Infection & Immunity (I&I) Mission statement The program aims to discover principles of infection and associated pathobiology at the cellular, organ, host, and population level with the goal to develop novel infection intervention and prevention strategies. Program outline Aim of the program Infectious diseases are a continuous threat to human, animal, and economic health. Control of the (re-)emergence and persistence of infectious agents requires detailed knowledge of all aspects of the entire infection chain, i.e. genome plasticity and virulence potential of infectious agents, cellular infection mechanisms, innate and adaptive host defense, and infection dynamics. The overall aim of this research program is to unravel principles of infection at the individual and community level to facilitate targeted development of novel infection intervention and prevention strategies. Research focus Principles of infection and intervention and prevention are investigated using infectious agents that have with zoonotic potential and/or are of major (future) veterinary, public health or economic relevance. Program design In general, infection cycles involve contact with and adaptation of infectious agents to distinct niches in the host, breaching of tissue barriers, multiplication, evasion of the innate and adaptive host defense, and spread to a new host. Development of novel infection intervention and prevention strategies thus requires detailed knowledge of the pathogen, its host, the interplay between pathogen and host, and the interaction of the host with the environment, including other hosts. Unraveling of principles of infection and the associated pathology requires a multidisciplinary approach involving microbiology, immunology, cell biology, biochemistry, pathology, animal health and epidemiology. The I&I program covers and integrates this expertise, providing the optimal setting to achieve our goals. To accommodate the complexity of the infection cycle and to facilitate the development of novel infection control tools, the research program is divided into three coherent research lines that each target key steps in the infection process:  Cellular infection mechanisms  Host defense  Infection dynamics The scope of the respective research lines is given below. I&I Research line 1: Cellular infection mechanisms Departments of I&I, PB, and B&C For most pathogens direct interaction with host cells and exploitation of host cell biology are essential in the establishment of an infection. This research line aims to dissect for selected sets of pathogens the molecular mechanisms that drive the cellular infection and associated changes in host cell biology. Objectives and approach 1. nidovirus and influenza infections Nidoviruses (corona-, toro-, and arteriviruses) and influenza virus cause infections in various animals and humans (SARS, influenza). Objectives are the unraveling of the mechanisms of (i) virus cell entry, (ii) replication, (iii) transcription, (iv) gene expression and (v) progeny virus assembly. Recent development of tools (infectious clones) and strategies (targeted RNA

recombination) for the genetic manipulation of viruses greatly facilitates fundamental studies as well as the development of vaccines and expression vectors. 2. Bacterial mucosal infections Campylobacter and Salmonella frequently colonize animals and are the major bacterial foodborne human pathogens. Objectives are to dissect (i) the bacterial virulence repertoire, (ii) relevant host cell receptors, (iii) the intracellular fate, and (iv) survival strategies for these pathogens in different host species (chicken, human). This approach adds the discovery of novel potential targets of infection intervention and candidate vaccine antigens. 3. host cell membrane dynamics Modulation of host cell membrane dynamics (e.g. interactions with membrane transport machinery and lipid rafts) is a typical trait of many pathogens. Objectives are to define cell membrane dynamics related to host-pathogen interactions via (i) characterization of microdomains from Golgi membranes, (ii) unraveling of the role of the Golgi complex in the membrane dynamics and pathogenesis, and (iii) elucidation of membrane dynamics involved in communication between immune-competent organelles including plasma membranes, phagosomes, and endosomes. This is accomplished using viral and bacterial cellular infection systems. Coherence of the research line The common theme of this research line is the interaction of infectious agents with host cells. Via a comparative infection biology approach using selected sets of different classes of infectious agents (bacteria, viruses), a comprehensive view of the cellular infection strategies of pathogens is obtained. Elucidation of host cell receptors, signaling pathways, virulence factors, and mechanisms via which pathogens modulate host response and cell membrane dynamics is essential for the rational design of novel infection intervention strategies and vaccines, and understanding of fundamental cell biology principles and associated pathology. A main challenge is to translate the molecular pathogen-host cell knowledge to the dynamics of the natural infection at the organand host-level (research line 3). Key publications  de Vries E, Tscherne DM, Wienholts MJ, Cobos-Jiménez V, Scholte F, García-Sastre A, Rottier PJ, de Haan CA (2011) Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway. PLoS Pathog 7: e1001329.  van der Meer-Janssen YP, van Galen J, Batenburg JJ, Helms JB (2010) Lipids in hostpathogen interactions: pathogens exploit the complexity of the host cell lipidome. Prog Lipid Res 49:1- 26.  Reggiori F, Monastyrska I, Verheije MH, Calì T, Ulasli M, Bianchi S, Bernasconi R, de Haan CA, Molinari M (2010) Coronaviruses hijack the LC3-I-positive EDEMosomes, ER-derived vesicles exporting short-lived ERAD regulators, for replication. Cell Host Microbe 7: 500508.  Zeng Q, Langereis MA, van Vliet AL, Huizinga EG, de Groot RJ (2008) Structure of coronavirus hemagglutinin-esterase offers insight into corona and influenza virus evolution. Proc Nat Acad Sci USA 105: 9065-9069. I&I Research line 2: host defense Departments of I&I, B&C, FAH The host immune response towards infectious agents is a key element of infection biology. Vice versa, unraveling of pathogen – host cell interactions leads to the discovery of novel molecular immunology principles. This research line focuses on the innate and adaptive host response and embodies the entire epithelial - DC - B/T cell axis. Understanding of the interplay between infectious agents and the host defense facilitates rational immunomodulation and successful vaccine design and immunotherapy.

Objectives and approach 1. Innate defense The innate host defense is increasingly appreciated as the first barrier for infectious agents and as a key element that orchestrates the adaptive immune response. This research field is rapidly gaining a rather central position in the molecular infection biology research program as it bridges the knowledge of the infectious agents per se with that of the host response, the susceptibility to infection, and infection-associated immunopathology. Primary objectives are identification of (i) innate sensing receptors for pathogens in different hosts (pigs, chicken, bovine, human, mouse), (ii) the regulation of the activation status and cross-talk between signaling pathways, and (iii) the nature and efficacy of innate effector molecules (e.g. antimicrobial peptides, collectins, cytokines, chemokines). Research includes cloning, identification and functional analysis of pathogen recognition receptors (PRR, e.g. Toll-like receptors, type C-lectin receptors, collectins) and their ligands, modulation of PRR function by environmental signals, and regulation of the adaptive immune response (T and B cell function) by innate signals. 2. Adaptive immune response Understanding of the mechanisms that direct the antigen-specific immune response is extremely valuable to vaccine design and understanding of pathogen-associated immunopathology. Primary objectives are unraveling of (i) regulatory mechanisms of antigen presentation by MHC class I and MHC class II, (ii) the role of exosomes in antigen presentation, memory, and tolerization, and their potential as immunomodulatory tools, (iii) pathogen-directed modulation of antigen-specific immunity, and (iv) the regulation of immunity by environment signals (cell stress, heat shock proteins, probiotics). Experiments involve both in vitro and in vivo analysis of immune cells of human, bovine, chicken and mouse species exposed to defined antigens and/or infected with the above-mentioned pathogens with zoonotic potential.

3. Immunomodulation Knowledge of the factors that direct the host defense provides the opportunity to modulate the immune response to our benefit. This is achieved by (i) identification of natural adjuvants of microbial origin (such as LPS) which adds rational vaccine design, (ii) targeted modification of the cell environment (e.g. via probiotics, antimicrobial peptides), and (iii) rational modulation of the immune response using defined microbial molecules. Analysis of immunopathology associated with infections further adds to unravel the principles of immune regulation. 4. Protection‑inducing antigens The main objective here is to (i) identify candidate vaccine antigens via analysis of immune responses during natural and experimental infections (FIPV, Campylobacter, M. paratuberculosis, influenza virus, PRRSV). This approach complements the targeted search for candidate vaccine antigens and key virulence components of research line 1. Coherence of the research line This research line shows strong coherence as innate and adaptive immunity, immunomodulation, immunopathology, and vaccine development are fully complementary but inherently dependent research topics. Key publications  de Zoete MR, Bouwman LI, Keestra AM, van Putten JPM (2011) Cleavage and activation of a Toll-like receptor by microbial proteases. Proc Natl Acad Sci U S A 108: 4968-4973.  de Jong A, Peña-Cruz V, Cheng TY, Clark RA, Van Rhijn I, Moody DB (2010) CD1aautoreactive T cells are a normal component of the human alpha beta T cell repertoire. Nat Immunol 11: 1102-1109.  Nolte-’t Hoen EN, Buschow SI, Anderton SM, Stoorvogel W, Wauben MH (2009) Activated T cells recruit exosomes secreted by dendritic cells via LFA-1. Blood 113:1977-1981.

I&I Research line 3: Infection Dynamics Departments of I&I, FAH, IRAS Infections result from a dynamic interplay between pathogen and host communities that live in distinct environments. Crucial in the establishment, persistence, and transmission of infectious agents are (i) the structure, biology, and adaptation potential of infectious agents, (ii) the natural architecture of the infection niche in the host, (iii) the genetic background and immune status of individuals and a population as a whole, and (iv) the environmental conditions outside the host. This research line addresses infection dynamics and aims to define common principles of (re) emergence, course, persistence, and cycles of infection at the organ, host, and population level as well as evaluation of novel infection prevention and intervention strategies. Objectives and approach 1. Pathogen plasticity The gene repertoire, mechanisms of environmental gene regulation, horizontal gene transfer, and the adaptive potential of infectious agents determine virulence, infection niche, persistence, and spread of infection including the crossing of species barriers. Objectives are to (i) identify relevant genetic differences between related strains and species, (ii) unravel mechanisms of phenotype variation that influences organ- and host-tropism, (iii) define the adaptation potential to changing host environments including intestinal ecology, and (iv) analyze mutations and gene flow (e.g. virulence and drug resistance genes) that influence infection and therapy. The work follows a genomics and molecular approach to identify stable markers of virulence that adds diagnostics, epidemiology, drug development, and vaccine design. 2. Complex infection models Infection dynamics becomes particularly evident in the setting of the natural infection when all players that determine the outcome of infection are present. Hence, important objectives are to (i) develop and (ii) apply well-defined infection models that enable translation of laboratory findings to the natural setting and vice versa, and (iii) to understand the infection-associated pathology in the context of the pathogen and its environment. Established infection models include FIPV in cats, Campylobacter in chicken, and M. paratuberculosis in livestock. 3. Population infection dynamics Detailed knowledge of the interaction between pathogen and host communities forms the basis of transmission studies and facilitates the development of concepts, strategies, and tools to prevent infection or to alleviate or cure the disease. Field observations may add to identify susceptible populations, transmission routes, host determinants that provide natural resistance against infection, and virulence determinants that contribute to the spread of an infection. Objectives are (i) to develop methodological (quantitative) tools to integrate knowledge and explain observed population effects, and (ii) to unravel transmission mechanisms underlying the occurrence, evolution, spread, and control of infectious diseases. 4. Molecular infection prevention and intervention Emerging infectious diseases and the steady increase in drug resistance urges development of novel molecular infection prevention and intervention methods (in addition to conventional measures to improve hygiene and prevent transmission). Primary objectives in this direction are (i) to evaluate the efficacy of probiotics, collectins, anti-microbial peptides and developed vaccines (see also research line 2), and (ii) to explain the effects on infection dynamics at the population, animal, organ and cellular level (iii) to explore the spread of antibiotic resistance and to develop alternative for the use of antibiotics. Coherence of the research line This research line focuses on infection dynamics at the molecular, animal and population level. This complementary approach benefits development as well as evaluation of novel infection intervention and prevention tools.

Key publications  van Duijkeren E, Ten Horn L, Wagenaar JA, de Bruijn M, Laarhoven L, Verstappen K, de Weerd W, Meessen N, Duim B (2011) Suspected horse-to-human transmission of MRSA ST398. Emerg Infect Dis 17: 1137-1139.  van Eijk M, Bruinsma L, Hartshorn KL, White MR, Rynkiewicz MJ, Seaton BA, Hemrika W, Romijn RA, van Balkom BW, Haagsman HP (2011) Introduction of N-linked glycans in the lectin domain of surfactant protein D: impact on interactions with influenza A viruses. J Biol Chem 286: 20137-20151.  Davis SA, Trapman JP, Leirs H, Begon M & Heesterbeek JAP (2008) The abundance threshold for plague as a critical percolation phenomenon. Nature 454: 634-637.  Juhász-Kaszanyitzky E, Jánosi S, Somogyi P, Dán A, van der Graaf-van Bloois L, van Duijkeren E, Wagenaar JA (2007) MRSA transmission between cows and humans. Emerg. Infect. Dis. 213, 630-632. Program organization The program combines research efforts of the Departments of Infectious Diseases and Immunology, Biochemistry and Cell Biology, Pathobiology, and Farm Animal Health, with links to the Institute for Risk Assessment Sciences. The program is embedded in the university research focus area ‘Infection and Immunity’, with links to the research focus area ‘Epidemiology’. I&I contact Prof.dr. H.P. Haagsman (program coordinator)