Work plan
The proposed project will address the replication and spread of medically highly relevant pathogens at increasing levels of cell and cell network complexity. Three multi-disciplinary teams will be implemented consisting of virologists, cell biologists, biophysicists, bioinformaticians and mathematicians. These teams will generate and analyze data in the different culture systems to allow cross-comparative mathematical modeling of pathogen replication and spread ranging from single-step growth cycles to complex spread in differentiated and polarized 3D cell systems (Fig. 28).


Comparative mathematical simulation of pathogen replication & spread  

































Team A (Bartenschlager, Frischknecht, Kaderali, Kräusslich, V. Lohmann, Müller, Urban) will analyze single round replication in standard monolayers, where individual replication stages will be quantified in a time-resolved manner. Two cell culture systems will be employed: human hepatocytes (cell lines Huh7 and HepaRG; primary hepatocytes) susceptible for HBV, HDV, HCV, DENV and Plasmodium and human T lymphocytes (cell lines Jurkat and SupT1; primary CD4+ T cells) susceptible for HIV and DENV. Experimental systems and methods will be harmonized to achieve optimal comparability. Quantitative data are already available for HCV, where mass action kinetics and ordinary differential equations have been used to describe viral replication and to explain differences in host cell permissiveness. This mathematical model will be used as basis for the other pathogens where analogous data will be produced. An important aspect is the stochastic nature of single cell permissiveness; cell-to-cell variations have so far largely been ignored, but crucially determine the outcome of an infection. Pathogen spread will be quantitatively analyzed using existing and newly developed fluorescent pathogens, and spatial models of spreading infection will be established and linked with single-cell models. Predictions from these models are fed into iterative cycles of model improvement and models will be validated in primary cell cultures. Targeted perturbations such as knock-down or over-expression of dependency or restriction factors will also be studied. Results for these factors will be integrated into pathogenand cell-type specific replication and spreading models, thus allowing in-silico comparative studies of load- and choke points of pathogen infection and potential drug effects.


Team B: (Bartenschlager, Fackler, Frischknecht, Höfer, Müller, Schwarz) will analyze pathogen spread in 3D culture models. For hepatotropic pathogens, this is based on porous microcarrier beads coated with extracellular matrix, which has been established for Plasmodium. It mimics cell differentiation, polarity and tight junctions, which are limitations for accessibility of entry sites due to cell-cell contact. For entry of hepatocytes, Plasmodia need to be motile before entering a host cell for differentiation. Motility can be quantitatively imaged on 2D substrates, in natural 3D environments and in micro-structured artificial environments. A stochastic model describing Plasmodium motility in micro fabricated obstacle arrays has been established that will be expanded to motility in natural environments, which are three-dimensional and disordered. The model will be expanded to formation and rupture of discrete adhesion sites. To describe the interaction of Plasmodium with hepatocytes, novel models for invasion and growth will be developed. The same or analogous hepatocyte- and T cell-derived 3D culture  models (e.g., cells embedded and imaged in collagen matrices) will be used for the other pathogens. Pathogen spread will be analyzed in a spatio-temporal manner and used to expand mathematical models from standard monolayer cultures. In addition, heterotypic 3D culture models, composed e.g. of hepatocytes and immuno-competent lymphocytes, will be established, thus limiting pathogen spread. Similar approaches will be undertaken in T lymphocyte cultures with the addition of dendritic cells or HIV-specific cytotoxic T cells.


Team C: (Fackler, Höfer, Keppler, Schirmacher, Schwarz, Urban) will validate insights from monolayer and 3D culture systems in ex vivo organotypic cultures. Cultures of human tonsil tissue have already been established as a primary target organ of lymphotropic infection. These cultures are permissive for HIV-1 and most likely also for DENV. Tonsils removed by routine tonsilectomy are cultured on sponge supports at the liquid-air interface as small tissue blocks, and retain their original multi-cell composition, tissue architecture and immune competence. Tissue blocks are amenable for direct monitoring of spread of (fluorescently labeled) pathogens by live imaging and for analysis of functional consequences on target and noninfected bystander cells by flow cytometry and histology. For selected aspects, tonsil tissue can be cultured as aggregates following tissue dispersion, allowing for manipulation of cell composition and cell-cell interactions. A similar model will be established for hepatotropic agents using cultured slices from human liver under conditions allowing pathogen infection and spread. At the highest level of complexity, model predictions will be validated using novel animal models available within CellNetworks (Keppler, Bartenschlager) or clinical data from patient cohorts within the new DZIF, where Heidelberg is a partner. Ultimately, these data should lead to individualized models allowing patient-specific predictions.