Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

A single workflow for multi-species blood transcriptomics.

BACKGROUND: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. RESULTS: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. CONCLUSIONS: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.

Development of Mass Spectrometry Imaging on skeletal muscle to characterize the local pro-inflammatory and pro-resolution lipid responses in a vaccination context.

Vaccine reactogenicity is well documented at the clinical level but the mechanism involved at the local or systemic level are still poorly understood. Muscular tissue where most vaccines are administered is the first place of interaction between the vaccine formulation and the host's immune cells. So far, this site of vaccine administration is not well documented from a mechanistic standpoint. The study of early molecular events at the injection site is crucial to understand the local response to vaccines. In this paper, we report a standardized workflow, from the injection of vaccine formulations in rabbit muscle, to the analysis by desorption electrospray ionization and histology staining to understand the role of lipids involved in the inflammation and its resolution on striated muscular tissue. The analysis of lipid mediators was optimized at the site of needle insertion to allow the spatial comparison of cellular infiltrates at the injection site. We showed that lipids were distributed across the spatial tissue morphology in a time-dependent manner. The MS imaging applied to vaccinology could pave the way to a better understanding of vaccine reactogenicity and mechanism of action.

Evaluation of safety and immuno-efficacy of a next generation live-attenuated yellow fever vaccine in cynomolgus macaques.

The increased demand for yellow fever (YF) vaccines over the last decade, along with insufficient availability of specific pathogen-free embryonated eggs required for timely vaccine production, has led to global YF vaccine shortages. A new live-attenuated YF vaccine candidate (generically referred to as vYF) cloned from a YF-VAX® vaccine (YF-17D vaccine) substrain adapted for growth in Vero cells cultured in serum-free media is currently in development. Here, we assessed the safety and immunogenicity of vYF, and its protective activity upon virulent challenge with wild-type yellow fever virus (YFV) Asibi, compared to licensed YF-17D vaccines in the translational cynomolgus macaque model. vYF was well tolerated with no major safety concerns. Vaccine-related safety observations were limited to minimal/minor microscopic findings at the injection sites and in the draining lymph nodes, consistent with expected stimulation of the immune system. vYF induced early differential expression of genes involved in antiviral innate immunity previously described in humans vaccinated with YF-17D vaccines, as well as YFV-specific IgM and IgG antibodies, high and sustained YFV neutralizing antibody titers from Day 14 up to at least Day 258 post-immunization, IgM+ and IgG+ memory B cells from Day 14 up to at least Day 221 post-vaccination, and Th1 interferon (IFN)-γ and interleukin (IL)-2 secreting effector and memory T cells. Additionally, vYF provided effective resistance to virulent challenge with wild-type YFV Asibi including complete protection against YFV-induced mortality, pathology, dysregulation of blood and liver soluble biomarkers, and a significant reduction in viremia and viral load to the limit of detection. These NHP data suggest that vYF would provide protection against YFV infection in practice, at least similar to that achieved with currently marketed YF-17D vaccines.

Characterization of prefusion-F-specific antibodies elicited by natural infection with human metapneumovirus.

Human metapneumovirus (hMPV) is a major cause of acute respiratory infections in infants and older adults, for which no vaccines or therapeutics are available. The viral fusion (F) glycoprotein is required for entry and is the primary target of neutralizing antibodies; however, little is known about the humoral immune response generated from natural infection. Here, using prefusion-stabilized F proteins to interrogate memory B cells from two older adults, we obtain over 700 paired non-IgM antibody sequences representing 563 clonotypes, indicative of a highly polyclonal response. Characterization of 136 monoclonal antibodies reveals broad recognition of the protein surface, with potently neutralizing antibodies targeting each antigenic site. Cryo-EM studies further reveal two non-canonical sites and the molecular basis for recognition of the apex of hMPV F by two prefusion-specific neutralizing antibodies. Collectively, these results provide insight into the humoral response to hMPV infection in older adults and will help guide vaccine development.

Göttingen Minipigs as a Model to Evaluate Longevity, Functionality, and Memory of Immune Response Induced by Pertussis Vaccines.

Evaluation of the short-term and long-term immunological responses in a preclinical model that simulates the targeted age population with a relevant vaccination schedule is essential for human vaccine development. A Göttingen minipig model was assessed, using pertussis vaccines, to demonstrate that vaccine antigen-specific humoral and cellular responses, including IgG titers, functional antibodies, Th polarization and memory B cells can be assessed in a longitudinal study. A vaccination schedule of priming with a whole cell (DTwP) or an acellular (DTaP) pertussis vaccine was applied in neonatal and infant minipigs followed by boosting with a Tdap acellular vaccine. Single cell RNAsequencing was used to explore the long-term maintenance of immune memory cells and their functionality for the first time in this animal model. DTaP but not DTwP vaccination induced pertussis toxin (PT) neutralizing antibodies. The cellular immune response was also characterized by a distinct Th polarization, with a Th-2-biased response for DTaP and a Th-1/Th-17-biased response for DTwP. No difference in the maintenance of pertussis-specific memory B cells was observed in DTaP- or DTwP-primed animals 6 months post Tdap boost. However, an increase in pertussis-specific T cells was still observed in DTaP primed minipigs, together with up-regulation of genes involved in antigen presentation and interferon pathways. Overall, the minipig model reproduced the humoral and cellular immune responses induced in humans by DTwP vs. DTaP priming, followed by Tdap boosting. Our data suggest that the Göttingen minipig is an attractive preclinical model to predict the long-term immunogenicity of human vaccines against Bordetella pertussis and potentially also vaccines against other pathogens.

Complementarity of assembly-first and mapping-first approaches for alternative splicing annotation and differential analysis from RNAseq data.

Genome-wide analyses estimate that more than 90% of multi exonic human genes produce at least two transcripts through alternative splicing (AS). Various bioinformatics methods are available to analyze AS from RNAseq data. Most methods start by mapping the reads to an annotated reference genome, but some start by a de novo assembly of the reads. In this paper, we present a systematic comparison of a mapping-first approach (FARLINE) and an assembly-first approach (KISSPLICE). We applied these methods to two independent RNAseq datasets and found that the predictions of the two pipelines overlapped (70% of exon skipping events were common), but with noticeable differences. The assembly-first approach allowed to find more novel variants, including novel unannotated exons and splice sites. It also predicted AS in recently duplicated genes. The mapping-first approach allowed to find more lowly expressed splicing variants, and splice variants overlapping repeats. This work demonstrates that annotating AS with a single approach leads to missing out a large number of candidates, many of which are differentially regulated across conditions and can be validated experimentally. We therefore advocate for the combined use of both mapping-first and assembly-first approaches for the annotation and differential analysis of AS from RNAseq datasets.

Identification of protein features encoded by alternative exons using Exon Ontology.

Transcriptomic genome-wide analyses demonstrate massive variation of alternative splicing in many physiological and pathological situations. One major challenge is now to establish the biological contribution of alternative splicing variation in physiological- or pathological-associated cellular phenotypes. Toward this end, we developed a computational approach, named "Exon Ontology," based on terms corresponding to well-characterized protein features organized in an ontology tree. Exon Ontology is conceptually similar to Gene Ontology-based approaches but focuses on exon-encoded protein features instead of gene level functional annotations. Exon Ontology describes the protein features encoded by a selected list of exons and looks for potential Exon Ontology term enrichment. By applying this strategy to exons that are differentially spliced between epithelial and mesenchymal cells and after extensive experimental validation, we demonstrate that Exon Ontology provides support to discover specific protein features regulated by alternative splicing. We also show that Exon Ontology helps to unravel biological processes that depend on suites of coregulated alternative exons, as we uncovered a role of epithelial cell-enriched splicing factors in the AKT signaling pathway and of mesenchymal cell-enriched splicing factors in driving splicing events impacting on autophagy. Freely available on the web, Exon Ontology is the first computational resource that allows getting a quick insight into the protein features encoded by alternative exons and investigating whether coregulated exons contain the same biological information.

Nucleosome eviction in mitosis assists condensin loading and chromosome condensation.

Condensins associate with DNA and shape mitotic chromosomes. Condensins are enriched nearby highly expressed genes during mitosis, but how this binding is achieved and what features associated with transcription attract condensins remain unclear. Here, we report that condensin accumulates at or in the immediate vicinity of nucleosome-depleted regions during fission yeast mitosis. Two transcriptional coactivators, the Gcn5 histone acetyltransferase and the RSC chromatin-remodelling complex, bind to promoters adjoining condensin-binding sites and locally evict nucleosomes to facilitate condensin binding and allow efficient mitotic chromosome condensation. The function of Gcn5 is closely linked to condensin positioning, since neither the localization of topoisomerase II nor that of the cohesin loader Mis4 is altered in gcn5 mutant cells. We propose that nucleosomes act as a barrier for the initial binding of condensin and that nucleosome-depleted regions formed at highly expressed genes by transcriptional coactivators constitute access points into chromosomes where condensin binds free genomic DNA.

Splicing misregulation of SCN5A contributes to cardiac-conduction delay and heart arrhythmia in myotonic dystrophy.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A. We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents.

The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.

In Vitro and In Vivo Modulation of Alternative Splicing by the Biguanide Metformin.

Major physiological changes are governed by alternative splicing of RNA, and its misregulation may lead to specific diseases. With the use of a genome-wide approach, we show here that this splicing step can be modified by medication and demonstrate the effects of the biguanide metformin, on alternative splicing. The mechanism of action involves AMPK activation and downregulation of the RBM3 RNA-binding protein. The effects of metformin treatment were tested on myotonic dystrophy type I (DM1), a multisystemic disease considered to be a spliceopathy. We show that this drug promotes a corrective effect on several splicing defects associated with DM1 in derivatives of human embryonic stem cells carrying the causal mutation of DM1 as well as in primary myoblasts derived from patients. The biological effects of metformin were shown to be compatible with typical therapeutic dosages in a clinical investigation involving diabetic patients. The drug appears to act as a modifier of alternative splicing of a subset of genes and may therefore have novel therapeutic potential for many more diseases besides those directly linked to defective alternative splicing.

Extended interaction network of procollagen C-proteinase enhancer-1 in the extracellular matrix.

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the ß-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 (KD=58.8 pM), collagen VI (KD=9.5 nM), TSP-1 (thrombospondin-1) (KD1=19.9 pM, KD2=14.5 nM), collagen IV (KD=49.4 nM) and endostatin, a fragment of collagen XVIII (KD1=0.30 nM, KD2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis.

Endothelial, epithelial, and fibroblast cells exhibit specific splicing programs independently of their tissue of origin.

Alternative splicing is the main mechanism of increasing the proteome diversity coded by a limited number of genes. It is well established that different tissues or organs express different splicing variants. However, organs are composed of common major cell types, including fibroblasts, epithelial, and endothelial cells. By analyzing large-scale data sets generated by The ENCODE Project Consortium and after extensive RT-PCR validation, we demonstrate that each of the three major cell types expresses a specific splicing program independently of its organ origin. Furthermore, by analyzing splicing factor expression across samples, publicly available splicing factor binding site data sets (CLIP-seq), and exon array data sets after splicing factor depletion, we identified several splicing factors, including ESRP1 and 2, MBNL1, NOVA1, PTBP1, and RBFOX2, that contribute to establishing these cell type-specific splicing programs. All of the analyzed data sets are freely available in a user-friendly web interface named FasterDB, which describes all known splicing variants of human and mouse genes and their splicing patterns across several dozens of normal and cancer cells as well as across tissues. Information regarding splicing factors that potentially contribute to individual exon regulation is also provided via a dedicated CLIP-seq and exon array data visualization interface. To the best of our knowledge, FasterDB is the first database integrating such a variety of large-scale data sets to enable functional genomics analyses at exon-level resolution.

Mapping of heparin/heparan sulfate binding sites on αvß3 integrin by molecular docking.

Heparin/heparan sulfate interact with growth factors, chemokines, extracellular proteins, and receptors. Integrins are αß heterodimers that serve as receptors for extracellular proteins, regulate cell behavior, and participate in extracellular matrix assembly. Heparin binds to RGD-dependent integrins (αIIbß3, α5ß1, αvß3, and αvß5) and to RGD-independent integrins (α4ß1, αXß2, and αMß2), but their binding sites have not been located on integrins. We report the mapping of heparin binding sites on the ectodomain of αvß3 integrin by molecular modeling. The surface of the ectodomain was scanned with small rigid probes mimicking the sulfated domains of heparan sulfate. Docking results were clustered into binding spots. The best results were selected for further docking simulations with heparin hexasaccharide. Six potential binding spots containing lysine and/or arginine residues were identified on the ectodomain of αvß3 integrin. Heparin would mostly bind to the top of the genu domain, the Calf-I domain of the α subunit, and the top of the ß subunit of RGD-dependent integrins. Three spots were close enough from each other on the integrin surface to form an extended binding site that could interact with heparin/heparan sulfate chains. Because heparin does not bind to the same integrin site as protein ligands, no steric hindrance prevents the formation of ternary complexes comprising the integrin, its protein ligand, and heparin/heparan sulfate. The basic amino acid residues predicted to interact with heparin are conserved in the sequences of RGD-dependent but not of RGD-independent integrins suggesting that heparin/heparan sulfate could bind to different sites on these two integrin subfamilies.

Protein interaction data curation: the International Molecular Exchange (IMEx) consortium.

The International Molecular Exchange (IMEx) consortium is an international collaboration between major public interaction data providers to share literature-curation efforts and make a nonredundant set of protein interactions available in a single search interface on a common website (http://www.imexconsortium.org/). Common curation rules have been developed, and a central registry is used to manage the selection of articles to enter into the dataset. We discuss the advantages of such a service to the user, our quality-control measures and our data-distribution practices.

Toward a systems level view of the ECM and related proteins: a framework for the systematic definition and analysis of biological systems.

Advances in high throughput 'omic technologies are starting to provide unprecedented insights into how components of biological systems are organized and interact. Key to exploiting these datasets is the definition of the components that comprise the system of interest. Although a variety of knowledge bases exist that capture such information, a major challenge is determining how these resources may be best utilized. Here we present a systematic curation strategy to define a systems-level view of the human extracellular matrix (ECM)--a three-dimensional meshwork of proteins and polysaccharides that impart structure and mechanical stability to tissues. Employing our curation strategy we define a set of 357 proteins that represent core components of the ECM, together with an additional 524 genes that mediate related functional roles, and construct a map of their physical interactions. Topological properties help identify modules of functionally related proteins, including those involved in cell adhesion, bone formation and blood clotting. Because of its major role in cell adhesion, proliferation and morphogenesis, defects in the ECM have been implicated in cancer, atherosclerosis, asthma, fibrosis, and arthritis. We use MeSH annotations to identify modules enriched for specific disease terms that aid to strengthen existing as well as predict novel gene-disease associations. Mapping expression and conservation data onto the network reveal modules evolved in parallel to convey tissue-specific functionality on otherwise broadly expressed units. In addition to demonstrating an effective workflow for defining biological systems, this study crystallizes our current knowledge surrounding the organization of the ECM.

MatrixDB, the extracellular matrix interaction database.

MatrixDB (http://matrixdb.ibcp.fr) is a freely available database focused on interactions established by extracellular proteins and polysaccharides. Only few databases report protein-polysaccharide interactions and, to the best of our knowledge, there is no other database of extracellular interactions. MatrixDB takes into account the multimeric nature of several extracellular protein families for the curation of interactions, and reports interactions with individual polypeptide chains or with multimers, considered as permanent complexes, when appropriate. MatrixDB is a member of the International Molecular Exchange consortium (IMEx) and has adopted the PSI-MI standards for the curation and the exchange of interaction data. MatrixDB stores experimental data from our laboratory, data from literature curation, data imported from IMEx databases, and data from the Human Protein Reference Database. MatrixDB is focused on mammalian interactions, but aims to integrate interaction datasets of model organisms when available. MatrixDB provides direct links to databases recapitulating mutations in genes encoding extracellular proteins, to UniGene and to the Human Protein Atlas that shows expression and localization of proteins in a large variety of normal human tissues and cells. MatrixDB allows researchers to perform customized queries and to build tissue- and disease-specific interaction networks that can be visualized and analyzed with Cytoscape or Medusa.

Interaction networks as a tool to investigate the mechanisms of aging.

Biological systems are made up of very large numbers of different components interacting at various scales. Most genes, proteins and other cell components carry out their functions within a complex network of interactions and a single component can affect a wide range of other components. Interactions involved in biological processes have been first characterized individually but this "reductionist" approach suffers from a lack of information about time, space, and context in which the interactions occur in vivo. A global, integrative, approach has been developed for several years, focusing on the building of protein-protein interaction maps or interactomes. These interaction networks are complex systems, where new properties arise. They are part of the emergent field of systems biology, which focuses on studying complex biological systems such as a cell or organism, viewed as an integrated and interacting network of genes, proteins and biochemical reactions. Aging is associated with many diseases, such as cancer, diabetes, cardiovascular and neurodegenerative disorders and this limits the investigation of the mechanisms underlying the aging process when focusing on a single gene or a single biochemical pathway. The integration of existing intracellular interaction networks with the extracellular interaction network we have developed (MatrixDB, http://matrixdb.ibcp.fr ) will contribute to provide further insights into the global mechanisms of aging.