Defense mechanisms on mucosal surfaces are obviously critical for two contradictive observations in different Lyssavirus hosts:
(a) in carnivores the complete different response to oral vaccination (fox, raccoon) and (b) the identification of bats as reservoir host for various emerging pathogens and their resistance against severe diseases induced by these pathogens in other hosts (Han et al., 2017; Schountz, 2014; Kohl and Kurth, 2014). This raises questions of the evolutionary adaptation in bat or carnivore defense mechanisms by innate resistance and adaptive immunity (Kevin et al., 2017).
As indicated by in-vitro data using localization specific cell lines from European bats the behavioral based transmission via aerosols led to specific, type I and type III interferon induced resistance mechanims on the respiratory and olfactory mucosa (He et al., 2014; He et al., 2013; Zhu, manuscript in preparation). However, due to the difficulties to perform in-vivo experiments using even a very few of the strictly protected European bats these data are not verified in-vivo yet.
The findings that a dog derived RABV virus induced the complete cycle from intramuscular infection to replication of virus within salivary glands and shedding via saliva opened the opportunity to use the mouse a “true model” (Eggerbauer, 2017).  However, this final stage was found not to be universally equal for specific viruses in potential hosts. In two studies with E. fuscus and E.serotinus only in one bat after subcutaneous infection virus shedding was confirmed (Freuling et al., 2009; Fooks et al., 2009). Summarizing these three studies we would like to use the same EBLV 1 isolate used to infect bats to investigate comparatively the induced innate immune mechanisms in-vitro as well as in a mouse trial in-vivo.
We would like to address the following questions in-vitro and in-vivo using fruit bats (Rousettus aegyptiacus), a carnivore species (Vulpes vulpes or Procyon lotor) and mice.
1.      Do innate resistance mechanisms on mucosal surface influence the adaptive immune response after vaccination or infection?
2.      How early immune mechanisms are regulated in different hosts and influence the adaptive immune memory after immunization/infection on mucosal surfaces?
3.      Is there a correlation between receptor expression (NCAM 120 versus 140) and susceptibility for Lyssaviruses in epithelial cells in mucosal surfaces?
4.      Which specific resistance and immunity is responsible for the evolutionary adaptation of bats to tolerate emerging viral pathogens without severe diseases seen in other hosts?
5.      In which way protect an innate, interferon type I and/or type III based response on aerogenic route against Lyssavirus infection? Is this IFN response different in different hosts?
The focus in this project is to investigate in comparative approaches the innate resistance mechanisms and the influence of the induced signaling pathways on the protective, adaptive immune memory in different host using,
(1) the established mouse model plus the excellent toolbox as “pathfinder” and
(2) the comparative analysis in two “natural” hosts (bats and carnivores) to identify different regulated responses on mucosal surfaces.
The expected in-vitro data using comparable location and cell type specific cells lines from different hosts (mouse, bats, and carnivores) will allow designing a very limited number of in-vivo trials to investigate the above-mentioned questions. In these in-vivo experiments we will comparatively analyze the early defense mechanism responsible for the different outcome after infection (a) with the same virus in different hosts, (b) in the same hosts after infection on different routes (intra-muscularly versus intra-nasally) or (c) with similar Lyssavirus clones displaying different virulence’s at different routes of infection (collaboration with S. Finke, Project 1). This could be a base for a rational vaccine design and vaccination protocols to stimulate defined immune mechanisms including both resistance and immunity against emerging pathogens.


Methoden:
During the last project period we cell used culture approaches and established cell lines from organs ‘along’ the aerogenic route from bats (He et al, 2014), carnivores (V. vulpes, P. lotor) and mouse (Schäfer, 2017). In the last months, we have further established about 40 cell lines from fruit bats (R. aegyptiacus; E. helvum; Glossophaga sec.) from mucosal and lymphoid organs. Recently, we analyze their mRNA pattern of cell type specific markers by qRT-PCR. We will use these cell lines for studies of early immune signaling pathways in single monolayer as well as polarized ‘air-liquid-interface’ cell cultures (ALI) to mimic the in-vivo situation on mucosal surfaces. Moreover, in insert culture systems we will study the influence of secreted regulatory molecules on the induction of anti-viral resistance stages.
The sequenced and functionally characterized type I and type III interferons and interferon-stimulated genes of European bats (He et al., 2014; Zhu et al., manuscripts in preparation) will be used to characterize the interaction of different IFN’s in response to Lyssavirus infection.   The identified by NGS type I IFN’s from different carnivores (V.vulpes, P. lotor) will be used in comparative studies to identify different regulatory mechanisms presumably responsible for the observed differences in resistance between bat and carnivores. The transcriptomes of lymphoid organs characterized from two bat species (M. myotis; E. serotinus) and from raccoon (P.lotor) by NGS and databases with identify immune genes will be used to develop quantitative analysis methods to define the transcriptional changes of immunoregulatory genes.
The in-vivo infection trials planned in mice are based on pathogenicity testes in the mouse model performed by /E. Eggerbauer, T.Müller and C. Freuling  were different outcome after infection on different routes were observed. The genome of these Lyssavirus clones is completely sequenced and their genomic variation is defined. This will allow to create comparative in-vivo experiments were defined Lyssavirus clones with known genomic variation will be used to differentiate and identify specific key pathways and key genes in the early immune recognition and regulation between different hosts after different routes of infection. To verify, whether such regulation is involved in different adaptive response after oral vaccination we will investigate the expression pattern of key genes and pathways in samples from previously performed vaccination trials in carnivores (Müller, Freuling).
Downstream analyses to quantify immunoregulatory molecules (IFN, ISG, cytokines, chemokines) in infected brains and other tissues by qRT-PCR and are established and validated in our lab and for a set of chemokines have already been established by S. Finke. Using specific monoclonal antibodies in flow cytometry based assays we will investigate the activation of leukocyte population mainly from mucosal surfaces but also form peripheral lymphoid organs by cytokine secretion.
Protocols to analyze the expression pattern of immunoregulatory cytokines and transcription factors in leukocytes from orally immunized carnivores will be established using cross reactive monoclonal antibodies or in-situ hybridization protocols (FlowFish; RNAScope). To define the humoral immune response we will analyze the antibody repertoire (specificity against the five Lyssavirus proteins) and the immunoglobulin-isotype response using the Immunproteome technology (in collaboration with Frank Schmidt, University Greifswald).


Anfangsdatum: 1. Oktober 2017

geschätzte Dauer: 36 Monate

Bezahlung: TVöD

Veröffentlichungen:
He X, Korytář T, Zhu Y, Pikula J, Bandouchova H, Zukal J, Köllner B. Establishment of Myotis myotis cell lines--model for investigation of host-pathogen interaction in a natural host for emerging viruses. PLoS One. 2014 Oct 8;9(10):e109795. doi: 10.1371
He X, Korytař T, Schatz J, Freuling CM, Müller T, Köllner B. Anti-lyssaviral activity of interferons κ and ω from the serotine bat, Eptesicus serotinus. J Virol. 2014 May;88(10):5444-54. doi: 10.1128
Zhu Y, He X, Schäfer A, Finke S, Müller T, Köllner B. The type I interferons of European bats. Molecular and functional characterization of bat specific anti-Lyssavirus resistance. Manuscript in preparation
Zhu Y, He X, Schäfer A, Finke S, Müller T, Köllner B. The type III interferons of European bats. Molecular and functional characterization of bat specific anti-Lyssavirus resistance. Manuscript in preparation
Schäfer A. 2017 Cellular immune response in brains of lyssavirus-infected mice. Master thesis, University Greifswald 2017


The offered position is part of a network project within FLI with total 5 groups.
We are seeking for a highly motivated young scientist with strong background in immunology and profound experiences in molecular biological techniques. The willingness to perform animal trials is mandatory.
We are a group with recently 4 PhD students working in different projects dealing with immune responses against different pathogens.
An open minded, friendly team player willing to work hard, to share knowledge and looking for an international team is very welcome.
very good knowledge in English is requested.

Please contact me in advance if you have any questions before you submit your application.

* science-jobs-de sublist: SJD-BIOLOGY