of ILC cell fate decisions.

Our recent analyses suggest that the genetic determinants of human neuroanatomical diversity are massively polygenic. Like other quantitative traits such as height – but also IQ or ASD risk – neuroanatomical diversity seems to result from the aggregated effect of thousands of frequent variants, each of small effect. GWAS should then require populations of hundreds of thousands of individuals to start to detect the individual variants. GCTA (genomic complex trait analysis) offers an alternative approach to obtain valuable neurogenetic information despite the current impossibility to detect enough individual variants to explaining any substantial part of the variability. We are currently pooling together neuroimaging genomics data from multiple international projects (in particular, IMAGEN, ENIGMA, UK Biobank) to replicate and extend our earlier analyses. We aim to: (1) Compute the amount of variance captured by genome-wide SNPs (SNP-heritability) for the several brain regions: ICV, BV, Hip, Th, Ca, Pa, Pu, Amy and Acc, (2) Compute the matrix of SNP-based genetic correlation among structures, (3) Partition the variance captured by SNPs among structural and functional sets: per chromosome, genic vs non-genic, low/medium/high minor-allele frequency, positive/negative selection, involved or not in neurodevelopment, etc. (4) Compare our results with those obtained using GWAS-based estimations (for example, those used in ENIGMA2). GCTA requires the computation of matrices of genetic relationship among all individuals, and thus, direct access to the genotyping data. Once the matrices are computed, the genotyping data is no longer required, and it is not possible to reconstruct an individual's genome from the matrices. Our analysis of the IMAGEN cohort was based on 1,765 Individuals, which gave us sufficient statistical power (80%) to detect only strong heritabilities (h2~45%), and the estimations had very large standard errors (~20%). A cohort of 4,000 subjects should allow us to decrease the standard error to ~8% (80% power to detect h2=22%), and a cohort of 8,000 subjects should decrease it to ~4% (80% power to detect h2=11%). In this way, we could obtain more accurate estimates, but also detect eventually more subtle effects related to functional genomic partitions.   References Yang et al (2010) Common SNPs explain a large proportion of heritability for human height. Nature Genetics, doi: 10.1038/ng.608 Davies et al (2011) Genome-wide association studies establish that human intelligence is highly heritable and polygenic. Molecular Psychiatry, doi: 10.1038/mp.2011.85 Gaugler et al (2014) Most genetic risk for autism resides with common variation. Nature Genetics, doi: 10.1038/ng.3039 Wood et al (2014) Defining the role of common variation in the genomic and biological architecture of adult human height, doi: 10.1038/ng.3097

After the behavioural characterisation of the Shank3 KO cohort, we extracted and dissected different region of the brain: cortex, hippocampus, striatum, cerebellum.

These regions were selected according to the expression level of Shank3 (Peça et al. 2011). Using QUIAGEN miRNAeasy with DNAse, miRNA-enriched RNA was extracted from the brain regions in order to perform RNAseq.

We will ask 3 questions:

• What is the pattern of Shank3 isoforms in different brain regions?

• What are the genes/pathways differentially expressed in wild-type and Shank3 knock-out mice?

• What are the genes/pathways associated with the severity of the self grooming behaviour ?

Beside data driven eperiments, we will test candidate genes/pathways such as glutamatergic receptors and <

Mycolactone, the lipid toxin produced by Mycobacterium ulcerans, has recently been shown to target the Sec61 transolcon, blocking protein translocation accross the ER membrane. As Sec61 has also been proposed as a critical mediator in cross-presentation in dendritic cells, this analysis aims to analyse mycolactone effects on the proteome of dendritic cells.

Our team is dedicated to understanding the mechanisms involved in cytokinesis. A screen has been performed and should help us better understand this process.

Lassa virus (LASV) is an arenavirus causing hemorrhagic fever in human. 300 000 to 500 000 cases of LASV infection are reported every year in western Africa, including 5 000 to 6 000 deaths. LASV is highly pathogenic, and no vaccine or treatment is available in endemic areas. LASV pathogenesis mechanisms are not well documented, and further investigations are needed to understand viral and immunological factors involved during infection. As previously shown by studies conducted on patients and non-human primates infected by LASV, type I interferon (IFN-I) is important for an effective response to LASV infection. As plasmacytoid DC are specialized in IFN-I response, they could be important for an efficient response to LASV infection.

Thanks to RNA-Seq experiments we identified  a new orphan DNA methylase (not associated with a restriction enzyme) that is highly upregulated in the presence of low concentrations of aminoglycosides in V. cholerae. The results allow us to consider a differential DNA methylation profile depending on the environment (with or without antibiotics), which can possibly correspond to a novel epigenetic control of the response.

We study the regulation of type I interferon (IFN) response in humans and in particular the functioning of a key negative feedback regulator, USP18. A recent article reported on a predicted 897nt-long LincRNA (long intergenic non coding RNA) that may target USP18. We have collected information for this LincRNA (genomic locus, putative transcript variants, sequence similarities with USP18 RNA, predicted ORFs, homologies with other genes, etc). To draw a guideline for wet lab experiments, we need to mine databases on this LincRNA. We expect to find it up-regulated in conditions of inflammation or in specific immune cell subsets, like macrophages. We wish to interrogate existing RNA-seq and Chip-seq human databases to obtain information on expression, transcriptional regulation, function, tissue-specificity or relation with pathological conditions of this LincRNA.

In recent years, large genome-wide association studies (GWAS) have been successful in identifying thousands of significant genetic associations for multiple traits and diseases1. In the course of this endeavor, sample size has proven to be the key factor for identifying new variants. For example, GWAS of body mass index (BMI), now including up to 350,000 individuals from more than 100 cohorts, have been able to identify genetic variant that explain as low as 0.02% of BMI variance2. While standard approaches for detecting new genetic variants associated with traits and diseases will go on as sample size increases, multivariate analyses have been proposed as an alternative strategy for both improving detection of new variants and exploring the multidimensional components of complex traits and diseases. Intuitively, multivariate analysis can be used to improve detection of variants displaying a pleiotropic effect3 by accumulating moderate evidence of association across multiple traits and diseases. Several recent examples have been published about not only GWAS hit overlap across related traits4, but also of genome-wide shared genetic effect5. Multivariate analyses of GWAS have also proven useful to understand shared genetics between diseases5, and potential causal relationship between phenotypes using Mendelian randomization (MR)6. Importantly, most of existing multivariate methods are based on GWAS summary statistics, while approaches based on individual-level data have been seldom considered because of major practical and ethical issues. In the continuity of ongoing work on multi-phenotype analysis (Aschard et al 20147, Aschard et al 20158), we developed an effective and robust multivariate approach of GWAS summary statistics that addresses the major barriers of existing approaches, i.e. the presence of correlation between studies that would exists when GWAS analyzed share sample9-16. Our approach consists in a robust omnibus multivariate test of GWAS summary statis

HIV infects and depletes CD4+ T cells, leading to a progressive loss of adaptive immune responses and ultimately to AIDS. In addition, HIV preferentially targets HIV-specific CD4+ T cells, resulting in an attrition of the very cells that should orchestrate the antiviral immune response. Due to this preferential depletion, HIV-specific CD4+ T cells are few and remain incompletely characterized. The emergence of single cell technologies opens the opportunity for an in depth analysis of the rare specific CD4+ T cells that persist in the circulation of chronically infected patients. We have sorted single CD4+ T cells specific for the most immunodominant epitope in HIV-1 capsid, using an optimized MHC class II tetramer labeling protocol. We now propose to analyze these single cells by multiplexed real time PCR in a microfluidics device, to define their transcriptional profile. We will analyze the differentiation status of HIV-specific CD4+ T cells in rare patients who naturally control HIV infection and in progressor patients who control HIV replication due to antiretroviral therapy. This project will help define the parameters of an efficient T cell response against HIV, and may provide insights into the type of responses that should be induced by candidate HIV vaccines.

The post-translational modification by SUMO is an essential regulatory mechanism of protein function that is involved in most challenges faced by eukaryotic cells. Gene expression is particularly regulated by sumoylation as many SUMO substrates are transcription factors and chromatin-associated proteins, including histones. The emerging paradigm for the proposed work is that sumoylation controls multiple aspects of chromatin structure and function in response to external cues. According to this view, sumoylation is expected to impact both global and specific transcriptional programs thereby affecting constitutive and inducible expression of both coding and non coding genes. Recently, we found SUMO as an integral and instructive component of chromatin in cell growth and senescence, thus establishing sumoylation as a new and paradigmatic chromatin modification. This work now paves the way for detailed understanding of the contribution of SUMO as a multifaceted modifier of chromatin.

Despite effective prevention against HBV infection, 300 million people worldwide are chronic HBV carriers, of whom 25% will die of liver cirrhosis or hepatocellular carcinoma (HCC). Current treatments for chronic hepatitis B (CHB) are inefficient to completely clear the virus and liver cancer is a lethal disease, thus representing an area of highly unmet medical need. Viral persistence is due to the maintenance, in the nuclei of infected cells, of the viral nuclear DNA : the cccDNA that is not targeted by the antiviral treatment and to the impairment of both the innate and adaptive immune responses that accompanies CHB infection. Viral replication depends on a balance between factors that benefit and those that restrict viral infection. However little is known about cellular factors that repress HBV replication. Using quantitative temporal viromics approach developed by P. Lenher in Cambridge we have performed a quantitative analysis of temporal changes in host and viral proteins in primary human hepatocytes through the course of HBV infection. We will in particular search for cellular factors involved in virus restriction and uncover the mechanism of viral escape.

Background: Dyslexia is characterized by difficulty with learning to read fluently and with accurate comprehension despite normal intelligence. It affects 5–10% of school-age children. Familial studies repeatedly showed that first-degree relatives of affected individuals have a 30–50% risk of developing the disorder. Twin studies showed that heritability was approximately 50% with a higher concordance rate for monozygotic twins compared to dizygotic twins. Although genetic factors contribute to dyslexia, very little is known on the genes associated with the condition. Preliminary data: Our project consists in the complementary analysis of (i) a cohort of 209 patients with dyslexia, 89 relatives and 95 very well phenotyped controls and (ii) an extended pedigree (Nantaise family) with 12 members diagnosed with dyslexia in three generations. For all the individuals of the project, we genotyped >600K SNPs in order to detect SNP association and copy-number variants (CNVs). For the extended pedigree, we also used linkage analysis and whole genome sequence (WGS). Our preliminary results indicate that a single region on chromosome 7q36 is segregating with dyslexia in the Nantaise family. The region is located within CNTNAP2, a gene previously proposed as a susceptibility gene, but without formal proof of its association. The WGS data of three affected and three unaffected individuals of the pedigree was performed to detect all the variants in the linkage region. Project: We proposed to use this unique resource in France to characterize the genetic profile of patients with dyslexia. We will (i) detect the CNVs present in the patients and (ii) detect the variants in the linkage region.

Mood disorders such as bipolar and major depressive disorders affects 2-5% of the population worldwide. They are relapsing, major psychiatric illnesses with poorly understood neurobiology, typically incomplete treatment responses, and often unsatisfactory clinical outcomes, with a high risk of premature mortality due to suicide and clinical comorbidities. Its complex manifestations include marked disturbances of emotional regulation, sensory-perception as well as major changes in mood, thinking, and many aspects of behavior. It is of crucial importance to be able to discriminate mood states in clinical practice, as pharmacological and psychological treatments as well as course of illness and outcome differ across the different mood states. By applying advanced mathematical approaches (with statistical/computational advice from a C3BI’s colleagues) this study aims to explore how sociodemographic, psychosocial and behavioral predictors may contribute for better characterizing patients with mood disorders. The study also aims to identify biological predictors of different mood states as well as of treatment response in mood disorders. Having a deeper understanding of phenomenology and pathophysiological mechanisms of mood disorders may help clinicians to provide more personalized interventions for individuals at-risk or suffering from mood disorders.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB), is the deadliest disease due to a single infectious agent. Despite considerable efforts to fight the disease, TB remains a major public health problem. Even more worrying for the future, multidrug resistant (MDR) strains of MTB are continually emerging and about 10% of people with MDR-TB have extensively drug-resistant TB (XDR-TB). Drug-sensitive TB can be cured by a 6-month treatment using 4 antibiotics, but MDR-TB and extensively drug-resistant XDR-TB require treatment for up to 2 years with more toxic and costly second- and third-line drugs. Toxicity of these drugs is well described; it includes hepatotoxicity, liver injury, skin reactions, gastrointestinal and neurological disorders. However how MTB drugs influence the host response to MTB infection has been poorly addressed. The main goal of project is to understand how drugs interact with the host in order to improve the treatment.

Innate lymphoid cells (ILCs) are the most recently identified components of the innate immune system. ILCs colonize different tissue sites and react promptly to microenvironmental perturbations. Due to their high plasticity, ILCs can shape their functional output in response to local cues. As such, ILCs play roles under homeostatic conditions and in the context of infection, chronic inflammation, metabolic diseases and cancer. Diverse ILC subsets (NK cells, ILC2) have been shown to regulate the metabolic homeostasis. Metabolic states affect cellular functions and have been shown to play an important role in the regulation of adaptive immunity. In contrast, almost nothing is known about innate lymphocytes metabolism and the importance of energy regulation for ILC function. This project will study metabolic profiles in human ILC subsets under diverse environmental conditions. Enhancing or interfering with ILC activity could ultimately represent a novel useful therapy for chronic inflammatory diseases.

Autism Spectrum Disorders (ASD) are heterogeneous neurodevelopmental disorders with a complex genetic architecture. They are characterized by impaired social communication, stereotyped behaviors and restricted interests and are frequently associated with comorbidities such as intellectual disability, epilepsy and severe sleep disorders. Hyperserotonemia and low melatonin levels are among the most replicated endophenotypes reported in ASD, but the genetic causes of such alterations remain largely unknown. Based on the biochemical profile of 717 individuals including 213 children with ASD, 128 unaffected siblings and 376 parents and other relatives, we aim to estimate the heritability of five quantitative traits associated with the melatonin synthesis pathway: whole-blood serotonin, platelet N-acetylserotonin (NAS) and plasma melatonin levels, as well as the two enzymes AANAT and ASMT activity measured in platelets.

Chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, spondyloarthritis (SpA) and psoriasis cause significant morbidity and are a substantial burden for the affected individuals and the society. An important obstacle to early diagnosis and the development of more specific and effective therapies is the very limited understanding of the pathogenesis of these diseases. In the past years genome-wide association studies have identified many genes that were not known to be involved in pathogenesis, and have linked several genes in immune pathways to inflammatory diseases, indicating that the immune system plays an essential role in the pathogenesis of these diseases. The current challenge is to correlate these genetic variants with the effector mechanisms implicated in pathogenesis, to allow translation of the genetic data into relevant diagnostics and innovative treatment strategies. To meet this challenge, we have designed a clinical study that examines the immune signaling pathways, the transcriptional networks and the genotype in the same SpA patient, in order to establish a link between genetic variation, cellular phenotype and function, and pathology. This approach will advance our understanding of the pathogenic mechanisms, and identify novel and relevant diagnostic tools, therapeutic targets and biomarkers. The long-term outcome of this strategy will be the rational design of specific therapies tailored to the genotype of the patient.

The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections, including Influenza, HIV and Zika viruses, by inhibiting virus-cell fusion. We showed that IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses. We also reported that amino-terminal mutants of IFITM3 preventing ubiquitination or endocytosis are more abundantly incorporated into virions and exhibit enhanced inhibition of HIV-1 fusion. An analysis of primate genomes revealed that IFITM3 is the most ancient antiviral family member of the IFITM locus and has undergone a repeated duplication in independent host lineages. Some IFITM3 genes in nonhuman primates, including those that arose following gene duplication, carry amino-terminal mutations that modify protein localization and function. Our aim is to analyze the RNA levels of the various members of the IFITM family, in various normal or pathological human or animal tissues.

Freezing is the most commonly used method for storing DNA extracts. However, that method is non-practical and expensive, since requiring freezers and back-up generators for storage, and specific conditions/reagents for transport. In addition, even when adequate procedures are followed, the frozen extracts integrity might suffer from repeated freeze-thaw cycles or residual microorganism activity. The Institut Pasteur’s ICAReB platform hosts the biological collection related to the CoSImmGEn cohorts (Cohort and Collection to Study the Immune System with its Genetic and Environmental determinants) since 2011 (see related team publication 1). Those cohorts have been designed for providing large, duly annotated, qualified blood-derived bio-resources such as blood peripheral mononuclear cells, DNA and RNA from healthy subjects or cases suffering from diseases such as Hidradenitis Suppurativa to support genetic studies linked to the immune system (see related team publication 2 et 3). To provide over the long term genomic DNA for any kind of future genetic studies searching for immune system etio-pathogenesis, the ICAReB platform has used a newly developed DNAshells® (Imagene) which ensure nondestructive, reliable and long-term stability of DNA at low-cost (Clermont et coll, Biopreserv Biobank, 2014). That technology involves encapsulation of the genomic material such that it can be stored dry at room temperature, in small, watertight, oxidation-proof metal capsules. The first aim of the present project is to determine if SNP genotyping allows the detection of DNA damage during storage in various conditions. The second aim of the project is to demonstrate that encapsulation allows an optimal storage of human blood derived DNA at room temperature.

Non human primates are an important reservoir for zoonotic disease. Here we analyze in Cameroon how human activities in the forest influence contact with non human primates to better understand processes of emergence.

Chronic inflammatory systemic diseases (CIDs) are a burden to humans because of life-long debilitating illness, increased mortality and high therapy costs. CIDs’ increasing prevalence in western countries has indeed placed them at the third rank of morbi-mortality causes. Unfortunately, available treatments are poorly targeted and non-curative. That is partly linked to a complex and largely ununderstood pathophysiology. Genetic susceptibility clearly plays a role. Genes linked to the immune system have been identified, but causal genes remain mostly unknown and other factors such as intestinal microbiota have also been implicated. The complexity of CIDs’ pathophysiology suggests that a holistic approach is the most susceptible to help make significant progress. Our project intends to take advantage of recent technical progress and development of informatics tools to set up a transversal approach. High-resolution sequencing technology indeed quickly produces large amounts of accurate data. Besides, new integrative informatics tools allowing storage and integrative analysis of this resulting high amount of data are now available. We intend to set-up a CID’s network allowing the gathering and extensive analysis of data related to immuno-genetic determinants, immune repertoire and microbiota from individuals suffering from one of the three major interlinked CIDs, namely Hidradenitis Suppurativa (HS), Crohn’s disease (CD) and Spondyloarthropathy (SpA) as compared to healthy volunteers.

It has been shown that methylation can act as a kind of memory of the immune system. For patients with co-infections, it is of particular importance to know when to begin an anti-retroviral therapy, especially if they are already infected with tuberculosis. The goal of this study is to find hyper or hypo methylated loci related to the reaction of HIV patients (co-infected or not) to different kind of treatments.

The human gene ANOS1, responsible for the X-chromosome-linked form of Kallmann syndrome (a developmental disease affecting the olfactory system), has been identified in 1991 by positional cloning. It is located on the X chromosome short arm (at Xp22.3), close to the STS gene and close to the boundary of the pseudoautosomal region (common to the X and Y sex chromosomes). Since then, orthologous genes have been identified in all animal species (including invertebrates), except in the mouse, rat, and other rodents (an orthologue in the naked mole-rat Heterocephalus glaber is however present in the GeneBank data base). The orthologous STS in the mouse has been identified in the mouse, and is located in the XY pseudoautosomal region in this species. The sequence of the mouse STS is unusually GC-rich and has markedly diverged from the human orthologous sequence, even though the amino acid sequence of the protein is highly conserved. The ANOS1 orthologous genes have not yet been identified in the mouse/rat, although we have been able to detect the encoded protein anosmin-1 in these species, with antibodies directed against the human protein (the orthologous proteins in the mouse and rat have the expected size in western blot analysis, i.e. about 690 amino-acids).

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by a biallelic mutation of the Ataxia Telangiectasia Mutated (ATM) gene. The ATM protein is involved in a very large number of cellular functions (over 700 substrates), mainly DNA repair, cell cycle regulation, mitochondrial and oxidative metabolism, as well as regulation of gene expression. The majority of hematological malignancies occurring in AT patients are non-Hodgkin's B lymphomas and Hodgkin's lymphomas. The association with the Epstein-Barr virus (EBV) is found in 50 to 100% of these cases according to the histological subtype. EBV is a widespread virus in human populations with a prevalence greater than 90% and is associated with the development of lymphomas in immunocompromised patients. We will study the transcriptome of EBV-infected cells in ATM patients, in order to determine the impact of ATM on EBV infection control.

Mood disorders such as bipolar and major depressive illnesses are among the most severe psychiatric disorders. They have high prevalence and chronic course, and are associated with significant mental and somatic comorbidities and high personal and societal costs (lost productivity and increased medical expenses). Patients with bipolar disorder (BD), for example, exhibit a reduced lifespan compared with the general population, a finding that cannot only be explained by high suicide risk, reduced access to medical care and lifestyle factors. However, the pathophysiological mechanisms of BD are poorly understood, and patients often have incomplete treatment response. Advanced mathematical approaches such as machine learning techniques are increasingly being used to generate predictions based on complex data, and it has been successfully used to detect a number of clinical outcomes and to predict behaviours. In combination with mobile technologies (e.g. smartphones, wearables) to collect behavioural, physiological and environmental data, these big data predictive approaches may provide a much richer and deeper understanding of phenomenology and pathophysiological mechanisms of mood and bipolar disorders. By taking advantage of the high-standard bioinformatics expertise offered by the C3BI, this multidisciplinary, collaborative project aims to explore how clinical and biological factors, may contribute for better characterizing BD patients as well as to identify predictors of treatment response in BD. Our project also aims to explore how daily behavioural and physiological parameters may influence mood and behaviour in individuals at-risk or suffering from mood disorders.

Hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematologic malignancies. The main therapeutic benefit derives both from the ability to treat patients with intensive chemotherapy and from a potent graft-versus-leukemia (GVL) effect mediated by donor T lymphocytes. Unfortunately, in some patients, donor T cells also attack host normal tissues, giving rise to graft-versus-host disease (GVHD). GVHD prevalence is between 40-80% depending on patient and transplantation characteristics and GVHD remains the main cause of non-relapse morbidity and mortality. Despite the advances in the field of HSCT and GVHD prophylaxis, disease processes in humans remain poorly understood, and the lack of biomarkers for the early diagnosis and prognosis of GVHD contributes to the high mortality of the disease. The objective of the study is to investigate the cellular and molecular mechanisms involved in the immune reconstitution after transplantation and to explore the mechanisms of acute GVHD. For three independent cohorts of donor-recipient pairs, blood samples were collected from the all the donors before transplantation and for the respective recipients either at GVHD onset or at the Day 30 or Day 90 for recipients that did not develop GVHD. Donors and recipients’ samples were analyzed using different approaches: spectral flow cytometry to investigate the cellular correlates of immune reconstitution after HSCT and of GVHD onset, gene expression analysis by NanoString technology to assess the molecular profile of immune cell populations important for GVHD development (CD4+ T cells, CD8+ T cells, NK cells and monocytes) as well as a metabolomics profiling of serum samples using mass spectrometry.

Cellular senescence is a complex stress response that durable (yet not irreversibly) arrests cell proliferation and is accompanied by widespread changes in chromatin structure, metabolism and gene exp

The central part of the intercellular bridge connecting the two daughter cells during cytokinesis is a highly dense structure named the Midbody first described by Flemming in 1891. Work in the past te

Anti-TNF therapy has revolutionized treatment of many chronic inflammatory diseases, including rheumatoid arthritis, Crohn’s disease and spondyloarthritis (SpA). However, clinical efficacy of TNF-inhi

A protein-protein interaction screen has been done beween viral proteins of influenza A viruses and a library of about 100 human factors invovled in RNA processing through RNA exonucleases activity. 2

Identification of alternative 5'UTR in an eukaryotic transcripts

Mitochondria are double-membrane bound organelles that are essential in every tissue of the body. They are metabolic hubs and signalling platforms that are deeply integrated into cellular homeostasis.

Our goal is to have a bioinformatic tool that performs the 2 x 2 comparison of matrices of numbers in matrix batches. Each matrix corresponds to the use of gene fragments (V or J) to create a re-arran

During infection with bacteria, secretion of toxin induces histone modifications in the host cell. One such modification is dephosphorylation of histone H3 on serine 10. We have characterized this mod

DNA Topoisomerase 1 (Top1) is a major regulator of gene expression with great impact on genome stability. Similarly, Guanine quadruplexes (G4) have recently emerged as critical elements for transcript

This project is integrated in the analysis of the gut ecosystem of children implicated in the AFRIBIOTA project, a translational project performed within a consortium of researchers and medical doctor

The SRC Kinase Adaptor Phosphoprotein 2 (SKAP2), a broadly expressed protein with some higher expression in haematological lineages recruiting protein partners to specific subcellular domains, plays a

A number of mammalian cell types are susceptible to Salmonella Typhimurium infection, including epithelial cells, fibroblasts and macrophages. It has recently been demonstrated that Salmonella display

Les modifications de la chromatine, au niveau de l’ADN ou des histones, jouent un rôle fondamental dans la régulation de l’expression des gènes chez les eucaryotes, en contrôlant l’accès de la machine

Cocaine is the most widely used illicit stimulant in Europe1, with a recent increase in use in the French general population. Cocaine addiction (CocAdd) is recognised as a public health priority world

Globally one out of four children under 5 years is affected by linear growth delay (stunting). This syndrome has severe long-term sequelae including increased risk of illness and mortality and delayed

Streptococcus gallolyticus sous espèce gallolyticus, autrefois dénommée Streptococcus bovis biotype I, est une bactérie de la flore intestinale qui constitue une cause émergente de septicémies et d’en

Disruption of GABAergic inhibitory circuits is one of the common alteration responsible for several psychiatric developmental disorders. Gephyrin (GPHN) is the common and main molecular organizer of i

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription regulator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level. In addition, structural parameters of the target DNA helix (curvature, flexibility and topology) are proposed to regulate IN selectivity at a local level. Our team is studying the role and molecular mechanisms associated with these various parameters (Botbol et al., 2008; Lesbats et al., 2011; Morchikh et al., 2013; Benleulmi et al., 2015; Naughtin et al.,). This project aims to define the role of cellular DNA topology during HIV-1 integration. We will first compare already mapped integration sites and superhelicity profiles and search for possible correlations between these two parameters. We will then modify topoisomerases activity in infected cells and study the consequences on viral replication and integration. Finally, we will study in vitro, the direct effects on integration of two parameters of DNA topology, the twist and writhe of the DNA helix. This project relies on complementary in vivo, in vitro and in silico approaches. Bio-informatics tools are crucial for the correlative and statistical analyses of integration sites and superhelicity maps.

HIV-1 replication requires the integration of the viral genome into the cell genome. A viral-encoded enzyme, integrase (IN), performs this critical step of infection and is a promising target for anti-viral therapeutics. If the catalytic properties of INs are well characterized, the mechanisms responsible for their site selectivity are still under investigation. Several cellular proteins, such as the LEDFGF/p75 transcription co-activator, the RNA polymerase II machinery, nuclear pore proteins and specific modified histones have been proposed to be involved in IN selectivity at a genomic level but the underlying molecular mechanisms remain to be demonstrated. In addition, structural parameters of the target DNA helix (curvature, flexibility, topology) are proposed to regulate IN selectivity at a local level. Our aims are to study the role of these different parameters of IN selectivity, using both in vitro and in vivo approaches. In vitro, we will map integration sites on various target DNA substrates (naked DNA or chromatin, minicircles, plasmids with different topologies, transcribed templates) and will test the effect of purified proteins suspected to regulate IN selectivity. In vivo, integration sites will be mapped in cells depleted of these suspected regulators or in cells incubated with drugs targeting enzymes involved in transcription, DNA topology or histone modifications. Integration sites will be mapped using published or “home-made” protocols and the sites will be compared with DNA structural parameters, nucleosome positions, histone modifications or transcriptional parameters (published maps). Bio-informatics tools are crucial for these correlative and statistical analyses of integration sites. Our project relies on complementary in vivo, in vitro and in silico approaches. It should establish molecular and mechanistic rules of HIV-1 integration selectivity that could serve in the development of new antiviral strategies and of safer gene therapy vectors.

Controlling virus replication by generating a strong CD8+ T cell response against HIV is one of the major goals in the development of an effective HIV vaccine candidate. Indeed, during the chronic phase of infection, HIV-specific CD8+ T cells were shown to have impaired functionality and fail to control viral replication. Understanding the profile of CD8+ T cells able to efficiently tackle HIV is therefore much needed. HIV controllers (HIC) are individuals who control viremia without antiretroviral therapy. CD8+ T cells seems to play a major role in their HIV control. We hypothesized that HIV-specific CD8+ T cells associated with control of infection bear particular transcriptional signature (cell cycle, survival, activation, cytolytic function…) when compared to HIV-specific CD8+ T cells not associated with control of infection sharing the same memory phenotype.

The objective of this study is to evaluate how the different programming of distinct CD4+ T cell subsets affected their susceptibility to HIV infection and the survival of infected cells. Dr. Saez-Cirion's Team evaluates how the metabolic state of CD4⁺ T cells and the regulation of cellular factors shape the distribution of the HIV reservoir in distinct CD4⁺ T cell subset. This project uses new technologies and reagents to analyze cells from uninfected donors, and from HIV patients in acute infection, chronic infection and HIV remission.

Les mutations de résistance aux traitements apparaissent sous l’effet de la sélection. Ces mutations sont transmises, les patients pouvant être infectés par des souches déjà résistantes à certains traitements. Ces mutations posent des problèmes considérables en limitant l’arsenal des traitements disponibles, pour les individus comme pour la population sur le long cours. Dans le cas du VIH, nous avons travaillé sur la transmission de ces mutations au sein de la population anglaise, et montré que la majorité de celles-ci étaient transmises par des patients naïfs ignorant leur infection (Mourad et al. AIDS 2015). La méthode employée était souvent ad-hoc et n’utilisait qu’une partie des données disponibles. L’objectif de ce projet est de mettre en place des modèles rigoureux et des méthodes efficaces d’estimation des paramètres (taux de transmission suivant les caractéristiques du donneur, taux de réversion, etc.).

We would like to be able to use IgBlast on the Galaxy platform. We are studying B cells in adaptive immune response, and are particularly interested in the antibodies termed as broadly neutralizing antibodies (bNAbs). By definition, these antibodies can neutralize most known HIV-1 strains, and are produced by rare infected individuals several years post-infection. We are currently investigating the bNabs immunoglobulin repertoire by focusing our NGS (454 pyrosequencing) analysis on  immunoglobulin sequences (V-domains) from HIV-infected patients who developed bNAbs. As immunoglobulin sequences result from the combinatorial rearrangement of 3 gene segments : V , (D) and J gene segments, we need a specific tool to analyze these sequences. Indentifying the germline genes which are involved in the rearrangment is an essential step. Two main tools are being widely used to analyze Immunoglobulins: IMGT and IgBlast. IgBlast has several advantages; it is based on BLAST (it is then possible for the user to build his own database), open source, can use protein or nucleotide sequences as input, and most of all, IgBlast is already installed on the Institut Pasteur's cluster as well as the germline genes database. As it would be very convenient for us to use the bic cluster and galaxy platform to run our analyzes, we would be grateful if IgBlast could be implemented in the Pasteur Galaxy Platform. In this regard, we are of course fully disposed to help in any ways. We also believe that it would be very useful to people working on immunoglobulin sequences in the immunology department by building specific pipelines. Thank you very much.

Integration of the viral reverse-transcribed genome into the genome of infected cells is an essential step of retroviral replication and is performed by a viral-encoded enzyme, named integrase (IN). In the case of HIV-1, IN is a new and efficient anti-viral target. The selectivity of this enzyme for its cellular genomic sites is also a major parameter of HIV replication and is regulated by several cellular parameters. One of them is chromatin, and different levels of this nucleoprotein complex are involved in the regulation of IN selectivity. Using in vitro integration assays, established by our team and collaborators, we have studied this regulation at two levels of chromatin architecture: large poly-nucleosome templates (Botbol et al., 2008; Lesbats et al., 2011; Benleulmi et al., 2015; Naughtin et al., 2015) or nucleosome-induced DNA curvature mimicked by DNA minicircles (Pasi et al., 2016). Our present project is to study IN selectivity into mononucleosomes (MN). These MNs will be used as target substrates of integration and the role of MN structure, histone modifications and IN cofactors will be studied. Results obtained in vitro, will be confronted to structural data obtained by molecular modeling and to integration sites observed in infected cells. This project will benefit from our expertise in integration in chromatin templates and a previous collaboration with the C3BI on the analysis of integration sites (Pasi, M., Mornico, D., S. Volant, S., et al., 2016). This project is funded by the ANRS.

We previously showed that humanized immune system (HIS) mice generated in Balb/c Rag2^-/-γ_c^-/- SIRP^NOD (BRGS) recipients are susceptible to HIV-1 infection (X4 and R5 isolates) and maintain circulating HIV-1 in the plasma, resulting in a dramatic depletion of human CD4⁺ T cells. We also characterized features of HIV physiopathology in this model. Human thymocyte subsets developing in the thymus of HIS mice appear phenotypically normal, but in the periphery the T cell repertoire is restricted compared with that of human peripheral blood T cells. This negatively impacts on the ability of HIS mice to generate antigen-specific human immune responses when mice are vaccinated with protein antigens or following infection with lymphotropic viruses such as HIV. One likely explanation for these functional deficiencies involves the fact that human T cells are selected intrathymically by mouse MHC molecules and that naïve T cells in peripheral lymphoid organs interact primarily with mouse DC (as human DC development in HIS mice is limited). As a first line of improvement, we recently generated a novel mouse model by crossing our BRGS mice with the HLA-A*02-HHD class I transgenic mice and the HLA-DRB1*15 class II transgenic mice, resulting in BRGS-A2DR2 mice. Following intra-hepatic injection of these mice with MHC-matched CD34⁺ stem cells we observed increased engraftment, with faster kinetics. Moreover BRGS-A2DR2 HIS mice have an increased T cell development leading to a more equilibrated B/T and CD4/CD8 phenotype. We showed that BRGS-A2DR2 HIS mice were able to sustain replication of HIV R5 virus as the BRGS hosts. Viremia was similar in a first phase and then lower in a second phase in BRGS-A2DR2 compared to BRGS HIS mice, which could be a consequence of a better quality of the immune response. However, the viremia reached a similar plateau in the last phase. We propose to study the impact of the immune res

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants underlying this response, we characterized the TCR repertoire directed at the most immunodominant CD4 epitope in HIV-1 capsid, Gag293. HIV Controllers from the ANRS CO21 CODEX cohort showed a highly skewed TCR repertoire characterized by a predominance of the TRAV24 and TRBV2 variable gene families. Controllers shared public clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control (Benati D. et al., J Clin Invest 2016).   We propose to test the generality of these findings by characterizing the TCRs specific for a series of immunodominant HIV Gag and Env epitopes, and comparing the frequencies of public clonotypes in groups of HIV Controllers and treated patients. We will then assay the functions of the most prevalent public clonotypes through lentivector-based TCR transfer, and correlate the panel of T cell functions to TCR affinity and frequency.

HIV infects and depletes CD4+ T cells, leading to a progressive loss of adaptive immune responses and ultimately to AIDS. In addition, HIV preferentially targets HIV-specific CD4+ T cells, resulting in an attrition of the very cells that should orchestrate the antiviral immune response. Due to this preferential depletion, HIV-specific CD4+ T cells are few and remain incompletely characterized. The emergence of single cell technologies opens the opportunity for an in depth analysis of the rare specific CD4+ T cells that persist in the circulation of chronically infected patients. We have sorted single CD4+ T cells specific for the most immunodominant epitope in HIV-1 capsid, using an optimized MHC class II tetramer labeling protocol. We now propose to analyze these single cells by multiplexed real time PCR in a microfluidics device, to define their transcriptional profile. We will analyze the differentiation status of HIV-specific CD4+ T cells in rare patients who naturally control HIV infection and in progressor patients who control HIV replication due to antiretroviral therapy. This project will help define the parameters of an efficient T cell response against HIV, and may provide insights into the type of responses that should be induced by candidate HIV vaccines.

Molecular markers of cervix lesions linked to HPV infections.

Because of their increasing incidence, dramatic severity, lack of treatment or vaccine, complicated diagnosis, misreading of the pathogenesis, and need for a maximum containment, Viral Hemorrhagic Fevers (VHF) constitute a major public health problem. There is therefore an urgent need to further study VHF to understand the pathogenesis of the severe disease and the host responses involved in their control or in the dramatic damages. Among VHF, Lassa fever (LF) is probably the most worrying one because of its endemicity and the large number of cases. LF is caused by the Old-World arenavirus Lassa virus (LASV). It is endemic to West Africa and is responsible for 300,000 cases and 5,000 to 6,000 deaths each year. We propose here to study the pathogenesis of VHF by using LF in cynomolgus monkeys as a paradigm, with a particular emphasis on the very early events. The viral tropism, pathophysiological mechanisms, and immune responses will be studied during the course of infection, including the incubation period. Powerful approaches will be used to (1) identify early biological markers of infection, to be able to confirm infection and isolate patients; (2) determine the viral tropism and dynamics during the course of infection to understand the natural history of virus into its host. (3) characterize the early pathogenic events that lead to the severe hemorrhagic syndrome to fully understand the pathophysiogenesis of VHF and identify new therapeutic targets. (4) identify the immune responses involved in the control of infection or in the fatal outcome, to reveal the involvement of immunopathological mechanisms and help to design a vaccine approach. This ambitious and unprecedented project will allow to develop therapeutic and prophylactic approaches but also to identify early biological markers of infection and improve the early diagnosis to optimize the management of outbreaks in the field and increase the survival rate in patients.

Non human primates are an important reservoir for zoonotic disease. Here we analyze in Cameroon how human activities in the forest influence contact with non human primates to better understand processes of emergence.