Serum and Salivary IgG and IgA Response After COVID-19 Messenger RNA Vaccination.

Importance: There is still considerable controversy in the literature regarding the capacity of intramuscular messenger RNA (mRNA) vaccination to induce a mucosal immune response. Objective: To compare serum and salivary IgG and IgA levels among mRNA-vaccinated individuals with or without previous SARS-CoV-2 infection. Design, Setting, and Participants: In this cohort study, SARS-CoV-2-naive participants and those with previous infection were consecutively included in the CoviCompare P and CoviCompare M mRNA vaccination trials and followed up to day 180 after vaccination with either the BNT162b2 (Pfizer-BioNTech) vaccine or the mRNA-1273 (Moderna) vaccine at the beginning of the COVID-19 vaccination campaign (from February 19 to June 8, 2021) in France. Data were analyzed from October 25, 2022, to July 13, 2023. Main Outcomes and Measures: An ultrasensitive digital enzyme-linked immunosorbent assay was used for the comparison of SARS-CoV-2 spike-specific serum and salivary IgG and IgA levels. Spike-specific secretory IgA level was also quantified at selected times. Results: A total of 427 individuals were included in 3 groups: participants with SARS-CoV-2 prior to vaccination who received 1 single dose of BNT162b2 (Pfizer-BioNTech) (n = 120) and SARS-CoV-2-naive individuals who received 2 doses of mRNA-1273 (Moderna) (n = 172) or 2 doses of BNT162b2 (Pfizer-BioNTech) (n = 135). The median age was 68 (IQR, 39-75) years, and 228 (53.4%) were men. SARS-CoV-2 spike-specific IgG saliva levels increased after 1 or 2 vaccine injections in individuals with previous infection and SARS-CoV-2-naive individuals. After vaccination, SARS-CoV-2-specific saliva IgA levels, normalized with respect to total IgA levels, were significantly higher in participants with previous infection, as compared with the most responsive mRNA-1273 (Moderna) recipients (median normalized levels, 155 × 10-5 vs 37 × 10-5 at day 29; 107 × 10-5 vs 54 × 10-5 at day 57; and 104 × 10-5 vs 70 × 10-5 at day 180 [P < .001]). In contrast, compared with day 1, spike-specific IgA levels in the BNT162b2-vaccinated SARS-CoV-2-naive group increased only at day 57 (36 × 10-5 vs 49 × 10-5 [P = .01]). Bona fide multimeric secretory IgA levels were significantly higher in individuals with previous infection compared with SARS-CoV-2-naive individuals after 2 antigenic stimulations (median optical density, 0.36 [IQR, 0.16-0.63] vs 0.16 [IQR, 0.10-0.22]; P < .001). Conclusions and Relevance: The findings of this cohort study suggest that mRNA vaccination was associated with mucosal immunity in individuals without prior SARS-CoV-2 infection, but at much lower levels than in previously infected individuals. Further studies are needed to determine the association between specific saliva IgA levels and prevention of infection or transmission.

Patient Derived Xenografts (PDX) Models as an Avatar to Assess Personalized Therapy Options in Uveal Melanoma: A Feasibility Study.

Uveal melanoma is the most common primary intraocular malignancy in adults. Up to 50% of UM patients develop metastatic disease, usually in the liver. When metastatic, the prognosis is poor, and few treatment options exist. Here, we investigated the feasibility of establishing patient-derived xenografts (PDXs) from a patient's tumor in order to screen for therapies that the patient could benefit from. Samples obtained from 29 primary tumors and liver metastases of uveal melanoma were grafted into SCID mice. PDX models were successfully established for 35% of primary patient tumors and 67% of liver metastases. The tumor take rate was proportional to the risk of metastases. PDXs showed the same morphology, the same GNAQ/11, BAP1, and SF3B1 mutations, and the same chromosome 3 and 8q status as the corresponding patient samples. Six PDX models were challenged with two compounds for 4 weeks. We show that, for 31% of patients with high or intermediate risk of metastasis, the timing to obtain efficacy results on PDX models derived from their primary tumors was compatible with the selection of the therapy to treat the patient after relapse. PDXs could thus be a valid tool ("avatar") to select the best personalized therapy for one third of patients that are most at risk of relapse.

Influence of FLG loss-of-function mutations in host-microbe interactions during atopic skin inflammation.

BACKGROUND: Loss-of-function mutations in the filaggrin (FLG) gene directly alter skin barrier function and critically influence atopic inflammation. While skin barrier dysfunction, Th2-associated inflammation and bacterial dysbiosis are well-known characteristics of atopic dermatitis (AD), the mechanisms interconnecting genotype, transcriptome and microbiome remain largely elusive. OBJECTIVE: In-depth analysis of FLG genotype-associated skin gene expression alterations and host-microbe interactions in AD. METHODS: Multi-omics characterization of a cohort of AD patients carrying heterozygous loss-of-function mutations in the FLG gene (ADMut) (n = 15), along with matched wild-type (ADWt) patients and healthy controls. Detailed clinical characterization, microarray gene expression and 16 S rRNA-based microbial marker gene data were generated and analyzed. RESULTS: In the context of filaggrin dysfunction, the transcriptome was characterized by dysregulation of barrier function and water homeostasis, while the lesional skin of ADWt demonstrated the specific upregulation of pro-inflammatory cytokines and T-cell proliferation. S. aureus dominated the microbiome in both patient groups, however, shifting microbial communities could be observed when comparing healthy with non-lesional ADWt or ADMut skin, offering the opportunity to identify microbe-associated transcriptomic signatures. Moreover, an AD core signature with 28 genes, including CCL13, CCL18, BTC, SCIN, RAB31 and PCLO was identified. CONCLUSIONS: Our integrative approach provides molecular insights for the concept that FLG loss-of-function mutations are a genetic shortcut to atopic inflammation and unravels the complex interplay between genotype, transcriptome and microbiome in the human holobiont.

Geniac: Automatic Configuration GENerator and Installer for nextflow pipelines.

With the advent of high-throughput biotechnological platforms and their ever-growing capacity, life science has turned into a digitized, computational and data-intensive discipline. As a consequence, standard analysis with a bioinformatics pipeline in the context of routine production has become a challenge such that the data can be processed in real-time and delivered to the end-users as fast as possible. The usage of workflow management systems along with packaging systems and containerization technologies offer an opportunity to tackle this challenge. While very powerful, they can be used and combined in many multiple ways which may differ from one developer to another. Therefore, promoting the homogeneity of the workflow implementation requires guidelines and protocols which detail how the source code of the bioinformatics pipeline should be written and organized to ensure its usability, maintainability, interoperability, sustainability, portability, reproducibility, scalability and efficiency. Capitalizing on Nextflow, Conda, Docker, Singularity and the nf-core initiative, we propose a set of best practices along the development life cycle of the bioinformatics pipeline and deployment for production operations which target different expert communities including i) the bioinformaticians and statisticians ii) the software engineers and iii) the data managers and core facility engineers. We implemented Geniac (Automatic Configuration GENerator and Installer for nextflow pipelines) which consists of a toolbox with three components: i) a technical documentation available at https://geniac.readthedocs.io to detail coding guidelines for the bioinformatics pipeline with Nextflow, ii) a command line interface with a linter to check that the code respects the guidelines, and iii) an add-on to generate configuration files, build the containers and deploy the pipeline. The Geniac toolbox aims at the harmonization of development practices across developers and automation of the generation of configuration files and containers by parsing the source code of the Nextflow pipeline.

QUARTIC: QUick pArallel algoRithms for high-Throughput sequencIng data proCessing.

Life science has entered the so-called 'big data era' where biologists, clinicians and bioinformaticians are overwhelmed with high-throughput sequencing data. While they offer new insights to decipher the genome structure they also raise major challenges to use them for daily clinical practice care and diagnosis purposes as they are bigger and bigger. Therefore, we implemented a software to reduce the time to delivery for the alignment and the sorting of high-throughput sequencing data.  Our solution is implemented using Message Passing Interface and is intended for high-performance computing architecture. The software scales linearly with respect to the size of the data and ensures a total reproducibility with the traditional tools. For example, a 300X whole genome can be aligned and sorted within less than 9 hours with 128 cores. The software offers significant speed-up using multi-cores and multi-nodes parallelization.

biogitflow: development workflow protocols for bioinformatics pipelines with git and GitLab.

The use of a bioinformatics pipeline as a tool to support diagnostic and theranostic decisions in the healthcare process requires the definition of detailed development workflow guidelines. Therefore, we implemented protocols that describe step-by-step all the command lines and actions that the developers have to follow. Our protocols capitalized on two powerful and widely used tools: git and GitLab. They address two use cases: a nominal mode to develop a new feature in the bioinformatics pipeline and a hotfix mode to correct a bug that occurred in the production environment. The protocols are available as a comprehensive documentation at https://biogitflow.readthedocs.io and the main concepts, steps and principles are presented in this report.

Analytical Platforms 1: Use of Cultured Cells and Fluorescent Read-Out Coupled to NormaCurve Normalization in RPPA.

The analytic platform described in this chapter uses proteins extracted from cultured cells as an infinite source of material to set up, validate, and quality control an RPPA platform. Readout of the arrays uses near-infrared fluorescence labeling and data normalization is performed using the bioinformatics package NormaCurve.In the first part, we will describe the advantages, drawbacks, and different applications of cell line material for RPPA. In the second part, we will describe how the staining protocol, the method of readout, and the normalization method applied afterward are interconnected and should be considered together. Finally, we will describe the NormaCurve package, which is freely available, and its requirements for implementation.Four protocols are provided in this chapter: (1) Protein lysis of cell lines using a homemade Laemmli buffer, (2) RPPA staining for fluorescent readout including a signal amplification step, (3) total protein staining in the visible spectrum for normalization purposes, and (4) total protein staining in the near-infrared spectrum for normalization purposes.

Microbe-host interplay in atopic dermatitis and psoriasis.

Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.

A Quantitative Multivariate Model of Human Dendritic Cell-T Helper Cell Communication.

Cell-cell communication involves a large number of molecular signals that function as words of a complex language whose grammar remains mostly unknown. Here, we describe an integrative approach involving (1) protein-level measurement of multiple communication signals coupled to output responses in receiving cells and (2) mathematical modeling to uncover input-output relationships and interactions between signals. Using human dendritic cell (DC)-T helper (Th) cell communication as a model, we measured 36 DC-derived signals and 17 Th cytokines broadly covering Th diversity in 428 observations. We developed a data-driven, computationally validated model capturing 56 already described and 290 potentially novel mechanisms of Th cell specification. By predicting context-dependent behaviors, we demonstrate a new function for IL-12p70 as an inducer of Th17 in an IL-1 signaling context. This work provides a unique resource to decipher the complex combinatorial rules governing DC-Th cell communication and guide their manipulation for vaccine design and immunotherapies.

Clinical and genetic landscape of treatment naive cervical cancer: Alterations in PIK3CA and in epigenetic modulators associated with sub-optimal outcome.

BACKGROUND: There is a lack of information as to which molecular processes, present at diagnosis, favor tumour escape from standard-of-care treatments in cervical cancer (CC). RAIDs consortium (www.raids-fp7.eu), conducted a prospectively monitored trial, [BioRAIDs (NCT02428842)] with the objectives to generate high quality samples and molecular assessments to stratify patient populations and to identify molecular patterns associated with poor outcome. METHODS: Between 2013 and 2017, RAIDs collected a prospective CC sample and clinical dataset involving 419 participant patients from 18 centers in seven EU countries. Next Generation Sequencing has so far been carried out on a total of 182 samples from 377 evaluable (48%) patients, allowing to define dominant genetic alterations. Reverse phase protein expression arrays (RPPA) was applied to group patients into clusters. Activation of key genetic pathways and protein expression signatures were tested for associations with outcome. FINDINGS: At a median follow up (FU) of 22 months, progression-free survival rates of this FIGO stage IB1-IV population, treated predominantly (87%) by chemoradiation, were65•4% [CI95%: 60•2-71.1]. Dominant oncogenic alterations were seen in PIK3CA (40%), while dominant suppressor gene alterations were seen in KMT2D (15%) and KMT2C (16%). Cumulative frequency of loss-of-function (LOF) mutations in any epigenetic modulator gene alteration was 47% and it was associated with PIK3CA gene alterations in 32%. Patients with tumours harboring alterations in both pathways had a significantly poorer PFS. A new finding was the detection of a high frequency of gains of TLR4 gene amplifications (10%), as well as amplifications, mutations, and non-frame-shift deletions of Androgen receptor (AR) gene in 7% of patients. Finally, RPPA protein expression analysis defined three expression clusters. INTERPRETATION: Our data suggests that patient population may be stratified into four different treatment strategies based on molecular markers at the outset. FUND: European Union's Seventh Program grant agreement No 304810.

ART-DeCo: easy tool for detection and characterization of cross-contamination of DNA samples in diagnostic next-generation sequencing analysis.

Next-generation sequencing (NGS) is routinely used for constitutional genetic analysis. However, cross-contamination between samples constitutes a major risk that could impact the results of the analysis. We have developed ART-DeCo, a tool using the allelic ratio (AR) of the Single Nucleotide Polymorphisms sequenced with regions of interest. When a sample is contaminated by DNA with a different genotype, unexpected ARs are obtained, which are in turn used for detection of contamination with a screening test, followed by identification and quantification of the contaminant. Following optimization, ART-DeCo was applied to 2222 constitutional DNA samples. The screening test was positive for 191 samples. In 33 cases (contamination percentages: 1.3% to 29.2%), the contaminant was identified and was mostly located in adjacent wells. Three other positive cases were due to barcoding errors or mixture of two DNA samples. Interestingly, the last contaminated sample corresponded to a bone marrow transplant recipient. Lastly, no contaminant was identified in 154 weakly positive ( < 4%) samples that were considered to be irrelevant to constitutional genetic analysis. ART-DeCo lends itself to mandatory quality control procedures, also highlighting the delicate steps of library preparation, resulting in practice improvement. Importantly, ART-DeCo can be implemented in any NGS workflow, from gene panel to genome-wide analyses. https://sourceforge.net/projects/ngs-art-deco/ .

Assessment of the novel online delineation workshop dummy run approach using FALCON within a European multicentre trial in cervical cancer (RAIDs).

BACKGROUND AND PURPOSE: Online delineation workshops (ODW) permit training of geographically dispersed participants. The purpose is to evaluate the methodology of an ODW using FALCON to harmonize delineation within a European multicentre trial on locally advanced cervical cancer (LACC). MATERIAL AND METHODS: Two ODW included 46 clinicians (14 centres). Clinicians completed baseline (C1), guideline (C2) and final contours (C3) for external beam radiotherapy (EBRT) and brachytherapy (BT) for LACC. Interobserver and intraobserver variability was evaluated quantitatively (using the DICE index) and qualitatively compared to expert contours. RESULTS: Nine clinicians submitted for EBRT and BT for C1-C3. Thirty-two sent any contour. Interobserver quantitative comparisons for EBRT showed significant improvement for C2 vs. C1 for bowel, CTV node, CTV-p and GTV node with significant detriment for GTV node (C3 vs. C1; C2), CTV-p (C3 vs. C2) and bowel (C3 vs. C2), showing in general an improvement in C2 vs. C1, with a detriment in C3 vs. C2 for two target volumes and an organ at risk. For BT there was significant improvement for C2 vs. C1 for bladder, GTV, HR-CTV and IR-CTV, with significant detriment for bladder (C3 vs. C2), thus overall improvement in C2 vs. C1, with only a detriment in C3 vs. C2 for bladder. Centres using MRI imaging for BT contouring did significantly better in the BT case for HR-CTV than those which used other techniques (C2 vs. C1: p<0.005; C3 vs. C1: p=0.02). Intraobserver quantitative comparisons showed significant improvement contouring a region of interest between C2 vs. C1, C3 vs. C1 and C3 vs. C2 for EBRT and between C2 and C1 for BT. CONCLUSIONS: ODW offer training, initial contouring harmonization and allow assessment of centres.

Spatiotemporal control of interferon-induced JAK/STAT signalling and gene transcription by the retromer complex.

Type-I interferons (IFNs) play a key role in the immune defences against viral and bacterial infections, and in cancer immunosurveillance. We have established that clathrin-dependent endocytosis of the type-I interferon (IFN-α/ß) receptor (IFNAR) is required for JAK/STAT signalling. Here we show that the internalized IFNAR1 and IFNAR2 subunits of the IFNAR complex are differentially sorted by the retromer at the early endosome. Binding of the retromer VPS35 subunit to IFNAR2 results in IFNAR2 recycling to the plasma membrane, whereas IFNAR1 is sorted to the lysosome for degradation. Depletion of VPS35 leads to abnormally prolonged residency and association of the IFNAR subunits at the early endosome, resulting in increased activation of STAT1- and IFN-dependent gene transcription. These experimental data establish the retromer complex as a key spatiotemporal regulator of IFNAR endosomal sorting and a new factor in type-I IFN-induced JAK/STAT signalling and gene transcription.

Precision medicine in cancer: challenges and recommendations from an EU-funded cervical cancer biobanking study.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality worldwide. CC pathogenesis is triggered when human papillomavirus (HPV) inserts into the genome, resulting in tumour suppressor gene inactivation and oncogene activation. Collecting tumour and blood samples is critical for identifying these genetic alterations. METHODS: BIO-RAIDs is the first prospective molecular profiling clinical study to include a substantial biobanking effort that used uniform high-quality standards and control of samples. In this European Union (EU)-funded study, we identified the challenges that were impeding the effective implementation of such a systematic and comprehensive biobanking effort. RESULTS: The challenges included a lack of uniform international legal and ethical standards, complexities in clinical and molecular data management, and difficulties in determining the best technical platforms and data analysis techniques. Some difficulties were encountered by all investigators, while others affected only certain institutions, regions, or countries. CONCLUSIONS: The results of the BIO-RAIDs programme highlight the need to facilitate and standardise regulatory procedures, and we feel that there is also a need for international working groups that make recommendations to regulatory bodies, governmental funding agencies, and academic institutions to achieve a proficient biobanking programme throughout EU countries. This represents the first step in precision medicine.

Whole-genome profiling helps to classify phyllodes tumours of the breast.

AIMS: The aim of this study was to analyse a series of borderline and malignant phyllodes tumours (PTs) of the breast by whole-genome profiling to identify genomic markers that could help to recognise potentially malignant tumours within borderline tumours. METHODS: We evaluated the genetic imbalances of a series of 53 PTs (30 borderline, 23 malignant) using the Human CNV370 BeadChip microarray (Illumina), containing 370 000 SNP markers and correlate this alterations with clinicopathological features. RESULTS: Forty-five PTs (85%) showed chromosome copy number variations (CNVs). Twenty PTs (37%) showed five or more chromosomal imbalances (8/30 borderline (27%) and 12/23 malignant (52%)). The large-scale genetic changes associated with malignant were+7p (9/23), +1q (8/23), -10p (8/23), -13q14 (7/23), +8q (6/23) and +10q (6/23) and borderline were+1q (13/30), -13q14 (9/30), -6q (8/30) and -10p (8/30). Losses in 9p21.3, encompassing CDKN2A/B gene, were present in three tumours (malignant), whereas deletions of 13q, with a minimal region in 13q14.2 encompassing the RB1 gene, were found in 9/30 borderline and 7/28 malignant tumours. High-level amplifications were seen in eight tumours (seven malignant and one borderline): in 7p in three tumours (including EGFR in two), 7q31.2 (including TFEC and MET), 8q24.21 (including MYC) and 8q23.3 (including CSMD3) in one tumour each. CONCLUSIONS: Whole-genome profiling by SNP arrays in PTs leads to identify a high number of CNV, gains of 7p and 8q, losses of 13q and 10, losses in 9p21.3 (CDKN2A/B) and the presence of amplifications, especially involving EGFR, as markers of potentially malignant tumours.

From prospective biobanking to precision medicine: BIO-RAIDs - an EU study protocol in cervical cancer.

BACKGROUND: Cervical cancer (CC) is -second to breast cancer- a dominant cause of gynecological cancer-related deaths worldwide. CC tumor biopsies and blood samples are of easy access and vital for the development of future precision medicine strategies. DESIGN: BIO-RAIDs is a prospective multicenter European study, presently recruiting patients in 6 EU countries. Tumor and liquid biopsies from patients with previously non-treated cervical cancer (stages IB2-IV) are collected at defined time points. Patients receive standard primary treatment according to the stage of their disease. 700 patients are planned to be enrolled. The main objectives are the discovery of -dominant molecular alterations, -signalling pathway activation, and -tumor micro-environment patterns that may predict response or resistance to treatment. An exhaustive molecular analysis is performed using 1° Next generation sequencing, 2° Reverse phase protein arrays and 3° Immuno-histochemistry. DISCUSSION: The clinical study BIO-RAIDs is activated in all planned countries, 170 patients have been recruited till now. This study will make an important contribution towards precision medicine treatments in cervical cancer. The results will support the development of clinical practice guidelines for cervical cancer patients to improve their prognosis and their quality of life. TRIAL REGISTRATION: Clinicaltrials.gov: NCT02428842 , registered 10 February 2015.

Using Transcriptional Signatures to Assess Immune Cell Function: From Basic Mechanisms to Immune-Related Disease.

Assessing human immune response remains a challenge as it involves multiple cell types in specific tissues. The use of microarray-based expression profiling as a tool for assessing the immune response has grown increasingly over the past decade. Transcriptome analyses provide investigators with a global perspective of the complex molecular and cellular events that unfold during the development of an immune response. In this review, we will detail the broad use of gene expression profiling to decipher the complexity of immune responses from disease biomarkers identification to cell activation, polarisation or functional specialisation. We will also describe how such data-driven strategies revealed the flexibility of immune function with common and specific transcriptional programme under multiple stimuli.

Combinatorial code governing cellular responses to complex stimuli.

Cells adapt to their environment through the integration of complex signals. Multiple signals can induce synergistic or antagonistic interactions, currently considered as homogenous behaviours. Here, we use a systematic theoretical approach to enumerate the possible interaction profiles for outputs measured in the conditions 0 (control), signals X, Y, X+Y. Combinatorial analysis reveals 82 possible interaction profiles, which we biologically and mathematically grouped into five positive and five negative interaction modes. To experimentally validate their use in living cells, we apply an original computational workflow to transcriptomics data of innate immune cells integrating physiopathological signal combinations. Up to 9 of the 10 defined modes coexisted in context-dependent proportions. Each interaction mode was preferentially used in specific biological pathways, suggesting a functional role in the adaptation to multiple signals. Our work defines an exhaustive map of interaction modes for cells integrating pairs of physiopathological and pharmacological stimuli.