Biology subjects

Seifert, J.

Publications and source records attributed to Seifert, J..

13 recordsLinked to original sources

bioRxiv2026

mRNA-based prime-and-pull vaccination combines benefits of subcutaneous and mucosal BCG vaccination

Pulmonary vaccination has been proposed as a strategy to improve protection against tuberculosis, due to the generation of immune cells that more efficiently survey and eliminate infected cells within the lung. However, uncontrolled replication and excessive inflammation associated with mucosal delivery of live-attenuated vaccines highlight the need for alternative strategies that balance efficacy and safety. Here, we describe a prime-and-pull vaccination approach in which systemic immunity is established by subcutaneous BCG vaccination, followed by the induction of local lung immunity through mucosal delivery of lipid nanoparticle-formulated multi-antigen mRNA. Proof-of-concept studies using a model antigen demonstrated the induction of polyfunctional antigen-specific T cells in the lung with minimal inflammatory cell infiltration, compared with mucosal BCG vaccination. Eight Mycobacterium tuberculosis- and BCG-derived proteins were subsequently selected to generate four multi-antigen mRNA constructs. In vivo vaccination and challenge experiments demonstrated that this prime-and-pull strategy is well tolerated and confers protective immunity against tuberculosis in a murine model. Collectively, these data support a modular mRNA-based prime-and-pull vaccination strategy as a translational approach that bridges the improved immunogenicity and efficacy of mucosal BCG vaccination with the safety profile of parenteral BCG administration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/739464v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c0635borg.highwire.dtl.DTLVardef@bb0357org.highwire.dtl.DTLVardef@1d87243org.highwire.dtl.DTLVardef@135bcd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2026

Pulmonary delivery of antigen-enhanced BCG overcomes safety barriers in immunocompromised hosts and protects against TB in the absence of adaptive immunity.

The low efficacy of Bacille Calmette-Guerin (BCG) in preventing pulmonary tuberculosis (TB) underscores the need for improved TB vaccines. Recombinant BCG (rBCG) strains secreting the virulence-associated effector molecule ESAT-6 from Mycobacterium tuberculosis (Mtb) markedly improve efficacy and immunogenicity in animal models of TB but have been considered unsuitable for clinical translation due to safety concerns identified in intravenous SCID mouse models. Here, we demonstrate that pulmonary delivery fundamentally reshapes the safety and protective efficacy of the ESAT-6-secreting rBCG strains, BCG::RD1 and BCG::ESAT6-PE25SS. In sharp contrast to intravenous delivery, pulmonary administration was markedly better tolerated, improved survival, and reduced systemic dissemination and brain pathology of severely immunocompromised mice. Strikingly, pulmonary rBCG vaccination also conferred superior protection against aerosol Mtb challenge in wild-type, type 2 diabetic, and adaptive immunity-deficient Rag1-/- and Rag2-/-Il2rg-/- mice, with BCG::RD1 showing the strongest adaptive immunity-independent protection. Mechanistically, pulmonary rBCG vaccination promoted lung innate immune activation, expansion of myeloid-biased progenitors in the bone marrow, and enhanced antimycobacterial activity of macrophages, consistent with trained innate immunity. Collectively, these findings reveal that pulmonary vaccination largely overcomes safety concerns of ESAT-6-secreting rBCG strains and provide preclinical evidence for a viable strategy to improve protection against TB in immunocompromised individuals.

immunology↗

bioRxiv2025

Spatial patterning of fibroblast TGFβ signaling underlies treatment resistance in rheumatoid arthritis

Treatment-refractory rheumatoid arthritis (RA) is a major unmet need, and the mechanisms driving treatment resistance are poorly understood. To identify molecular determinants of RA non-remission, we performed spatial transcriptomic profiling on pre- and post-treatment synovial tissue biopsies from treatment naive patients who received conventional DMARDs or adalimumab for 6 months. In the baseline biopsies of non-remission patients, we identified significant expansion of fibrogenic fibroblasts marked by high expression of COMP, a fibrosis-associated extracellular matrix protein. COMPhi fibroblasts localized to perivascular niches that, unexpectedly, served as transcriptional hubs for TGF{beta} activity. We identified endothelial-derived Notch signaling as an upstream regulator of fibroblast TGF{beta} signaling via its dual role in driving TGF{beta} isoform expression and suppressing TGF{beta} receptors, generating a proximal-distal gradient of TGF{beta} activity. Further, disruption of steady-state Notch signaling in vitro enabled fibrogenic fibroblast activation. Analysis of post-treatment biopsies revealed marked expansion of COMPhi fibroblasts in non-remission RA patients, despite evidence of successful immune cell depletion, suggesting a spatiotemporal process of fibrogenic remodeling linked to treatment resistance. Collectively, our data implicates targeting of TGF{beta} signaling to prevent exuberant synovial tissue fibrosis as a potential therapeutic strategy for refractory RA.

immunology↗

bioRxiv2025

Critical Assessment of MetaProteome Investigation 2 (CAMPI-2): Multi-laboratory assessment of sample processing methods to stabilize fecal microbiome for functional analysis

BackgroundFecal samples are widely used as a proxy for studying gut microbiome composition in both human and animal research. Fecal metaproteomics provides valuable insights by tracking changes in the relative abundance of microbial taxa and their protein functions. To ensure reliable results, it is crucial to minimize alterations in the metaproteome occurring from sample collection to protein extraction. Therefore, employing effective stabilization methods is essential to preserve the integrity of the fecal metaproteome from sample collection to laboratory analysis, particularly over long distances or when rapid freezing options are not readily available. In line with these needs, the second edition of the Critical Assessment of MetaProteome Investigation (CAMPI-2) was specifically focused on testing sample stabilization protocols to be applied before metaproteomic analysis. ResultsThis collaborative multicenter study assessed the ability of five different stabilization methods, based on two commercial devices and three specific reagents (acetone, lithium dodecyl sulfate, and an RNAlater-like buffer), respectively, to stabilize the fecal metaproteome during room-temperature storage (14 days) and shipment to mass spectrometry facilities. The five methods were tested simultaneously by eight different laboratories across Europe, using aliquots from the same fecal sample. After protein extraction and digestion, duplicate aliquots of the resulting peptides were analyzed independently by two mass spectrometry facilities at distinct international locations. Analysis of the mass spectrometric data using two different search engines revealed that the fecal metaproteome profile differed considerably depending on the stabilization method used in terms of richness, alpha and beta diversity, reproducibility, and quantitative distribution of main taxa and functions. Although each method showed unique strengths and weaknesses, a commercial swab-based device stood out for its remarkable reproducibility and ranked highest for most of the metrics measured. ConclusionsCAMPI-2 allowed a robust evaluation of five different methods for preserving fecal metaproteome samples. The present investigation provides useful data for the design of metaproteomics and multi-omics studies where fecal sampling cannot be immediately followed by long-term storage at -80{degrees}C. Further optimization of the tested protocols is necessary to improve stabilization efficiency and control bias in the taxonomic and functional profile of the gut microbiome.

microbiology↗

bioRxiv2024

Lung epithelial progenitor-mediated release of TGF-β regulates induction and persistence of lung CD8+ TRM cells following mucosal BCG vaccination

A principal reason for the high global morbidity and mortality of tuberculosis (TB) is the lack of efficacy of the only licensed TB vaccine, Bacillus Calmette-Guerin (BCG), as parenteral BCG does not induce local pulmonary immune memory. Animal studies have shown that mucosal BCG vaccination provides superior protection against TB due to generation of lung resident memory T cells (TRM). Here, we demonstrated that following mucosal vaccination with the genetically modified virulent BCG strain, BCG::RD1, distal airway epithelial progenitors were mobilized to assist with restoration of alveolar epithelium. By way of their migration-mediated activation of latent TGF-{beta}, lung CD8+ TRM differentiation was induced. Mucosal vaccinations using nonvirulent strains of BCG in which airway epithelial progenitors were not mobilized, as well as genetic inhibition of migration-mediated activation of TGF-{beta}, resulted in significantly lower numbers of lung CD8+ TRM. In addition, we discovered CD8+ cells with ex-lung and stem-like TRM phenotypes that persisted in the lung-draining mediastinal lymph nodes for up to four months following mucosal BCG vaccination. These results link airway epithelial progenitor-mediated repair of injured lung tissue with induction of resident T cell memory and delineate why persistence of TRM in the lung is short-lived. These findings may explain why mucosal vaccination with virulent BCG strains is more protective against TB and thus have notable implications for future TB vaccine development. One Sentence SummaryFollowing lung damage due to inhalation of virulent BCG, distal airway epithelial progenitor cells interact with lung CD8+ T cells to induce their differentiation into resident memory T cells via migration-mediated activation of TGF-{beta}.

immunology↗

bioRxiv2024

A modular self-assembling and self-adjuvanting multiepitope peptide nanoparticle vaccine platform to improve the efficacy and immunogenicity of BCG

After more than a century since its initial development, Bacille Calmette-Guerin (BCG) remains the only licensed vaccine against tuberculosis (TB). Subunit boosters are considered a viable strategy to enhance BCG efficacy, which often wanes in adolescence. While many studies on booster subunit vaccines have concentrated on recombinant proteins, here we developed a novel modular peptide-based subunit vaccine platform that is flexible, cold-chain independent and customizable to diverse circumstances and populations. Each individual peptide building block consists of a linear arrangement comprising a 15-leucine self-assembly inducer moiety, a Mycobacterium tuberculosis (Mtb) target epitope and an HLA-E binding moiety, with each moiety separated by a triple lysine spacer. The building blocks, in any combination, were able to form a multiepitope nanoparticle. Six Mtb epitopes were selected to produce the self-assembling and self-adjuvanting peptide-based TB nano-vaccine candidate PNx6. In vivo vaccination-challenge experiments demonstrated that subcutaneous boost of parenteral BCG immunization with PNx6 significantly enhanced its immunogenicity and improved its protective efficacy in a murine model of TB by more than 5-fold. Our study present evidence that purely amphiphilic peptides self-assemble into self-adjuvanting nanoparticles with appropriate size and morphology for TB vaccination with great potential for a multitude of other diseases.

microbiology↗

bioRxiv2024

Antiviral defence systems in the rumen microbiome

The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanism. However, the diversity and prevalence of antiviral defence systems in complex communities is still unknown. We therefore investigated the diversity and abundance of viral defence systems in 3038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defence systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defence systems in the genomes. However, the number of defence systems, defence system families and systems density varied widely from genome to genome. Additionally, the number of defence system correlated positively with the number of defence systems families and the genome size. Restriction modification, Abi and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defence systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage-host interaction. ImportancePhages may act antagonistically at the cell level but have a mutualistic interaction at the microbiome level. This interaction shapes the structure of microbial communities and is mainly driven by defence mechanism. However, the diversity of such mechanism is larger than previously thought. Because of that, we described the abundance and diversity of antiviral defence system of a collection of genomes, from the rumen. While defence mechanisms seem to be prevalent among bacteria and archaea, only a few were common. This suggests that most of these defence mechanisms are not present in many rumen microbes but could be shared among different members of the microbial community. This aligned with the pan-immune system model, which appears to be common across different environments.

microbiology↗

bioRxiv2023

Deep immunophenotyping reveals circulating activated lymphocytes in individuals at risk for rheumatoid arthritis

Rheumatoid arthritis (RA) is a systemic autoimmune disease with currently no universally highly effective prevention strategies. Identifying pathogenic immune phenotypes in At-Risk populations prior to clinical disease onset is crucial to establishing effective prevention strategies. Here, we applied mass cytometry to deeply characterize the immunophenotypes in blood from At-Risk individuals identified through the presence of serum antibodies to citrullinated protein antigens (ACPA) and/or first-degree relative (FDR) status (n=52), as compared to established RA (n=67), and healthy controls (n=48). We identified significant cell expansions in At-Risk individuals compared with controls, including CCR2+CD4+ T cells, T peripheral helper (Tph) cells, type 1 T helper cells, and CXCR5+CD8+ T cells. We also found that CD15+ classical monocytes were specifically expanded in ACPA-negative FDRs, and an activated PAX5low naive B cell population was expanded in ACPA-positive FDRs. Further, we developed an "RA immunophenotype score" classification method based on the degree of enrichment of cell states relevant to established RA patients. This score significantly distinguished At-Risk individuals from controls. In all, we systematically identified activated lymphocyte phenotypes in At-Risk individuals, along with immunophenotypic differences among both ACPA+ and ACPA-FDR At-Risk subpopulations. Our classification model provides a promising approach for understanding RA pathogenesis with the goal to further improve prevention strategies and identify novel therapeutic targets.

genomics↗

bioRxiv2023

Novel taxonomic and functional diversity of bacteria from the upper digestive tract of chicken

Strategies to increase the production rate of chicken for human consumption alter the natural process of microbial colonisation and the nutritional performance of the animal. The lack of sufficient reference genomes limits the interpretation of sequencing data and restrain the study of complex functions. In this study, 43 strains obtained from crop, jejunum and ileum of chicken were isolated, characterised and genome analysed to observe their metabolic profiles, adaptive strategies and to serve as future references. Eight isolates represent new species that colonise the upper gut intestinal tract and present consistent adaptations that enable us to predict their ecological role, expanding our knowledge on the adaptive functions. Molecular characterisation confirmed the classification of Clostridium anaerovisceris sp. nov. (DSM 113860= LMG 32675), Clostridium butanolproducens (DSM 115076= LMG 32878), Ligilactobacillus hohenheimius sp. nov. (DSM 113870= LMG 32876), Limosilactobacillus gallus sp. nov. (DSM 113833 =LMG 32623), Limosilactobacillus avium sp. nov. (DSM 113849= LMG 32671), Limosilactobacillus pullum sp. nov. (DSM 115077 =LMG 32877), Limosilactobacillus digestus sp. nov. (DSM113835= LMG 32625) and Limosilactobacillus difficile sp. nov. (DSM 114195= LMG 32875). Strains of Limosilactobacillus were found to be more abundant in the crop, while Ligilactobacillus dominated the ileal digesta. Isolates from crop encode a high number of glycosidases specialized in complex polysaccharides compared to strains isolated from jejunum and ileum. These results represent the first approach for the isolation and detection of bacteria from the chickens upper digestive tract and describe the functional diversity of bacteria inhabiting these regions, improving the potential handling of chicken microbiota with biotechnological applications.

microbiology↗

bioRxiv2023

Role of the NO-GC/cGMP signaling pathway in platelet biomechanics

Cyclic guanosine monophosphate (cGMP) is a second messenger produced by the NO-sensitive guanylyl cyclase (NO-GC) enzyme. In platelets, the NO-GC/cGMP pathway inhibits aggregation. One aspect of the inhibitory mechanism involves changes in the cytoskeleton; however, the molecular mechanisms underlying platelet inhibition and its correlation with cytoskeletal cellular stiffness are poorly understood. We measured the cellular stiffness of individual platelets after treatment with the NO-GC stimulator riociguat or the NO-GC activator cinaciguat, using scanning ion conductance microscopy (SICM). We quantified changes in platelet shape using deep learning-based platelet morphometry. Cytoskeletal actin polymerization and platelet activation were measured by co-immunostaining F-actin and P-selectin, respectively. To test for clinical applicability of NO-GC stimulators in the context of increased thrombogenicity risk, we investigated the effect of riociguat on platelets from human immunodeficiency virus (HIV)-positive patients taking abacavir sulphate (ABC)-containing regimens, compared with HIV-negative volunteers. Stimulation of human and murine platelets with the NO-GC stimulator riociguat or with the NO-GC activator cinaciguat downregulated P-selectin expression, decreased F-actin polymerization, and decreased cellular stiffness by {approx}50%, compared to vehicle control. In addition, platelets became more circular, indicating decreased activation. Riociguat did not cause any change in platelet aggregation or circularity in HIV-positive patients taking ABC-containing regimens. These results corroborate a functional role of the NO-GC enzyme in platelet biomechanics (cellular stiffness) in correlation with the inhibition of platelet activation and morphological changes. The observed changes in stiffness and platelet shape therefore demonstrate the possibility of pharmacologically targeting the NO-GC/cGMP pathway.

biochemistry↗

bioRxiv2022

Dual IgA/IgG family autoantibodies from individuals at-risk for rheumatoid arthritis identify an arthritogenic strain of Subdoligranulum

The mucosal origins hypothesis of rheumatoid arthritis (RA) proposes a central role for mucosal immune responses in the initiation and/or perpetuation of the systemic autoimmunity that occurs with disease. However, the connection between the mucosa and systemic autoimmunity in RA remains unclear. Using dual IgA/IgG family plasmablast-derived monoclonal autoantibodies obtained from peripheral blood of individuals at-risk for RA, we identified cross-reactivity between RA-relevant autoantigens and bacterial taxa in the closely related families Lachnospiraceae and Ruminococcaceae. After generating bacterial isolates within Lachnospiraceae/Ruminococcaceae genus Subdoligranulum from the feces of an individual, we confirmed monoclonal antibody binding as well as MHC class II dependent CD4+ T cell activation in RA cases compared to controls. Additionally, when Subdoligranulum isolate 7 but not isolate 1 colonized germ-free mice, it stimulated Th17 cell expansion, serum RA-relevant IgG autoantibodies, and joint swelling reminiscent of early RA with histopathology characterized by antibody deposition and complement activation. Systemic immune responses were likely due to the generation of colon isolated lymphoid follicles (ILFs) driving increased fecal and serum IgA by isolate 7, as B and T cell depletion not only halted intestinal immune responses but also eliminated detectable clinical disease. In aggregate, these findings demonstrate one mucosal mechanism in RA through which an intestinal strain of bacteria can drive systemic autoantibody generation and joint-centered antibody deposition and immune activation. One-sentence summarySubdoligranulum spp. targeted by rheumatoid arthritis (RA)-relevant autoantibodies activate T cells in individuals with RA, and in mice stimulate autoantibodies and joint swelling associated with antibody deposition and complement activation.

immunology↗

bioRxiv2020

Mapping cellular nanoscale viscoelasticity and relaxation times relevant to growth of living Arabidopsis thaliana plants using multifrequency AFM

The shapes of living organisms are formed and maintained by precise control in time and space of growth, which is achieved by dynamically fine-tuning the mechanical (viscous and elastic) properties of their hierarchically built structures from the nanometer up. Most organisms on Earth including plants grow by yield (under pressure) of cell walls (bio-polymeric matrices equivalent to extracellular matrix in animal tissues) whose underlying nanoscale viscoelastic properties remain unknown. Multifrequency atomic force microscopy (AFM) techniques exist that are able to map properties to a small subgroup of linear viscoelastic materials (those obeying the Kelvin-Voigt model), but are not applicable to growing materials, and hence are of limited interest to most biological situations. Here, we extend existing dynamic AFM methods to image linear viscoelastic behavior in general, and relaxation times of cells of multicellular organisms in vivo with nanoscale resolution, featuring a simple method to test the validity of the mechanical model used to interpret the data. We use this technique to image cells at the surface of living Arabidopsis thaliana hypocotyls to obtain topographical maps of storage E’ = 120 − 200 MPa and loss E’’= 46 − 111 MPa moduli as well as relaxation times τ = 2.2 − 2.7 µs of their cell walls. Our results demonstrate that cell walls, despite their complex molecular composition, display a striking continuity of simple, linear, viscoelastic behavior across scales–following almost perfectly the standard linear solid model–with characteristic nanometer scale patterns of relaxation times, elasticity and viscosity, whose values correlate linearly with the speed of macroscopic growth. We show that the time-scales probed by dynamic AFM experiments (milliseconds) are key to understand macroscopic scale dynamics (e.g. growth) as predicted by physics of polymer dynamics.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2017

Rice paddy Nitrospirae encode and express genes related to sulfate respiration: proposal of the new genus Candidatus Sulfobium

Nitrospirae spp. distantly related to thermophilic, sulfate-reducing Thermodesulfovibrio species are regularly observed in environmental surveys of anoxic marine and freshwater habitats. However, little is known about their genetic make-up and physiology. Here, we present the draft genome of Nitrospirae bacterium Nbg-4 as a representative of this clade and analyzed its in situ protein expression under sulfate-enriched and sulfate-depleted conditions in rice paddy soil. The genome of Nbg-4 was assembled from replicated metagenomes of rice paddy soil that was used to grow rice plants in the presence and absence of gypsum (CaSO4x2H2O). Nbg-4 encoded the full pathway of dissimilatory sulfate reduction and showed expression thereof in gypsum-amended anoxic bulk soil as revealed by parallel metaproteomics. In addition, Nbg-4 encoded the full pathway of dissimilatory nitrate reduction to ammonia, which was expressed in bulk soil without gypsum amendment. The relative abundance of Nbg-4-related metagenome reads was similar under both treatments indicating that it maintained stable populations while shifting its energy metabolism. Further genome reconstruction revealed the potential to utilize butyrate, formate, H2, or acetate as electron donor, with the Wood-Ljungdahl pathway being expressed under both conditions. Comparison to publicly available Nitrospirae genome bins confirmed that the pathway for dissimilatory sulfate reduction is also present in related Nitrospirae recovered from groundwater. Subsequent phylogenomics showed that such microorganisms form a novel genus within the phylum Nitrospirae, with Nbg-4 as a representative species. Based on the widespread occurrence of this novel genus, we propose for Nbg-4 the name Candidatus Sulfobium mesophilum, gen. nov., spec. nov.\n\nImportanceRice paddies are indispensable for food supply but are a major source of the greenhouse gas methane. If not counterbalanced by cryptic sulfur cycling, methane emission from rice paddy fields would be even higher. However, the microorganisms involved in this sulfur cycling are little understood. By using an environmental systems biology approach of Italian rice paddy soil, we could retrieve the population genome of a novel member of the phylum Nitrospirae. This microorganism encoded the full pathway of dissimilatory sulfate reduction and expressed it in situ under sulfate-enriched and anoxic conditions. Phylogenomics and comparison to environmental surveys showed that such microorganisms are actually widespread in freshwater and marine environments. At the same time, they represent a yet undiscovered genus within the little explored Nitrospirae. Our results will be important to design enrichment strategies and postgenomic studies to fully understand the contribution of these novel Nitrospirae to the global sulfur cycle.

microbiology↗