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 expression including the production and secretion of a plethora of inflammatory factors. Cellular senescence plays beneficial roles during embryonic development, tissue regeneration, and tumor suppression. Paradoxically, it is also considered a major contributor to aging and age-related diseases, the latter mostly through its inflammatory phenotype, the so-called SASP (senescence-associated secretory phenotype). The proposed work aims at integrating time-resolved transcriptome, ChIP-seq, and ATAC-seq datasets into a comprehensive understanding of senescence-associated gene regulation.

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 ten years revealed that the midbody is a platform that concentrates essential proteins involved in cytokinetic abscission. After abscission, the midbody is cut on both sides, thus generating a midbody remnant (named MBR). The MBR usually interacts with the cell surface of one of the two daughter cells, before being engulfed in a phagocytic-like manner. We also found that the MBR can be easily released from cells before their engulfment by calcium chelation. Of note, MBRs at the cell surface might act as pro-proliferative, signalling entities but the proteins involved and the mechanisms of MBR anchoring are unknown. A previous proteomic study of the midbody conducted by Skop purified intercellular bridges from cell lysates recovered after cell synchronization, microtubule stabilization and detergent treatment. This pioneer proteomic study, although informative, did not allow the recovery of many key known proteins of the midbody. Here, we set up an experimental protocol to purify intact, detergent-free MBRs in order to have the full proteome of this organelle. Quantitative, label-free proteomics enabled us to identify 529 proteins enriched at least 2 times as compared to whole cell lysates, that we named the “Flemmingsome”. Besides known and well-established proteins of the midbody (MKLP1, MgcRacGAP, AuroraB, INCENP, MKLP2, Rab8, Rab11, Rab35, Citron Kinase, ESCRTs…), we identified new and promising candidates potentially involved in cytokinetic abscission. In addition, we identified 27 transmembrane proteins that are excellent candidates for mediating interactions between the MBR and the receiving daughter cells after cytokinetic abscission. We are also currently exploring whether newly identified candidates could participate in the signalling mediated by the MBRs. We would thus like to create a website that recapitulates the findings of our screen. The proteins discovered represent new candidates for the understanding of cytokinesis and tumorigenesis. This should be instrumental in the field as the previous websites are not updated (Microkits, Uniprot) and do not focus on this particular step of cytokinesis.

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-inhibitors (TNFi) is limited by a high rate of non-responsiveness (30-40%) both in SpA and other diseases, exposing a substantial fraction of patients to important side-effects without any clinical benefit. Despite the extensive use of TNFi since many years, it is still not possible to determine which patients will respond to TNFi before treatment initiation. In this study, we have tested the hypothesis that the functional analysis of immune responses may not only improve our understanding of the molecular mechanisms of TNF-blocker activity, but also identify correlates of therapeutic responses in SpA patients. To facilitate the potential translation of our findings into a clinical setting, we have used standardized whole-blood stimulation assays (“TruCulture” assays, Duffy et al., Immunity 2014), and have minimized sources of pre-analytical variability, implementing a highly sensitive and robust pipeline to assess immune functions in patients. To investigate the concept that the immune status of a patient will define their response to TNFi treatment, we have used machine-learning algorithms to identify, in whole-blood stimulation assays, immunological transcripts that correlate with clinical efficacy of TNFi. Our results obtained with a cohort of 67 SpA patients demonstrate that high expression, before treatment initiation, of molecules associated with leukocyte invasion/migration and inflammatory processes predisposes to favorable outcome of anti-TNF therapy, while high-level expression of cytotoxic molecules was associated with poor therapeutic responses to TNF-blockers. These findings may suggest that SpA patients whose immune response is characterized by strong, NF-kB-mediated inflammation are more likely to benefit from TNFi treatment than patients with an active T/NK-cell component. Unfortunately our manuscript describing these results has been rejected by Nature Medicine. However, in her letter the editor mentioned, “Should future experimental data allow you to demonstrate that the identified gene signatures predict response to treatment and outperform previously reported approaches in an independent cohort, we would be happy to look at a new submission…”. We have recruited additional SpA patients over the summer and we are currently in the process of performing the gene expression analysis. The goal of this bioinformatic analysis will be to identify transcripts in stimulated immune cells that predict therapeutic outcome in a training set of patients using machine-learning algorithms and validate the findings in a replication cohort.

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. 20 factors were identified as interactors of viral proteins. To see if this targeting corresponds to specific RNA metabolism focntions of the cell, we analysed using Cytoscape the first neighbors of these factors in the human ORFeome. We then compared using the ClueGo pluging the functional enrichment of the factors targeted by viral factors and the functional enrichment of the non-interacting subset. We need now a statistical analysis of the results the functional enrichment comparison, which is required for further progress of the ongoing research project.

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. The functions of mitochondria are intimately linked to their form, which is regulated by a balance of membrane fusion and fission: dynamin-like GTPases OPA1 and MFN1/2 perform membrane fusion and DRP1 regulates membrane fission. Mutations in mitochondrial genes cause a pleiotropic spectrum of clinical disorders whose underlying genetic, morphological and biochemical defects can be easily studied in skin fibroblasts generated from patient biopsies. The morphology of mitochondria is inextricably linked to its many essential functions in the cell and we are interested in understanding the relationship between mitochondrial shape changes and metabolism in the context of acquired and inborn human diseases. Balanced fusion and fission events shape mitochondria to meet metabolic demands and to ensure removal of damaged organelles. Mitochondrial fragmentation occurs in response to nutrient excess and cellular dysfunction and has been observed in mitochondrial genetic diseases and is thought to play an important role in the development of disease. The physiological relevance of mitochondrial morphology and the mechanisms that regulate mitochondrial dynamics are incomplete and so we have set out to find ways to rebalance mitochondrial dynamics in genetic diseases. We recently developed imaging and informatics pipelines to allow for the automated, rapid, reliable quantification of mitochondrial morphology in human fibroblasts. We applied this new technology in the context of genome-wide siRNA screens in immortalized fibroblasts from an OPA1 patient with dominant optic atrophy and control fibroblasts to identify candidate genes able to reverse the mitochondrial fragmentation phenotype associated with mitochondrial dysfunction in patient cells. We have performed the same genome-wide screen in healthy immortalized control fibroblasts. Together, these studies will help us identify lists of genetic modifiers and therapeutic targets that can be investigated further using cell biology and biochemical tools in the lab.

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-arranged gene (VJ) encoding a specific antibody, identified by droplet microfluidic or flow cytometry techniques followed by high throughput sequencing (NGS). ). The matrices are made for the two chains encoding the specificity of an antibody: the heavy chain (VH) or the light chain (VL). The goal is to compare VH matrices one against another, and VL matrices one against another for antibody gene rearrangements in mice, llama, and humans.

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 modification and the bacterial factors that are important for inducing it. The bacterial toxin listeriolysin O is one such factor. However the role of this modification is unknown. Genome wide analysis of the genes or genetic regions marked with dephosphorylated H3 will help us reveal the function of this modification during infection.

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 transcription regulation and potential threats to the genome integrity. Studies of Top1 and G4 have many medical implications since their mutations or deregulations are associated with several pathologies, such as cancers, neurodegenerative diseases or viral infections. A few studies, including ours, have revealed physical and functional interactions between these two elements and their involvement in transcriptional regulation of viral and cellular genes. Our research hypothesis is that this Top1-G4 interaction is involved in a new mechanism of transcriptional regulation. This project aims to test this hypothesis and to determine, genome-wide, the contribution of this interaction on human gene transcriptional regulation. This will be tested using genomic and transcriptomic data obtained on the Top1-dependent transcriptional regulation of cellular genes (RNA-seq data), on the localization of Top1 along the cellular genome (ChIP data) and on the presence of G4 structures at specific foci of this genome (G4Hunter prediction and ChIP data). Results obtained by this bio-informatics analyses, should help us to design new experiments to test our hypothesis, on both retroviral and cellular promoters. In long-term perspectives, this project should reveal new mechanisms of gene transcriptional regulation that could serve for the development of new therapeutic strategies.

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 doctors from the Central African Republic, Madagascar, France and Canada (see https://research.pasteur.fr/fr/program_project/the-afribiota-project/). AFRIBIOTA aims at understanding the risk factors and pathophysiological changes underlying chronic child malnutrition as manifested through delayed growth. Within the project submitted, we aim at assessing a possible link between asymptomatic pathogen carriage, gut inflammation and stunted growth of children included in the study site in Antananarivo, Madagascar.

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 central role in multiple physiological processes in relation with cell migration. We have linked this gene in interaction with CNTNAP5 to susceptibility to Dengue Shock Syndrome in a cohort of Vietnamese patients. By coupling a yeast-two-hybrid screen with a luciferase complementation assay and mutagenesis, we would like to understand how are the different functions of SKAP2 modulated by protein-protein interactions (PPI). We will also study SKAP1 paralog, which has similar but non-redundant functions, to compare their PPIs. The hub will have to analyze these multidimensional data to statistically validate which function is affected by each PPI and SKAP2 mutation and define common patterns. One goal of these analyses will be to define new hypotheses in particular for characterizing binding sites that will be secondary tested.

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 specific lifestyles in a host cell type-specific manner, where these lifestyles are remarkable for the subcellular localization and replication rate of Salmonella. During epithelial cells infection, Salmonella are first uptaken by the host cell and being encapsulated in a unique endocytic compartment termed Salmonella-containing vacuole (SCV). Within the SCV, Salmonella could remodel the SCV into a viable replicative niche or rupture the SCV and hyper-replicate in the host cytosol. Salmonella replicate at distinct rates in the two localizations, the Salmonella replication in SCV commences from 5 hours post-infection (pi) and infected host cell harbors less then 10 bacteria at 8h pi. While the cytosolic Salmonella begins replication within the first hour of infection at a rate of 30 minutes per division and accumulates up to over 50 bacteria in the host at 8h pi. To understand how Salmonella achieves the two distinct intracellular lifestyles as well as the pathophysiological impacts of the two lifestyles, the primitive goal of my project is to develop a bacterial-borne fluorescent reporter that clearly depicts the subcellular localization and replication rate of Salmonella. With this multiplex fluorescent reporter, we will set to explore the fundamental biological questions: to determine the immune response of epithelial cells against the two Salmonella lifestyles.

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 machinerie transcriptionnelle aux séquences promotrices. Des études récentes ont mis en évidence que modifier la chromatine est l’un des moyens par lesquels les bactéries pathogènes interfèrent avec le programme transcriptionnel des cellules hôtes. Cependant, les mécanismes moléculaires sous-jacents sont très peu caractérisés. Ainsi que les rôles de ces modifications pour l’hôte ou pour la bactérie et l'impact à long terme des changements induits chez l’hôte restent mal définis. Les colonisateurs naturels ou les bactéries commensales n'ont pas été étudiés pour leur capacité à modifier les histones de l’hôte. Dans notre projet, nous utilisons le model bactérien, Streptococcus pneumoniae, pour nous focaliser sur les modifications d’histones importantes pour un colonisateur naturel de l’épithélium respiratoire et un pathogène opportuniste redoutable, dans le but de caractériser précisément les modifications des histones qui persistent après la colonisation afin d’évaluer la réponse de la mémoire. Nos résultats préliminaires montrent que cette bactérie induit bien des modifications d’histones qui sont conservés à long terme. Pour identifier les gènes affectés par ces modifications d’histones observées et comprendre leurs roles, nous voulons réaliser du ChIPseq.

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 worldwide, affecting 3% of the US general population, with high burden for individuals and societies1 (4 to 8-fold increase in standardised mortality rates). Clinically, (cocaine) addiction (or substance use disorder) is defined as the compulsive use of a substance causing clinically and functionally significant impairment (health problems, disability, and failure to meet major responsibilities at work, school, or home). These features constitute the basis of diagnostic criteria. There is no approved medication to treat CocAdd, despite significant advances regarding the mechanisms underpinning the neurobiology of chronic cocaine self-administration in rodents. CocAdd is defined as a maladaptive and compulsive reward-seeking behaviour related to cocaine. The loss-of-control over drug use and associated urges (craving) is such that 75-90% people with CocAdd report that they have relapsed within one year after inpatient detoxification. It is thus crucial to develop innovative and precise biomarkers to predict the liability to relapse if we are to develop efficient treatment strategies against this devastating disorder. Relapse is a key player in the chronicity of addiction in general and, as such, is the core therapeutic target of any therapy against addiction . Attached to an ongoing study of the neuroimaging signature of CocAdd relapse, we implemented an auxiliary study to draw blood samples from patients with CocAdd undergoing hospital detoxification and followed for up to three months after, so as to collect genetic material. The 'OBSCOC' protocol thus provides biological samples at entry and 15, 30 and 90 days after. These samples are aimed for characterising the whole RNome, miRNome and ChipSeq profile of these patients, who all provided written informed consent for the genetic study and for extensive clinical and sociodemographic assessment as well. We hypothesised that specific changes of gene expression between time points could either predict increased risk for/precocity of or announce imminent relapse. Thus they could serve, on the longer-term, as biomarkers and/or therapeutic targets.

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 psychomotor development. Stunting is a syndrome that is linked to poor nutrition and repeated infections. To date, the treatment of stunted children is challenging as the underlying etiology and pathophysiological mechanisms remain elusive. We hypothesize that pediatric environmental enteropathy (PEE), a chronic inflammation of the small intestine, plays a major role in the pathophysiology of stunting, failure of nutritional interventions and diminished response to oral vaccines, potentially via changes in the composition of the pro- and eukaryotic intestinal communities. The main objective of AFRIBIOTA is to describe the intestinal dysbiosis observed in the context of stunting and to link it to PEE. Secondary objectives include the identification of the broader socio-economic environment and biological and environmental risk factors for stunting and PEE as well as the testing of a set of easy-to-use candidate biomarkers for PEE. We also assess host outcomes including mucosal and systemic immunity and psychomotor development. AFRIBIOTA is a case-control study for stunting recruiting children in Bangui, Central African Republic and in Antananarivo, Madagascar. In each country, 460 children aged 2–5 years with no overt signs of gastrointestinal disease are recruited (260 with no growth delay, 100 moderately stunted and 100 severely stunted). We compare the intestinal microbiota composition (gastric and small intestinal aspirates; feces), the mucosal and systemic immune status and the psychomotor development of children with stunting and/or PEE compared to non-stunted controls. We also perform anthropological and epidemiological investigations of the children’s broader living conditions and assess risk factors using a standardized questionnaire. To date, the pathophysiology and risk factors of stunting and PEE have been insufficiently investigated. AFRIBIOTA will add new insights into the pathophysiology underlying stunting and PEE and in doing so will enable implementation of new biomarkers and design of evidence-based treatment strategies for these two syndromes. These analyses comprise the child development aspects of AFRIBIOTA.

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’endocardites chez les personnes âgées. Depuis plus de 50 ans, les études épidémiologiques indiquent l’existence d’une forte association entre les infections invasives à S. gallolyticus et le CCR (cancer colorectal) (Pasquereau-Kotula et al., 2018). Néanmoins, le rôle exact de S.gallolyticus dans le développement du cancer colorectal n'est toujours pas déterminé. Dans notre projet, nous voulons déterminer si S.gallolyticus, une bactérie commensale du colon, est à l’origine du cancer colorectal comme Helicobacter pylori est l’agent étiologique du cancer de l’estomac. Précédemment, notre l’équipe a montré que cette bactérie est aussi capable de profiter de l’environnement tumoral pour se multiplier au détriment d’autres bactéries présentes dans le microbiote de souris comme Enterococcus faecalis (Aymeric et al., 2018). L'hypothèse alternative étant que cette bactérie profite de l’environnement tumoral pour se multiplier au détriment des autres populations bactériennes et provoquer des endocardites suite au relâchement de la barrière intestinale provoquée par la tumeur. L’objectif principal de ce projet de recherche est de mieux comprendre le rôle de S. gallolyticus dans les CCR. L’étude transcriptomique va nous permettre de déterminer impact de S. gallolyticus sur les cellules intestinalles non transformées vs cellules tumorales. Cette l’étude aidera à identifier les gènes altérés par S. gallolyticus qui pourraient à long terme contribuer à la progression de CCR et/ou à oncogénique des cellules normales. La réponse transcriptionnelle induite par S.gallolyticus sur les cellules humaines (normale et canceresuse) permettra de mieux comprendre les mécanismes moléculaires qui sous-tendent le rôle de S.gallolyticus dans le développement du cancer colorectal.

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 inhibitory synapses. It acts as a hub under the postsynaptic membrane for the multiple protein-protein interactions. Intriguingly, inhibitory synapses are highly heterogeneous, bearing various inhibitory postsynaptic potential (IPSP) properties and also specific subcellular localization on their target neuron. The molecular mechanism responsible of this diversity is still unknown although it could result, in part, of alternative splicing regulation that will produce specific GPHN isoforms carrying versatile properties. Interestingly, exons alternatively included in Gphn transcripts are proposed to change the binding of GPHN protein with inhibitory receptor as well as its oligomerization. Thus, alternative splicing regulation of Gphn expression intuitively provides a potential molecular mechanism to finely regulate several aspect of inhibitory synapse development, however this regulation step is still largely unexplored. In collaboration with Fabrice Ango (IGF-Montpellier), we have designed an experimental approach to sequence GPHN transcripts using the technologies from Pacific Bioscience and Oxford Nanopore. It was applied to samples prepared from mouse and human tissues. To date, we got sequences from Pacific Bioscience sequencing and our interaction with E. Kornobis allow us to get preliminary data that have revealed a high level of complexity in alternative transcripts expressed by GPHN in Mouse brain samples. However, we are fighting to cluster these sequences and pulling together alternative GPHN transcripts with a bioinformatic pipeline able to decipher properly between light variation of sequences and sequencing errors associated to long read sequencing. Results obtained from currently available solutions, such as PacBio IsoSeq3 analysis pipeline, led us to believe that a more suitable software solution is needed, especially to properly characterize Gephyrin splicing diversity. We propose to build a new bioinformatic pipeline to analyze our data and usable to long read sequencing obtained with Pacific Bioscience and Oxford Nanopore technologies.

The provision of human biological material collected, processed and stored under optimal conditions is crucial to ensure the quality of research carried out downstream. These optimal conditions must be determined by a rational method validation approach taking into account the critical parameters of the different biological sample management processes that are likely to have an impact on their quality.

The therapeutic anti-IgE antibody Omalizumab is used for the treatment of severe asthma, and is known to trigger anaphylaxis in some patients. Since Omalizumab is a humanized IgG1, so we hypothesized that Omalizumab could trigger anaphylaxis through the alternative FcgR-mediated pathway. Indeed, we observed that Omalizumab can trigger anaphylaxis in genetically modified mice expressing human FcgRs. We further produced a mutant version of Omalizumab which can still block IgE but cannot bind FcgRs, and demonstrated that this mutant anti-IgE does not induce FcgR-mediated anaphylaxis.

The quality of the samples stored in biolocial resources centers is a real concern at the international level. Recent scientific publications highlighted difficulties for reproducing published scientific results which constitute a brake to consequent developpment of medical applications.. The quality of the samples used in experiments may partly explain those difficulties. Within the ICAReB platform, samples dating from 1950 are conserved. The question of their quality and usability is posed. Our main objective is therefore to idnetify universal markers of quality for blood derived samples which constitutes the most frequent biological material stored in biobanks.

Despite the success of anti-retroviral therapy (ART) to treat HIV-1-infected individuals, the persistence of a viral reservoir remains an obstacle to a cure. Characterization of the antibody repertoire in patients led to the identification of broadly neutralizing antibodies (bNAbs) targeting the viral envelope glycoproteins and suppressing HIV-1 infectivity with unprecedented potency. Passive administration of bNAbs in animal models or in infected humans revealed their capacity to decrease viral loads and to delay viral rebound after ART cessation. bNAbs also activate the immune system and mediate functions that go well beyond neutralization, such as killing of infected cells or enhancement of T and B cell responses. Immune cells with cytotoxic, phagocytic or immunomodulatory activities express Fc Receptors (FcR) that recognize the constant Fc region of antibodies. The complement cascade is also initiated through binding to the Fc. The molecular and cellular mechanisms underlying the activation of Fc-dependent effector functions of antibodies, including bNAbs, are incompletely understood. Our aim is to analyze the interactions between HIV-1 specific antibodies, infected cells, and the immune system

Deconvolution of the polyclonal antibody response targeting HIV-1 allowed the identification of broadly neutralizing monoclonal antibodies (bNAbs) targeting the viral envelope. Infusion of bNAbs protects against HIV-1 acquisition and decreases viral load. bNAbs kill HIV-1-infected cells, modulate host immune responses, and, when associated to latency-reversal agents, delay viral rebound in animal models. These antiviral activities are mediated by the Fc region, which is required for optimal in vivo efficacy. Thus, the landscape of Fc effector functions triggered by bNAbs is under intense investigation. Our aim is to decipher the molecular mechanisms underlying the sensitivity of HIV-1-infected cells to Fc effector functions.

Adaptive immune cells play important role in pathogenesis of multiple sclerosis by infiltrating the central nervous system, thereby contributing to demyelination and maintenance of an inflammatory microenvironment. In particular, autoreactive T cells, CD4+ T helper and CD8+ T cells exert a detrimental effect, while CD4+ regulatory T cells (Treg) have impaired suppressive function. Interferon b (IFNb), a commonly prescribed treatment for relapsing-remitting multiple sclerosis (RR-MS) decreases relapse rates and brain disability progression. However, around 30% of patients are or become non-responsive to treatment. Our aim is to determine the immune signatures associated with clinical efficacy of IFNb treatment in RRMS patients

Urinary Tract Infections (UTIs) represent approximatively 150 to 250 million cases per year, thus are of major public health concern. 80% of community-acquired UTIs are caused by UPECs and about 40% of UPECs are known to produce the Cytotoxic Necrotizing Factors1 (CNF1). The CNF1 is a Rho-activating toxin that catalyzes a specific deamidation of a glutamine of Rho GTPases for cells invasion. The deamidation maintains the GTPases constitutively activated and the constant activation of RhoGTPases by CNF1 leads to proteasomal degradation through K48 ubiquitination. However, the change in proteins expression levels, the rate of modification induced by the CNF1 on the cellular proteome and the outcomes of host responses have not yet been investigated. By conducting quantitative mass spectrometry–based proteomics approaches, we intend to describe changes of the cellular proteome during cell invasion by UPEC producing CNF1.

We are interested in Spondyloarthritis. Spondyloarthritis is a chronic inflammatory rheumatism. Currently 2 biologic treatments are available : anti TNF and anti IL-17A. We are analyzing how these treatments modify the immune system of the patients. The goal of this part of the project is to describe how biologics treatments in Spondyloarthritis modifiy the frequence of immune cell populations.

Our hypothesis is that gut microbiota could define predictive markers of response and tolerance to biologics. A. Preliminary results: gut bacteria predicting response to TNF blockers. A proof-of-concept study has been performed on 58 patients who were recruited according to the following criteria: active disease despite NSAIDs intake; no history of inflammatory bowel disease; no antibiotics intake within 3 months prior recruitment. Bacterial 16S rRNA gene sequencing region was performed on stools samples before and after TNF-blocker treatment. Diversity metrics and custom LefSe were used to explore the relationship between the composition of the intestinal microbiota and the efficacy of TNF-blockers. A lower alpha diversity at baseline was unexpectedly associated with better treatment response, HLA-B27 genotype and smoking behavior. Meanwhile, beta diversity was associated with smoking behavior and HLA-B27 genotype before and after treatment. Beta diversity at baseline was associated with the BASDAI index after treatment, and the response to the treatment. These results indicate a potential regulatory role for the gut microbiota on the underlying mechanisms involved in the response to TNF-blockers. Moreover, a LefSe-like approach identified 6 bacterial species as potential biomarkers for the treatment response, despite the absence of global changes (beta diversity) in the microbiota composition following a 3-month TNF-blockers intake. B. Current project: ITS2 fungal rDNA sequencing analyses In order to establish a causal link between host-microbe interactions and clinical efficacy of anti-TNF, we expect the following research endpoints from our experimental and translation approach (cohorts/clinical trials): 1. Defining the impact of anti-TNF on fungal microbiota and on the relative representation of fungal/bacterial components 2. Defining correlations between gut fungal composition and clinical outcome, with the aim of identifying stool microbial fingerprint of durable responses and/or primary resistance to anti-TNFα in SpA patients. The analyses will be performed on the same 58 SpA patients that were previously analyzed for their 16S bacterial component. These patients perfectly described regarding their disease characteristics, demographics, ongoing treatments, response to anti-TNF after a 3-month treatment period.

The rare patients who spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient T cell response. We aim at characterizing the molecular determinants underlying this efficient antiviral response. We have previously shown that CD4+ T cells from HIV controllers express T cell receptors (TCRs) of particularly high affinity for Gag peptides/ MHC II complexes. Furthermore, HIV controllers shared Gag-specific TCR clonotypes at higher frequencies than treated patients, suggesting the implication of particular TCRs in HIV control. To extend these studies, we propose to characterize the TCR repertoire and the transcriptional pattern of HIV-specific CD4+ T cells at the single cell level. We will compare HIV-specific CD4+ T cells obtained from HIV controllers included in the ANRS CO21 CODEX cohort to those from patients receiving antiretroviral therapy.

Glioblastoma cells interconnect forming a functional and resistant network. We are investigating the role of these connections in the cancer relapse and treatment-resistance

The aim of this project is to understand how inherited APC gene mutations affect the immune system of familial adenomatous polyposis patients, and to evaluate the potential contribution of altered immune responses to polyposis and intestinal cancer development in these patients. (see https://research.pasteur.fr/en/project/projet-de-recherche-clinique-impact-des-mutations-du-gene-apc-adenomatous-polyposis-coli-dans-les-reponses-immunes-anti-tumorales/)

The enteropathogen, Shigella, is highly virulent and remarkably adjusted to the intestinal environment of its almost exclusive human host. Key for Shigella pathogenicity is the injection of virulence effectors into the host cell, via its type three secretion system (T3SS), initiating disease onset and progression. T3SS-mediated host cell targeting is associated with Shigella invasion and dissemination in epithelial cells. Yet, our group reported the direct targeting of human lymphocytes by an injection-only mechanisms, i.e. the injection of Shigella T3SS effectors without subsequent cell invasion. To further investigate the scope and selectivity of this targeting mechanism, we utilised human lamina propria mononuclear cells (LPMCs) isolated from colonic explants and peripheral blood mononuclear cells (PBMCs) and assessed the mode of Shigella targeting by the use of a T3SS reporter stain and multiparametric CyTOF technology. In human LPMCs, a population of CD38hi plasma cells/ plasmablasts was identified as primarily targeted and presented up to 80% of all targeted LPMCs. Interestingly, plasma cells constitute the primary IgA-producing cell type and IgA has been previously identified as important mediator of Shigella pathogenicity. The functional implications on plasma cell targeting by Shigella is subject of current investigations.

The transcriptome signature during rabies infection will be analyzed both in animal model (mouse) and in human, at the central nervous system level, to identify the main canonical pathways involved during the infection.

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. We set up an experimental protocol to purify intact, detergent-free MBRs in order to have the full proteome of this organelle. Quantitative, label-free proteomics enabled us to identify MBR proteins, that we named the “Flemmingsome”, and 489 of them were found enriched as compared to whole cell lysates, thus named \\\"Enriched Flemmingsome\\\" (Addi et al. manuscript submitted). Proteins composing the Enriched Flemmingsome were individually browsed with their Protein and Gene names for their function in cytokinesis or midbody localization on PubMed and the reference indicated on the flemmingsome website. This project aims at keeping this database up-to-date with the literature.

Viruses have evolved powerful countermeasures to evade host innate immunity, which produces immediate, but non-specific, immune response during infection. Among viruses possessing RNA genomes, the order of negative-single-strand viruses (Mononegavirales) encompasses many human and animal pathogens that cause severe disease, including measles virus, mumps virus and rabies virus. Rabies virus is known for its neurotropic retrograde progression from the site of transmission to brain parenchyma, and towards salivary glands, different organs linked through parasympathetic nervous system. In the cases of human infection, the exhibited symptoms such as hallucination, diplopia, hydrophobia, unsteadiness or paralysis all indicate that there is causality between rabies virus infection and dysfunction of neural activity. However, lack of pathological brain lesions observed at the point of autopsy or noncytolytic propagation devoid of apoptosis suggest that rabies virus possesses mechanisms to evade or delay immune responses and cell death at least for the duration of replication and transmission. Even though the details in molecular perspective of these discoveries are well laid out now, how these proteins work in coordination or if there are hidden components which connect them all together leading toward deterioration of neural cells on the benefit of virus is largely unclear. Moreover, considering the complexity of brain cell composition and how important the neighboring cells are to shape one neuron’s specialization and dependency onto others in homeostasis, which result in the astounding heterogeneity of gene expression, an integrated and holistic approach is mandatory to get a fully comprehensive view of the mechanisms involved. Consequently, we performed an RNASeq analysis in human interneuron cells derived from induced pluripotent stem cells and infected by two recombinant rabies viruses (Tha virus, isolated from a dog in Thailand and Th4M, a less pathogenic virus which is mutated on 4 different residues of the M gene; this virus can no longer escape the NF-KB pathway) in order to obtain transcriptome data by comparison with uninfected cells, and to have an overview of the temporal dynamics of the genes expression.

Energy demands vary widely depending on cellular activation states. Here we perform an integrated bioenergetic analysis (epigenetic, transcriptomic and metabolomic) of the human CD56Br and CD56Dim natural killer (NK) cell subsets under steady-state conditions and after cytokine activation. We found that CD56Dim NK cells were metabolically active at steady state, but that both subsets increased bioenergetic properties upon priming with IL-15, with metabolic profiles similar to tonsillar NK cells. In contrast, IL-12/IL-18-induced cell activation promoted a dichotomous response with an elevated respiratory and functional capacity in CD56Br NK cells versus a metabolic switch towards glycolysis and reduced IFN-γ production in CD56Dim NK cells. The latter was associated with mitochondria fragmentation; inhibition of which increased IFN-γ production. Polarized mitochondria were identified with the CD57+ CD56Dim NK cell subset, thereby identifying a potential signature for NK cell functional maturation. We propose mitochondrial remodeling as a key regulator of NK cell function.

Parvimonas micra is a gram+ anaerobic coccus bacteria associated with colorectal cancer (CRC). The stool of patients with CRC has higher Parvimonas micra concentration compared to healthy individuals. It is also present in the colonic mucosa of CRC patients. Even if we find it in the adjacent healthy tissue, it is predominantly found in the tumor. Furthermore, it is correlated with the CpG island methylator phenotype (CIMP), a particular subtype of CRC. More specifically, it is associated with the hyper-methylation of tumor suppressor gene promoters. To find out whether this bacteria is involved in the initiation of CRC, we have developed a model of a differentiated epithelial monolayer of human colonic primary cells. After co-culture with this microorganism, we found a significant increase in the global DNA methylation level of the cell genome compared to the bacteria-free control. Now we would like to determine whether this increase in DNA methylation is specific to certain regions of the genome. Therefore, we will use Illumina's Infinium MethylationEPIC BeadChip Kit.

The RIG-I like receptors (RLRs) play a major role in sensing viral infection to initiate and modulate antiviral immunity. These are three RNA helicases entitled RIGI, MDA5 and LGP2 that interact with particular signatures of viral RNA molecules in cytoplasm of infected cell triggering a downstream signalling cascade resulting in the expression of type-I IFNs, proinflammatory cytokines and divers set of antiviral genes. We have recently generated stable cell lines expressing tagged RLRs. By applying affinity chromatography purification of RLR ribonucleoprotein complexes followed by RNA extraction, we observed that short-length RNAs were enriched on RLRs. We have previously sequenced small RNAs of RLRs (PF2 Pasteur) and now need to adjust our new bioinformatic analysis to these RNAseq data with the help of C3BI.

Institut Pasteur and institut Imagine will coordinate a french collection for genome-wide association study of COVID suceptibility and severity. This collection will contribute to the ongoing major international efforts addressing this question (https://www.covid19hg.com), but it will also be a major platform for a range of other national and international collaborations addressing host biology questions, and for integrative approaches including host genomics data (metabolomic, transcriptomic…), epidemiologic data, and virus data. The project is to develop and publish a webpage presenting the collection.

Background : The Immunoregulation Unit is composed at present of: Lab Head, one senior staff scientists, two “ingegnieurs” (one IP, one Fondation APHP), two PhD students. The focus of the research is the study of immuno-mechanisms in the pathogenesis of chronic inflammatory diseases, and of the molecular basis of response to treatment. All members of the lab have expressed an interest in acquiring basic R skills for the analysis of large gene expression data sets, collected from the study of patients’ samples. Only one PhD student in the lab is currently using R tools for data analysis, the other members have all received some previous instructions in biostatistics and/or R, but have not been using R tools currently. Requirements : Lab members have expressed a specific need to be instructed in the following areas: 1.refresh basic notions of R language, with a particular focus on handling large gene expression data sets 2.introduction to graphic tools for data representation (ggplot) 3.introduction to tools for differential gene expression analysis (limma) 4.data exploration using Principal Component Analysis Constraints : 1. For the course, it has been decided to use data generated by the lab. Placing the course in the lab’s data context has the advantage of ensuring that the course content is adapted to the “real-life” situations that lab members face in the analysis of their own data. 2.The present situation of “confinement” due to the Covid19 pandemic offers some opportunities (time availability of all members to follow the instruction), but obvious restriction. To maintain interactivity, classes are held at a distance, using Skype group meetings.

Hepatitis C virus (HCV) infection is a slow and asymptomatic progressive liver disease leading to metabolic disorders such as steatosis, an accumulation of lipid droplets (LDs) in hepatocytes. We have assessed the interplay between HCV variants recovered from chronically infected patients and LD content in infected hepatoma cells, through quantitative imaging approaches.

Chlamydia trachomatis is an intracellular bacterium. We have observed that some of the epigenetic changes in the host that occur in infection can be reversed by modifying the culture conditions. We will measure the impact this has on the global transcriptional response of the host to infection.

Paleo(meta)genomics is an emerging and rapidly growing field where most is yet to be done. In most cases, it consists in the analysis of ancient DNA high-throughput sequencing data obtained from archaeological material or historical samples, and the goal is to retrieve and interpret the genomic information from species that from the past (microbial, eukaryotic, etc.) It combines tools borrowed from different fields, such as genomics, computational biology, microbial ecology, phylogenetics, population genetics, etc. However, at the moment there are no well-established tools for the analysis of this type of data. Hence, each lab must develop custom solutions, combining existing tools or developing new ones to meet the goals of their research programs. As a recently established lab, the microbial paleogenomics unit will spend the upcoming months setting up diverse pipelines and analysis tools for the different projects that will be developed in the coming years, many of which have been already used but need to be re-written in an understandable languaje and structure.

The key role of CD8+ T-cells in controlling HIV replication has been particularly well established. Functional and metabolic properties of HIV specific CD8+ T cells appear to be essential for an effective control of the virus, as shown in HIV-1 and HIV-2 controllers. Mounting of functional CD8+ T cell responses is dependent on the effective priming of optimal antigen specific memory like CD8+ T-cells. However, factors that may dampen or promote effective priming of CD8+ T-cell responses during HIV infection remain largely unknown. Our aim is to investigate potential factors that may help us improving the induction or reprogramming of robust CD8+ T cells against HIV. We propose to study functional and metabolic attributes of HIV specific CD8+ T cells from HIV controllers and non controllers and determine if defects in CD8+ T cells form non controllers can be corrected with small molecules targeting the molecular program of the cells; de novo priming of HIV specific CD8+ T cells can be altered by antigen presenting cell-derived factors. The results of this project will have direct implications for our understanding of natural immune control of HIV, as well as the optimization of vaccine strategies or immune therapies against HIV.

With an estimated 1031 particles on earth, bacteriophages are the most abundant genomic entities across all habitats and important drivers of microbial communities. Growing evidence suggest that they play roles in intestinal human microbiota homeostasis, and recent metagenomics studies on the viral fraction of this ecosystem have provided crucial information about their diversity and specificity. However, the bacterial hosts of this viral fraction, a necessary information to characterize further the balance of these ecosystems, remain poorly characterized. Here we unveil, using an enhanced metagenomic Hi-C approach, a large network of 6,651 host-phage relationships in the healthy human gut allowing to study in situ phage-host ratio. We notably found that half of these contigs appear to be sleeping prophages whereas ¼ exhibit a higher coverage than their associated MAG representing potentially active phages impacting the ecosystem. We also detect different candidate members of the crAss-like phage family as well as their bacterial hosts showing that these elusive phages infect different genus of Bacteroidetes. This work opens the door to single sample analysis and concomitant study of phages and bacteria in complex communities.

Immune thrombocytopenia (ITP) is an autoimmune condition characterized by an autoantibody response against platelets, driven by pathogenic plasma cells often found in the spleen. This autoreactive plasma cell population remains poorly characterized, especially in terms of antibody affinity and anatomical distribution. In order to tackle this problem, we have developed a microfluidic platform to characterize platelet-specific plasma cells isolated from ITP patients, in terms of their IgG secretion rate and affinity for a platelet autoantigen at the single-cell level.

Chronic inflammatory diseases (CID) are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. Genome-wide association studies (GWAS), together with mouse models of autoimmune disease, demonstrated the implication of the IL-23 cytokine pathway in several CID. The overall goal of our work is to improve our understanding of the role of IL-23 in the pathophysiology of CIDs, such as axial spondyloarthritis, psoriatic arthritis, and psoriasis. The specific question we want to address with this project is how is expression of the IL-23 receptor regulated in immune cell populations.

Anti-TNF therapy has proven effective to reduce inflammation and clinical symptoms in SpA, however, the high rate of non-responsiveness (30-40%) to TNFi exposes a substantial fraction of patients to side effects without clinical benefit, and it is still not possible to determine which patients will respond to TNF inhibitors (TNFi) before treatment initiation. The recent introduction of antibodies blocking IL-17A has expanded the therapeutic options for axial SpA (axSpA), as well as psoriasis and psoriatic arthritis. It is therefore important to develop tools to guide treatment decisions for patients affected by SpA and other chronic inflammatory diseases, to optimize clinical care and contain health care costs. This project builds on our recent work performed in collaboration with the Bioinfo Hub, in which we have defined the mechanisms of action of TNF-blockers and identified immune signatures correlating with therapeutic responses to anti-TNF therapy in SpA patients (Menegatti et al., 2020). We have measured whole blood immune responses to microbial and pathway-specific stimuli using TruCulture assays in 20 axSpA patients before or after treatment with IL-17A-inhibitors and 20 patients before and after anti-TNF therapy. Proteins in supernatants have been measured by Olink technology for protein profiling. Gene expression in cell pellets has been analyzed by RNA sequencing at the Biomics platform. A preliminary analysis of the RNA-Seq data has been performed by Etienne Kornobis and Thomas Cokelaer.

The LabEx Milieu interieur (MI) project is a clinical study aiming to define the natural variability of the human immune response. Fibroblasts were prepared from skin biopsies of 300 of the 1000 healthy donors (30 men and 30 women in each of 5 decades from 20 to 69 years) by Genethon. Each primary line is annotated by metadata derived from the systematic genotypic-phenotypic analysis Labex-MI (serology, genomic analyzes, proteomics, transcriptome, microbiota, clinical data and epidemiological selection criteria). The objective is to characterize the innate immune response of fibroblasts from healthy donors. We have developed approaches to obtain standardized measures of the immune response and to identify interindividual variance (Chansard et al., 2021) on a subset of sixteen primary fibroblast lines.. We observed that cell responses to LPS stimulation were distributed between “low” and “high” responders, while inter-donor variability of the response to poly I: C was very low. Poly I: C and LPS activate pathways from TLR3 and TLR4 receptors, respectively. We set up a real-time quantitative PCR assay of the 16 cells in parallel using the BioMark automated PCR system (Fluidigm) for comparative quantification of mRNAs baseline expression in the donors’ fibroblasts to check if low responses to LPS were due to a different expression of TLR4 and other factors of the pathway. We will extend the analysis to stimulated cells by Poly I: C ,LPS and other agonists of the innate immune response and also in parallel to cells infected by Leptospira interrogans.

Le projet a pour objectif de développer une page web pour présenter les travaux de modélisation de la pandémie de la COVID-19 au grand public.

JASS is a python package that handles the computation of the joint statistics over sets of selected GWAS results, and the interactive exploration of the results through a web interface (https://jass.pasteur.fr/index.html). The functionalities of the current web interface include: the interactive visualization of multi-trait GWAS results, the sharing of results through a permanent link and generation of static summary plots. This work and its accompanying tools has been described in a publication (Julienne et al, 2020). However, the web interface present several shortcomings that may impede its wider adoption throughout the scientific community. Key additional information could help contextualize the multi-trait GWAS results such as the gene positions or the linkage disequilibrium with the lead SNPs. The interface ergonomics is overall perfectible as we would like to allow for user with a specific biological question to launch computation and visualize results only on their region of interest.

Myotonic dystrophy type 1 (DM1 or Steinert disease) is a monogenic neurodegenerative disorder due to the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene on chromosome 19. We developed a strategy to induce a DNA double-strand break (DSB) within the expanded repeat in order to contract it below the pathological range in humans. This approach was successful in yeast and mouse cells and we are now trying to apply it to human cells from a patient affected by DM1. This patient was diagnosed with a large CTG repeat expansion (≈ 2000 triplets) and we estimated the size of the repeat tract by Southern blot to be 1980 triplets. These cells (ASA cells) were immortalized by overexpression of the telomerase catalytic unit, theoretically allowing to grow them for 100 generations (instead of ca. 20 generations for primary cells). A TALEN directed to the expanded CTG repeat was expressed in ASA cells for several weeks and the population was subcloned. Trinucleotide repeat length was analyzed in many clones, by Southern blotting. This showed that the repeat was partially contracted in most clones, the others showing no size change (or a small expansion). Ten clones were chosen to be sequenced by PacBio, in addition to a non-treated ASA clone that will be used as the reference for future work on ASA cells.

There are papain treated and papain+CPG treated group. From mouse lung tissue, I'd like to find out the target cell and their key molecule through scRNAseq and receptor-ligand analysis. When I get the target cell, I want to do ATACseq (or scATACseq) of target cell to see if memory of previous exposure to inflammatory stimuli represented by chromatin accessibility and which has something to do with 2nd exposure of same allergen.

Déploiement sur l'infra-structure de l'Institut Pasteur du LIMS permettant la gestion de la collection “ORFeome humain” à partir d'une base de code open-source contenant deux projets (une API et une interface web). Cet outil informatique permet la gestion de la collection ainsi que des sous-collections qui en découlent, il permet également d'effectuer des recherches multi-critères et d'éditer des listes de cherry-picking dans un format directement utilisable sur la plateforme robotique TECAN. Il s'agit donc d'un interface bioinformatique indispensable à l'utilisation de cette collection de gènes humains.

The chronic inflammatory diseases (CID), as psoriasis, psoriatic arthritis (PsA) and axial spondyloarthritis (SpA), are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. GWAS strongly suggests that the IL-23/IL-17 pathway plays a key role in several CID. The successful treatment of psoriasis, PsA and axial SpA with IL-17A inhibitors has been validated. Interestingly, in literature, a strong GWAS association of IL23R with SpA was shown and IL-17A was shown to be downstream of IL-23 in murine CD4+ T cells. Surprisingly, a recent phase 2 study testing the IL-23 inhibitor risankizumab did not show any clinically improvement compared to placebo in patients with active axial SpA, whereas it has proven effective for the treatment of psoriasis and PsA. The reasons underlying the failure of IL-23 blocking in axial SpA are not known. Our understanding of the pathogenic mechanisms of these diseases is therefore limited, and the role of IL-23 needs to be studied. The specific question we want to address with this project is what are the gene induced by IL-23 in immune cell populations.

Nous étudions la toxine CNF1 des E. coli uropathogènes. Cette toxine active des protéines ayant des activités pro-oncogéniques et pro-inflammatoires. Ces E. coli peuvent être en portage asymptomatique dans la flore intestinale de patients sains. Par des mesures quantitative du taux d’anticorps anti-CNF1 sérique nous cherchons à définir l’exposition de ces patients à cette toxine. Ici nous cherchons à définir si les différences mesurées entre groupes de patients sont significatives.

La pandémie de la COVID-19 est un défi médical inédit en particulier du fait de la nature systémique de cette pathologie. En effet, la COVID-19 affecte à la fois plusieurs organes et systèmes. L’atteinte cérébrale s’effectue de plusieurs façons : infection directe des cellules nerveuses par le SRAS-CoV-2, inflammation du système nerveux central avec, entre autre, invasion de lymphocytes T et activation de la microglie, inflammation systémique sévère qui inonde le cerveau d'agents pro-inflammatoires et endommage ainsi les cellules nerveuses, ischémie cérébrale globale liée à une insuffisance respiratoire, accidents thromboemboliques liés à une augmentation de la coagulation intravasculaire et stress psychologique sévère. En conséquence, la COVID-19 se manifeste parfois par des symptômes neurologiques et neuropsychiatriques tels que des vertiges, des troubles du sommeil, des déficits cognitifs, un délire, ou une dépression sévère. La perte soudaine de l'odorat est un symptôme fréquemment associé à la COVID-19 et l'infection par le SARS-CoV-2 des neurones du système olfactif a été signalée à la fois chez le hamster et l'humain. La grande majorité des patients atteints de la COVID-19 retrouvent leur fonction olfactive en quelques semaines. Cependant une minorité significative de personnes infectées (1 sur 5 cas), souffre toujours de troubles olfactifs (anosmie, hyposmie et/ou parosmie) plusieurs mois après la primo-infection. Ces troubles olfactifs sont fréquemment associés à un comportement dépressif et à des plaintes cognitives. En TEP, il est même possible de corréler ce dysfonctionnement cognitif à un hypométabolisme de certaines régions cérébrales, y compris le gyrus olfactif. Ce projet propose d'évaluer et de suivre l’évolution, durant un an, des capacités olfactives de patients atteints de trouble persistant de l’odorat depuis un an (+/- 3 mois) des suites de la COVID-19. Il permettra de mieux distinguer si les troubles olfactifs de longue durée sont liés à une atteinte du système olfactif périphérique (en raison d’une persistance virale locale et/ou d’une inflammation chronique de l’épithélium olfactif nasal), ou du système nerveux central. En particulier, l’enregistrement des potentiels évoqués olfactifs permettra d'objectiver les capacités olfactives, de manière non déclarative. Cette étude pourra servir de base au développement d’études visant à évaluer les risques cliniques neurologiques et/ou psychiatriques associés à la forme chronique des troubles de l’odorat des suites de la COVID-19.

This project addresses two main questions: does Chlamydia trachomatis modify glycolysis and/or oxidative phosphorylation in the host, and do the bacteria need these metabolic pathways to complete their developmental cycle.

Bacterial toxins can induce a wide range of life threatening diseases and are at risk of bioweapon development due to their extreme toxicity and ease of production. Some AB-like toxins exploit the host cell endocytic machinery to enter into the cell cytosol where they exert their poisoning effects. Host-targeted therapy involving the development of small molecules interfering with key vesicular trafficking components hijacked by bacterial toxins offers to counteract toxin action. We have identified the small compound C910, in a cell-based High-throughput screen (HTS) followed by orthogonal screens described in (Mahtal N et al., 2018), for its cell-protective activity against CNF1 toxin from pathogenic Escherichia coli, as well as diphtheria and Shiga toxins. Therefore, to characterize C910 mechanism of action and find its cellular target we have realized an siRNA wide-genome screening on primary human cells. This screening aims to find genes that can interfere with the trafficking of the CNF1 toxin.

Chronic inflammatory diseases (CID) are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. Genome-wide association studies (GWAS), together with mouse models of autoimmune disease, demonstrated the implication of the IL-23 cytokine pathway in several CID. The overall goal of our work is to improve our understanding of the role of IL-23 in the pathophysiology of CIDs, such as axial spondyloarthritis, psoriatic arthritis, and psoriasis. The specific question we want to address with this project is how is expression of the IL-23 receptor regulated in T cell populations.

In recent years, immune dysfunctions, including auto-immune mechanisms and peripheral inflammation, have been clearly associated with severe neuropsychiatric disorders like bipolar disorders (BD) and schizophrenia (SZ). These findings are supported by an extensive litterature highlighting a higher risk to develop neuropsychiatric disorders in patients suffering from auto-immune diseases and also the presence of low-grade inflammation and abnormal immunoglobulin rates in patients with psychosis. It is now well-established, since the description of paraneoplastic and auto-immune encephalitis, that antibodies targeting self-antigens such as membrane receptors or intracellular proteins could trigger psychiatric symptoms and severe inflammation. Therefore, an in-depth characterisation of circulating auto-antibodies and their clinical and/or biological implications has become a critical issue to stratify specific subgroups of patients with auto-immune psychosis in order to adjust and adapt the treatment and care, and develop novel approaches. Nicotinic acetylcholine receptors (nAChRs) have been linked to severe neuropsychiatric disorders by clinical and genome-wide association studies (GWAS). In addition to their key roles in neuronal function, nAChRs are also involved in the complex regulation of immuno-inflammatory processes both in the brain and the periphery, making them prime candidates to study the link between inflammation and major psychiatric disorders. Furthermore, nAChRs have already been identified as the target of autoimmune mechanisms leading to the destruction of the neuromuscular junction in the well-described autoimmune disease, myasthenia gravis. Based on these findings, we have started to dissect auto-immune mechanisms against nAChRs involved in SZ and BD. In brief, anti-nAChR auto-antibodies contribute to cognitive dysfunction and psychotic symptoms through peripheral inflammation. Here, our goals are (1) to extend the current knowledge by dissecting the relationship between clinical features and peripheral inflammation (cytokines, chemokines, ...) (2) to stratify patients with anti-nAChR auto-immune psychosis by using cluster analyse

The majority of approved drugs target proteins, which are encoded in a very small fraction of the human genome. When a pathology is associated with so-called undruggable proteins, an alternative strategy should be sought. In the last twenty years, non-coding RNA molecules have been shown to perform a variety of crucial biological functions, including regulating gene expression, protecting chromosomes from foreign nucleic acids, and guiding telomers synthesis. In this context, targeting either mRNA molecules that are translated into undruggable protein targets or biologically relevant non-coding RNA molecules with small molecules is emerging as a promising therapeutical approach in pathologies such as cancer, viral infections, and neurodegenerative disorders. However, the number of approved drugs that target RNA molecules is still very limited and the existing examples have mostly been found by costly and time-consuming screening experiments. In this project, we aim at building a computational framework to guide the rational design of drugs targeting RNA. To this end, we created a database containing all the experimentally-determined structures of RNA-small molecule complexes deposited in the PDB database. The entries containing drug-like compounds were selected and annotated based on the different biological entities interacting with the ligands. Our database, freely accessible via a web interface, will facilitate i) mapping the chemical space of the small molecules known to bind RNA, ii) understanding the nature of the interactions that drive ligand/RNA recognition, and iii) benchmarking existing tools for in silico protein drug design with RNA targets.

We observed that Chlamydia trachomatis infection results in deep changes in the histone methylation profile of the infected cells.

Myotonic dystrophy type 1 (DM1 or Steinert disease) is a monogenic neurodegenerative disorder due to the expansion of a CTG trinucleotide repeat in the 3' UTR of the DMPK gene on chromosome 19. We developed a strategy to induce a DNA double-strand break (DSB) within the expanded repeat in order to contract it below the pathological range in humans. This approach was successful in yeast and mouse cells and we are now trying to apply it to human cells from a patient affected by DM1. This patient was diagnosed with a large CTG repeat expansion (? 2000 triplets) and we estimated the size of the repeat tract by Southern blot to be 1980 triplets. These cells (ASA cells) were immortalized by overexpression of the telomerase catalytic unit, theoretically allowing to grow them for 100 generations (instead of ca. 20 generations for primary cells). A TALEN directed to the expanded CTG repeat was expressed in ASA cells for several weeks and the population was subcloned. Trinucleotide repeat length was analyzed in many clones, by Southern blotting. This showed that the repeat was partially contracted in most clones, the others showing no size change (or a small expansion). Ten clones were chosen to be sequenced by PacBio, in addition to a non-treated ASA clone that will be used as the reference for future work on ASA cells.

Measles virus (MeV) impairs the mitochondrial network. This leads to the fusion of mitochondria and simultaneous release of mitochondrial DNA (mtDNA). We have shown that released mtDNA is recognized as a danger signal, capable of stimulating signaling pathways and inducing the production of pro-inflammatory cytokines. The expression of human cytidine deaminase APOBEC3A is highly upregulated by interferon responses. This enzyme catalyzes the deamination of cytidine to uridine in single-stranded mitochondrial DNA substrates, resulting in the catabolism of edited DNA. Deep sequencing analyses of cytosolic mtDNA mutations revealed an APOBEC3A signature predominantly in the 5’TpCpG context. Using cell lines deprived of mtDNA, we have demonstrated the implication of mtDNA in the production of interferon and APOBEC3A expression during viral infection. We have shown that MeV induces mitochondrial network fragmentation and confirmed the implication of RNA polymerase III/RIG-I signaling in the capture of cytosolic mtDNA.

Cytokinesis is the last step of cell division and leads to the physical separation of the daughter cells. The purpose of this project is to infer the timing of abscission in various cell types by using few microscopic images of live cells and fluorescent markers.

Chronic inflammatory diseases (CID) are clinically heterogeneous conditions that share common inflammatory pathways and derive from aberrant immune responses. Genome-wide association studies (GWAS) demonstrated that Th17 cells play a pivotal role in the pathogenesis of these diseases and several loci involved in the IL-23/IL-17 are associated with the CID (IL23R, IL12B, IL6R, IL1R2, RORC, RUNX3, TYK2, JAK2, CARD9). IL-23 is important for the expansion and the functional activity of the Th17 cell subset and several studies have suggested that IL-23 may also regulate the function of IL-17-producing innate immune cells, which express the IL-23 receptor (IL-23R). The overall goal of this project is to improve our understanding of the so-called “IL-23/IL-17 axis” in the pathophysiology of CID and in particular to understand the effects of IL-23 on distinct cell populations and how is the expression of the IL-23 receptor regulated.

Oncodash is an open-source software developed within the DECIDER H2020 project, which aims at improving clinical decisions via integrating multiple data to overcome chemotherapy resistance in high-grade serous ovarian cancer. The corresponding DECIDER workpackage is led by Johann Dreo and Benno Schwikowski. The current state of the software can be checked at: https://github.com/oncodash/oncodash

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.

HPV RNA-Seq, a novel and unique molecular test that allows detection of pre-cancerous lesions of the cervix from cervical samples.

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.