Scientific Approach
The planned scientific approach of the FluZooMark project is divided into 6 work packages (WP1-6). A brief description of each is given below, and their internal relationships are visualized in the figure.
Aim
To identify genetic markers of the IAV genome associated with infectivity, host adaptation, transmissibility and severity of IAV infections in humans and pigs
Work plan
Deep full genome sequencing of archived and contemporary IAVs collected from humans and pigs from 2009 and onwards. The sequences will be analyzed using advanced bioinformatics to identify viral genetic markers related to infectivity, host adaption, transmissibility, and severity of IAV. If needed, the sequences will be augmented with samples from the Influenza Research Database (IRD). The applied approach will be based on established bioinformatics pipelines and will include analysis of point mutations and their relation to epidemiological data, phylogenetic analysis of key genomic elements to explore potential transmission including sub-strains and re-assortment events of viral genes. Composition of viral quasi-species will also be assessed together with additional exploratory techniques on modelling the data utilizing e.g. artificial intelligence.
Deliverables
Identification of specific viral genomic molecular markers related to zoonotic transmission, human/swine host adaptation and disease severity. A Post Doc and PhD project on bioinformatics will be completed.
Aim
To identify porcine host factors associated with susceptibility and severity of infection after experimental exposure of swine to swine and human IAV strains, respectively. In this WP, the pigs act as both a host and as a large animal model for human IAV infection.
Work plan
Tissue samples including nasal swaps, mucosa and blood sample will be taken repeatedly from pigs infected with swine- and human-adapted IAV strains. At termination, pathological and histopathological examination of upper and lower respiratory tissues will be performed, and samples from lung, trachea and nasal cavities will be collected. All samples will be analyzed for the gene expression of e.g. pattern recognition receptors, cytokines, and interferons, and will be subjected to proteomic and degradomic profiling, that will identify host proteins decisive for susceptibility, infectivity and severity of infection. The relative quantification of various innate and adaptive immune cell subsets will also be determined.
Deliverables
Identification of local and systemic host response markers of IAV infection susceptibility and severity in swine. This WP will educate one PhD student in early host response modifiers of IAV infection and disease severity in pigs and another in identification and quantification of human and porcine host proteases decisive for IAV infection. This 'One Health' approach will contribute further to our understanding of host factors decisive for IAV infection. Putative targets for development of new antiviral therapies based on inhibitions of virus activating host proteins may well be an additional outcome of the later PhD project.
Aim
To identify host immune responses in human systems infected with zoonotic and seasonal strains of influenza virus.
Work plan
This work plan is designed to complement and integrate specifically with data generated in WP2. While WP2 will focus on swine host systems, WP3 will concentrate on human systems. Seasonal and pandemic strains of influenza virus (overlapping substantially with those used in WP2) will be used to infect primary differentiated epithelial cells and/or macrophages and respiratory organoids, and the innate immune responses will be characterized. To enhance comparison with WP2, many of the same methods will be employed to look at transcriptional responses. The same targets will be explored as detailed in WP2. Depending on the results of the analyses of bulk transcriptional profiling, we will also undertake 10X genomics studies to determine the transcriptional profiles of individual cells. This will allow us exactly to identify which cell types are producing the innate immune mediators and to what extent this subset of cells overlaps with virus-infected cells. In addition to in vitro analysis, we will also look at the innate responses in material derived from infected individuals. Serum and nasal swab samples will be collected from seasonal influenza virus-infected individuals. We will measure cytokine responses and undertake proteomic and degradomic profiling at DTU on human material collected and extracted in the US by shared PhD and postdocs.
Deliverables
Identification of host response markers of IAV infection in humans, which will be correlated with viral and host markers identified in WP1 and WP2, respectively. Identification of host innate markers that correlate with exacerbated disease and therefore will be targets of host-factor mediated treatments.
Aim
To generate modified influenza A viruses (IAV) harboring the genetic markers in the virus genomes identified in WP1.
Work plan
A plasmid system will be used to create influenza viruses modified with the viral genetic markers identified in WP1. The mutations will either be introduced into the gene of interest by site directed mutagenesis or by purchase of plasmids harboring the mutations. The constructs will be verified by full genome sequencing and the function of each plasmid confirmed by exclusion rescue where each modified plasmid will be tested in already established control systems.
Deliverables
Production of genetically modified IAVs for in vitro, ex vivo and in vivo test in WP5. A PhD student will be involved in the molecular work using reverse genetics, which will include a research stay of 6 months at St.
Aim
Functional and phenotypic characterization of wild type and genetically modified influenza A viruses (IAV) and proof-of-concept for host factors using knockout mice.
Work plan
Growth dynamics of genetically modified IAVs will be characterized in already established human- and pig-derived cell lines, primary cell cultures and ex vivo explants of porcine and human nasal and tracheal epithelia. Quantities of antiviral immune factors and levels of inflammation will be measured as host cell response to viral infections. In situ methods to visualize viral and host factors on fixed cell cultures and organ tissues will be established and “real life” virus infection will be investigated in vitro by confocal microscopy. In vivo studies in pigs and ferrets will be completed to investigate the infectivity, pathogenicity, and transmissibility of the genetically modified viruses. Proof-of-concept for the effect of identified host factors of importance will be performed in genetically modified mice.
Deliverables
The phenotypic traits of the genetically modified IAVs are evaluated to confirm the impact of the viral genetic markers and correlated immunological host factors identified. One PhD will be involved in the establishment of in vitro/ex vivo methods and one PhD in establishment of in situ methods for virus and host factor visualization.
Aim
To use the knowledge obtained in WP1-5 to design more effective vaccines.
Work plan
Virus factors identified in WP1 and WP4 to be associated with infectivity, severity and transmissibility will be cloned into expression plasmids and their importance as influenza vaccine components will be tested in pigs.
Adjuvant components targeting the host factors identified in WP2 and WP3 to be of importance for protection or influencing disease severity will be added or inhibited during vaccination and/or during experimental infection. To access the broadness of the influenza A plasmid DNA vaccine, the antisera generated will be screened against a panel of archived and contemporary human and pig virus strains identified in WP1. Phylogenetic analyses performed in WP1 will be used to identify important type-specific and cross-reactive virus epitopes for improved vaccine designs against also potentially new pandemic viruses.
Induced protective immunity, other than the antibodies measured (e.g. cellular mediated immunity), will be tested in selected in vivo challenge experiments by measuring degree of protection and immune responses to identify possible parameters and correlations with protection.
Deliverables
Efficacy measures of different DNA vaccines and immune parameters against circulating virus strains including potentially new pandemic virus strains. Proof-of-concept for the effect on the identified viral and host factors of importance using different combinations of genetic elements of both virus and host. A PhD student will be involved and perform the analyses on the broadness of the vaccine constructs.