Biology subjects

Graw, F.

Publications and source records attributed to Graw, F..

13 recordsLinked to original sources

bioRxiv2025

Dynamics of interactions between three major respiratory pathogens in reconstituted human epithelium

Respiratory viral coinfections pose a substantial global health burden, yet the underlying virus-virus interactions remain incompletely understood. Here, we systematically examined the interplay among influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV) using a reconstituted human airway epithelium model. We monitored viral replication dynamics and host transcriptional responses under both simultaneous and sequential infection conditions. Our results reveal complex, asymmetric interactions strongly influenced by infection timing and sequence. IAV exerted a pronounced inhibitory effect on SARS-CoV-2, mediated mainly by a robust type III interferon response. However, transient early enhancement of SARS-CoV-2 by IAV and subsequent bidirectional inhibition were also observed. Transcriptomic profiling identified coinfection-specific gene expression signatures enriched for metabolic and cell death pathways. In IAV-RSV coinfections, IAV generally suppressed RSV replication; strikingly, prior RSV infection significantly enhanced subsequent IAV replication, potentially through RSV-induced cellular remodeling or syncytia formation. Overall, host responses to coinfection were integrated and non-additive, with distinct transcriptional programs suggesting activation of unique cellular pathways beyond canonical antiviral responses. Together, these findings highlight the pivotal role of innate immunity and the order of infection in determining the outcome of respiratory viral coinfections, providing mechanistic insights with implications for clinical management and epidemic modeling. Author SummaryRespiratory viruses often circulate simultaneously; however, their interactions within the same host remain poorly understood. We used a reconstituted human airway epithelium model to examine coinfections involving influenza A virus (IAV), SARS-CoV-2, and respiratory syncytial virus (RSV). Our findings reveal that these viruses do not simply compete or coexist: their interactions are dynamic, asymmetric, and highly dependent on the order in which infections occur. IAV generally suppresses SARS-CoV-2 replication through a strong type III interferon response, although brief early enhancement and later mutual inhibition can occur. Transcriptomic profiling reveals that coinfection triggers a complex array of host responses, including alterations in metabolic and cell-death pathways. In IAV/RSV coinfections, IAV consistently inhibits RSV; however, prior RSV infection markedly boosts subsequent IAV replication--likely due to RSV-induced cellular remodeling, such as syncytia formation. These transcriptional signatures point to altered cell adhesion and motility as key features of such interactions. Overall, our results demonstrate that innate immunity and the sequence of infection shape coinfection outcomes in non-additive ways, driving distinct cellular responses beyond classical antiviral pathways. Understanding these mechanisms is essential for improving clinical management and guiding the development of targeted antiviral strategies.

microbiology↗

bioRxiv2025

Geometry of disordered porous environments regulates cell migration

Cell migration is a dynamic process that is of critical importance to various aspects of living organisms, including organogenesis, wound healing, and immune responses. Several external factors are known to influence and direct active cell movement, such as chemokine gradients or the composition and mechanical properties of the extracellular matrix (ECM). While progress has been made in elucidating some of the biochemical pathways that control cell migration, little is known about the impact of the porous structure of the ECM on active cell motion. Here, by combining computational modelling and theory, we reveal how porous environments, as represented by the ECM, determine cell migration dynamics. Simulating cell movement in a 3D cellular Potts model accounting for amoeboid-like cell shape dynamics, we show that cell migration within disordered porous environments is characterized by distinct transient motility regimes that deviate from persistent motion and are best described by hopping of cells between traps. Using theory, we are able to show how these motility regimes and large scale transport properties are linked to geometrical properties of the microstructure. Importantly, our analyses reveal that spatial heterogeneities in the porosity lead to non-homogeneous cell distributions and effectively guide cell movement towards regions of low porosity, an effect which we here term as porotaxis. Overall, our work reveals the porosity of the ECM as an important control parameter that shapes cell migration and cellular distribution, and provides a conceptual framework to relate experimentally observed cell motility modes to tissue structures and vice versa. This connection between geometry and cell motility could enhance our understanding of how structural elements shape cell migration and tissue organization in various conditions, such as chronic inflammation, immunity, and cancer.

biophysics↗

bioRxiv2024

Quality of vaccination-induced T cell responses is conveyed by polyclonality and high, but not maximum, antigen receptor avidity

While the quantity of vaccination-induced T cells represents a routine immunogenicity parameter, the quality of such responses is poorly understood. Here, we report on a clinical cohort of 29 human healthy individuals who received three mRNA vaccinations against SARS-CoV-2 before any breakthrough infection. We characterized the magnitude, phenotype and clonal composition of CD8 T cell responses against 16 epitope specificities by ELISpot, flow cytometry as well as single-cell RNA, TCR and surface protein sequencing. To test the functionality of identified clonotypes, 106 T cell receptors (TCR) from five epitope-specific repertoires were re-expressed and tested for peptide sensitivity. While recruited repertoires were overall enriched for high-avidity TCRs, differential clonal expansion was not linked to fine avidity differences. Instead, maintenance of polyclonality ensured robustness in counteracting mutational escape of epitopes. Our findings on the induction and maintenance of high-functionality polyclonal T cell repertoires shed light on T cell quality as a neglected criterion in the assessment of vaccine immunogenicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/620795v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@f48079org.highwire.dtl.DTLVardef@1eaa46org.highwire.dtl.DTLVardef@13cfb93org.highwire.dtl.DTLVardef@a8a6e5_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2024

Morphology-dependent entry kinetics and spread of influenza A virus

Influenza A viruses (IAV) display a broad variety of morphologies ranging from spherical to long filamentous virus particles. These diverse phenotypes are believed to allow the virus to overcome various immunological and pulmonary barriers during entry into the airway epithelium and influence the viral entry pathway. Remarkably, lab-adapted IAV strains lost this morphological variance and exhibit preferred spherical morphology. However, it remains unclear which factors lead to this lab-adapted preference and which pulmonary defense factors are responsible for the preferred filamentous morphology in physiological settings. In this study, we established fluorescent reporter viruses with spherical or filamentous morphology but with the same surface glycoproteins. We developed a correlative fluorescence and scanning electron microscopy workflow to analyze the impact of viral morphology on cell-to-cell spread and identify conditions under which IAV with either spherical or filamentous morphology confer an advantage. Our findings demonstrate that filamentous IAV cell-to-cell spread is significantly slower in various cell lines, which can explain the predominant spherical morphology in lab-adapted strains. This observation is consistent with delayed entry kinetics of filamentous viruses structurally analyzed by cellular cryo-electron tomography. We found that cellular junction integrity and mucin do not exert morphology-dependent inhibition of IAV cell-to-cell spread. On the other hand, filamentous virions confer an advantage under the pressure exerted by neutralizing antibodies against hemagglutinin.

microbiology↗

bioRxiv2024

Accurate, fast and memory efficient quantification of immune cell phenotypes in cytometry using machine learning

To achieve accurate and reproducible cytometry data analysis, we benchmarked 19 machine learning algorithms for supervised and unsupervised cell classification. The underlying data encompassed 138 million cells from seven independent datasets including conventional flow cytometry, spectral flow cytometry and mass cytometry. We found that tree-based classifiers and in particular Decision Trees, outperformed other approaches in classification accuracy, speed and memory use. High accuracy was achieved even for cell populations rarer than 1% using decision trees. We validated our decision tree-based approach in a clinical setting using diagnostic blood T cell phenotyping of 107 patients. Automatic quantification of CD4 helper T cell phenotypes achieved 99 % accuracy compared to manual expert assessment. Finally, we combined automated data transformation, supervised and unsupervised gating, an application program interface and a user-friendly desktop-application into FACSPy and FACSPyUI, a fast and scalable open-source toolbox for the analysis and visualization of cytometry data.

bioinformatics↗

bioRxiv2023

An Environmental Restriction impairs HIV-1 virion fusion and triggers innate immune recognition

In vivo, HIV-1 replicates within tissue, but how three-dimensional (3D) tissue-like environments influence viral spread and pathogenesis remains largely unknown. We previously identified an Environmental Restriction to cell-free Virus Infectivity (ERVI), imposed by collagen-rich 3D extracellular matrix (ECM), which diminishes HIV-1 particle infectivity. Here we report that ERVI is broadly implemented by various adhesive ECM components assembled into tissue-like 3D scaffolds. This restriction rapidly reduces infectivity within minutes, is saturable, and affects diverse primary HIV-1 strains and virions with distinct viral glycoproteins by impairing their ability to fuse with target cells. Importantly, particles subjected to ERVI also trigger pronounced pro-inflammatory cytokine secretion by monocyte-derived macrophages. Mechanistic analyses reveal that transient contact with collagen fibers induces conformational changes in the viral envelope glycoprotein (Env) and enhance its recognition by toll-like receptor (TLR) 2. This recognition promotes routing of viral particles into TLR8-positive endosomes, amplifying innate immune sensing of viral RNA genomes. ERVI thus acts via a dual mechanism: directly limiting the fusogenicity of cell-free virions and sensitizing them for innate immune detection. These findings highlight the biophysical properties of the ECM as a previously unrecognized, tissue-intrinsic barrier that restricts HIV-1 spread and promotes local inflammation representing a novel, broadly acting arm of antiviral innate immunity.

immunology↗

bioRxiv2023

Population context drives cell-to-cell variability in interferon response in epithelial cells

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to the neighboring cells) of human intestinal epithelial cells affects single cell response to interferons. We identified that cells at the edge of a cellular colony are significantly more responsive than cells embedded within this colony. We determined that this spatial heterogeneity in IFN response was the result of the polarized basolateral distribution of the IFN receptors making cells located in the center of a cellular colony not responsive to ectopic IFN stimulation. We could demonstrate that this population context driven cell-to-cell variability influences the outcome of viral infection as cells embedded in a cellular colony are not protected by interferons and therefore more susceptible to infection. Our data highlights that the behavior of individual isolated cells does not directly translate to their behavior in a population, placing the population context as a key driver of cell-to-cell heterogeneity in IFN response.

immunology↗

bioRxiv2023

FitMultiCell: Simulating and parameterizing computational models of multi-scale and multi-cellular processes

MotivationBiological tissues are dynamic and highly organized. Multi-scale models are helpful tools to analyze and understand the processes determining tissue dynamics. These models usually depend on parameters that need to be inferred from experimental data to achieve a quantitative understanding, to predict the response to perturbations, and to evaluate competing hypotheses. However, even advanced inference approaches such as Approximate Bayesian Computation (ABC) are difficult to apply due to the computational complexity of the simulation of multi-scale models. Thus, there is a need for a scalable pipeline for modeling, simulating, and parameterizing multi-scale models of multi-cellular processes. ResultsHere, we present FitMultiCell, a computationally efficient and user-friendly open-source pipeline that can handle the full workflow of modeling, simulating, and parameterizing for multi-scale models of multi-cellular processes. The pipeline is modular and integrates the modeling and simulation tool Morpheus and the statistical inference tool pyABC. The easy integration of high-performance infrastructure allows to scale to computationally expensive problems. The introduction of a novel standard for the formulation of parameter inference problems for multi-scale models additionally ensures reproducibility and reusability. By applying the pipeline to multiple biological problems, we demonstrate its broad applicability, which will benefit in particular image-based systems biology. AvailabilityFitMultiCell is available open-source at https://gitlab.com/fitmulticell/fit. Contactjan.hasenauer@uni-bonn.de Supplementary informationSupplementary data are available at https://doi.org/10.5281/zenodo.7646287 online.

systems biology↗

bioRxiv2022

A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes, that do not include the highly prevalent gt1b, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, but cell-to-cell spreading efficiency was not higher as in other isolates like JFH-1. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is the first efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Author summaryChronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibly to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating the first efficient full-replication cycle cell culture model for highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is not only an important novel development facilitating future efforts in vaccine development. It also provides novel perspectives towards our understanding how liver transplantation drives the evolution of viral isolates with high replication capacity, which might contribute to direct pathogenesis of HCV infection.

microbiology↗

bioRxiv2022

Inferring cell motility in complex environments with incomplete tracking data

Cell motility has important influence on cell interactions and functionality for various biological aspects. Deciphering these dynamics often relies on live-cell microscopy measurements, which partly have to deal with limitations that could impair a reliable quantification of their motility. Especially given complex environments and tissue structures, limited observation periods, cells moving in and out of focus and impaired calibration of observation axes often lead to loss of cell tracks and insufficient tracking of motility within several dimensions. However, a reliable quantification of cell motility dynamics is essential when aiming at extrapolating the observed dynamics in order to understand cell population dynamics at larger temporal and spatial scales using appropriate simulation environments. To analyze how incomplete observations affect interpretation and parameterization of cell motility, we combined experimental observations with computational models. Studying individual cell dynamics within 3D collagen environments, we found that the gradual loss of cell tracks leads to an underestimation of several motility parameters with the effect dependent on the collagen density. By extending the automated fitting strategy FitMultiCell to account for cell track loss, we show that we are able to retrieve the actual cell dynamics and, thus, to reliably parameterize cell motility from such incomplete data. Applying our approach to the analysis of CD4+ T cells within 3D collagen environments that were infected with HIV-1, we could show that despite a considerable loss of cell tracks, the data still contained sufficient information to compare individual cell motilities by inferring and simulating their dynamics. Thereby, the analysis allowed us to disentangle the effect of HIV-1 infection and collagen density on individual cell motility. Our extended FitMultiCell-approach presented here provides a solution for the elimination of artifacts from cell track data analysis to robustly infer cell motility dynamics.

systems biology↗

bioRxiv2021

Constant rate of Plasmodium falciparum sexual commitment promotes variable gametocyte proportions as infection progresses over time

Plasmodium falciparum infections persist through long dry seasons at low parasitaemia without causing malaria symptoms and thus remain untreated. In asymptomatic children, increased circulation of infected erythrocytes without adhering to the vascular endothelium is observed during the dry months, compared to febrile malaria in the wet season. However, alterations of parasite sexual commitment and gametocytogenesis have not been investigated. Here, we compared the expression of genes related to sexual commitment and gametocytogenesis, the proportion and density of P. falciparum gametocytes, and the blood concentration of phospholipids in dry season asymptomatic individuals versus symptomatic subjects in the wet season. Additionally, we adapted a within-host mathematical model considering asexual and sexually-committed parasites and gametocytes to understand the dynamics of gametocyte number and proportion as infections progress. Compared to clinical malaria cases, transcripts of late-stage gametocytes were predominantly upregulated in the dry season, associating with increased proportions of mature gametocytes; while transcription of genes related to parasite sexual commitment was unaltered throughout the year. Our data suggest that gametocyte density and proportion diverge as infections progress from recent transmission to chronic carriage, without alterations in the sexual commitment rate over time.

microbiology↗

bioRxiv2021

HCV spread kinetics reveal varying contributions of transmission modes to infection dynamics

Hepatitis C virus (HCV) is capable of spreading within a host by two different transmission modes: cell-free and cell-to-cell. Although viral dissemination and diffusion of viral particles facilitates the infection of distant cells, direct cell-to-cell transmission to uninfected neighboring cells is thought to shield the virus from immune recognition. However, the contribution of each of these transmission mechanisms to HCV spread is unknown. To dissect the contribution of these different transmission modes to HCV spread, we measured HCV lifecycle kinetics and used an in vitro spread assay to monitor HCV spread kinetics after low multiplicity of infection in the absence and presence of a neutralizing antibody that blocks cell-free spread. By analyzing these data with a spatially-explicit mathematical model that describes viral spread on a single-cell level, we quantified the contribution of cell-free and cell-to-cell spread to the overall infection dynamics and show that both transmission modes act synergistically to enhance the spread of infection. Thus, the simultaneous occurrence of both transmission modes likely represents an advantage for HCV that may contribute to the efficient establishment of chronic infection. Notably, the relative contribution of each viral transmission mode appeared to vary dependent on different experimental conditions and suggests that viral spread is optimized according to the environment. Together, our analyses provide insight into the transmission dynamics of HCV and reveal how different transmission modes impact each other. ImportanceHepatitis C Virus can spread within a host by diffusing viral particles or direct cell-to-cell transfer of viral material between infected and uninfected cells. To which extend these cell-free and cell-to-cell transmission modes contribute to HCV spread, establishment of chronicity and antiviral escape is still unknown. By combining in vitro experimental HCV spread data with a multi-scale mathematical model we have disentangled the contribution and interplay of cell-free and cell-to-cell transmission modes during HCV infection. Our analysis revealed synergistic effects between the two transmission modes, with the relative contribution of each transmission mode varying dependent on the experimental conditions. This highlights the adaptability of the virus and suggests that transmission modes might be optimized dependent on the environment, which could contribute to viral persistence.

microbiology↗

bioRxiv2021

Locally confined IFNγ production by CD4+ T cells provides niches for murine cytomegalovirus replication in the salivary gland

Cytomegalovirus (CMV) has evolved a unique virus-host relationship in the salivary glands (SGs) to sustain prolonged viral replication and hence chances for horizontal transmission. Previous reports have established a decisive role for IFN{gamma} producing CD4+ T cells to control murine CMV (MCMV) infection in the SGs; however, micro-anatomical information regarding their mode of action is largely missing. Here, we provide a spatiotemporal analysis of defined antiviral immune actions that eventually culminate in control of lytic MCMV replication in this preferred mucosal niche. CXCR3-mediated guidance of CD4+ T cells towards CXCL9 and CXCL10 expressing cells resulted in discrete clusters close to infection foci where they reported TCR engagement and produced IFN{gamma}. Of note, these clusters occasionally contained CD11c+ antigen-presenting cells with engulfed virus-associated remnants, most likely apoptotic bodies derived from previously infected cells, enabling antigen presentation to CD4+ T cells. The induced IFN{gamma} production within these CD4+ T cell accumulations triggered IFN{gamma}R signaling in a confined perimeter, thereby inducing local, but not organ-wide protection, and allowing MCMV replication to continue at not yet protected sites. Combining experimental data with a mathematical model of the spatiotemporal dynamics of infection and CD4+ T cell dynamics revealed a scenario, in which ultimate MCMV control is achieved through accumulating sites of regionally-confined tissue protection.

immunology↗