Contact Details

E: david.tscharke@anu.edu.au T: (+61 2) 6125 3020 F: (+61 2) 6125 0313

Main Research interests

Viral immunology, poxviruses and herpesviruses, CD8+ T cells

Teaching Activities

BIOL2162: Molecular Biotechnology (Co-Convenor) Medical School: Virology

Awards and Fellowships

NHMRC Howard Florey Centenary Fellowship, 2003-2005 2006 NSW/ACT Young Tall Poppy Science Award and Agilent Award for Life Science, Australian Institute for Policy and Science. NHMRC Career Development Award (RD Wright), 2007-2011

Research Group - Viral Immunology Laboratory

From left in the photo: Yang Wang – NHMRC CJ Martin (postdoctoral) Fellow Stuart Smith – Research technician and lab manager Tracy Yuen – Honours student Yik Chun (Michael) Wong – Honours student David Tscharke Inge Flesch – Postdoctoral Fellow Chien-Wei (Leon) Lin – PhD student

Research Activities

The viral immunology laboratory studies large DNA viruses such as poxviruses and herpesviruses and how they are recognised and fought by the immune system. Our main focus is vaccinia virus, which is best known as the vaccine used to eliminate smallpox from the world. More recently genetically engineered (or recombinant) vaccines based on vaccinia virus have been developed and are being tested in human clinical trials to prevent diseases as diverse as malaria, HIV/AIDS and some cancers. Despite this history and potential future in human medicine, the functions of many of the approximately 200 genes of vaccinia virus remain unknown. Furthermore, many aspects of immunity to vaccinia virus are poorly understood. Immunodominance in CD8+ T cell responses to vaccinia virus One main aim is to understand and manipulate CD8+ T cell responses to viruses and genetically engineered virus vaccines. CD8+ T cells are an important weapon used by humans and other mammals to fight infection. We use vaccinia virus as a model to dissect the factors that contribute to the strength and specificity of anti-viral T cell responses. T cells play a critical role in the immune response to many pathogens and malignancies. In the case of virus infection, CD8+ T cells recognize infected cells and either kill them or secrete anti-viral factors. This recognition of infected cells is mediated by the interaction of the T cell receptor (on the CD8+ T cell) with small portions of virus proteins (peptides of around 8-12 amino acids, referred to as epitopes) presented by MHC class I on the surface of infected cells. One might expect that the immune system would target as many epitopes from a pathogen as possible, but this is not the case. CD8+ T cells specific for only a few peptide/MHC complexes dominate the response to pathogens that potentially display hundreds (if not thousands) of different epitopes. In addition the epitopes that are targeted can be ranked, with those that elicit the greatest number of T cells at the top and those that elicit the fewest at the bottom. This phenomenon is called immunodominance and while the reasons it occurs are not well understood, it has clear implications for vaccine design and anti-viral immunity. Recent work by ourselves and collaborators has defined many of the vaccinia virus proteins and epitopes that are targeted by CD8+ T cells. With this epitope information in hand, our ongoing work aims to understand how virus genetics, the properties of individual viral proteins and infection/vaccination routes affect anti-viral and vaccine-elicited CD8+ T cell responses. Viral infections and eczema Eczema (atopic dermatitis) is a frequent allergy in Australia and around the world. People with eczema can suffer very severe skin infections with some viruses including herpes simplex virus (the virus that causes cold sores) and vaccinia virus but we do not know why this happens. A newly discovered protein called TSLP is now known to be made by skin affected by eczema and there is evidence that TSLP may interfere with the way the body fights viruses. This collaborative project with Frank Carbone’s group at the University of Melbourne will examine whether TSLP programs the immune system so that it is less able to fight herpes and poxviruses Molecular virology Vaccinia virus is predicted to have around 200 genes but the function of around a quarter of these is entirely unknown. Large viruses such as poxviruses have many genes that are not essential for the basic replication of the virus, but rather are important for growth and persistence of the virus in their animal host. Some of these genes modulate the immune response to viruses, others protect infected cells from various anti-viral mechanisms. We are interested in characterising these classes of genes and the role they play in virus pathogenesis and immunity. Vaccination and infection in mice with mutations that affect anti-viral responses A genome-wide screen of mice for mutations that affect their ability to fight viral infections is being done by Drs Guna Karupiah, Edward Bertram and Prof Chris Goodnow at the John Curtin School of Medical Research. As mice of interest are identified by this work, in collaboration with these groups we will test them for their responses to vaccinia virus vaccines and their ability to resist vaccinia and herpes simplex virus infections. Work in the viral immunology lab is funded by grants from the NHMRC and US National Institutes of Health.

Key Collaborators

Guna Karupiah, John Curtin School of Medical Research Edward Bertram & Chris Goodnow, John Curtin School of Medical Research Aude Fahrer, BaMBi Alessandro Sette, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Frank Carbone, Dept. Microbiology and Immunology, University of Melbourne, Vic, Australia

Publications

2009 Flesch, I. E., Y.-P. Woo, Y. Wang, V. Panchanathan, Y.-C. Wong, N. L. La Gruta, T. Cukalac, and D. C. Tscharke. 2009. Altered CD8+ T cell immunodominance after vaccinia virus infection and the naïve repertoire in inbred and F1 mice. J. Immunol. In Press. Yang Wang, Inge E.A. Flesch and David C. Tscharke (2009). Vaccinia virus CD8+ T cell dominance hierarchies cannot be altered by prior immunization with individual peptides. J. Virol. (in press) Lutzky, V. P., J. E. Davis, P. Crooks, M. Corban, M. C. Smith, M. Elliott, L. Morrison, S. Cross, D. Tscharke, B. Panizza, W. Coman, M. Bharadwaj, and D. J. Moss. 2009. Optimisation of LMP-specific CTL expansion for potential adoptive immunotherapy in NPC patients. Immunol. Cell Biol. In Press. 2008 Haeryfar SMM, Hickman HD, Irvine KR, Tscharke DC, Bennink JR, and Yewdell JW. (2008). Terminal deoxynucleotidyl transferase establishes and broadens anti-viral CD8+ T cell immunodominance hierarchies. J. Immunol. 181:649-59. Tellam J, Smith C, Rist M, Webb N, Cooper L, Vuocolo T, Connolly G, Tscharke DC, Devoy MP, and Khanna R. (2008). Regulation of Protein Translation through mRNA Structure Influences MHC Class I Loading and T cell Recognition. PNAS In Press. Oseroff C, Peters B, Pasquetto V, Moutaftsi M, Sidney J, Panchanathan V, Tscharke DC, Maillere B, Grey H, and Sette. A. (2008). Dissociation between epitope hierarchy and immunoprevalence in CD8 responses to vaccinia virus Western Reserve. J. Immunol. In Press. Assarsson E, Greenbaum JA, Sundstrom M, Schaffer L, Hammond JA, Pasquetto V, Oseroff C, Hendrickson RC, Lefkowitz EJ, Tscharke DC, Sidney J, Grey HM, Head SR, Peters B, and Sette A. (2008). Kinetic analysis of a complete poxvirus transcriptome reveals an immediate-early class of genes. PNAS 105:2140-5. 2007 Moor RJ, Morrison LE, Moss DJ, and Tscharke DC. (2007). Use of CD107-based cell sorting ex vivo to enrich subdominant CD8+ T cells in culture. Immunol Cell Biol 85:546-50. Fischer MA, Tscharke DC, Donohue KB, Truckenmiller ME, and Norbury CC. (2007). Reduction of vector gene expression increases foreign antigen-specific CD8+ T-cell priming. J. Gen. Virol. 88:2378-86. Tscharke DC. (2007) Adaptive immunity to vaccinia virus: revisiting an old friend. Future Virology 2 (2):163-172 Tellam J, Fogg MH, Rist M, Connolly G, Tscharke D, Webb N, Heslop L, Wang F, and Khanna R. (2007). Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes. J. Exp. Med. 204:525-32. 2006 Moutaftsi M, Peters B, Pasquetto V, Tscharke DC, Sidney J, Bui HH, Grey H, and Sette A. (2006). A consensus epitope prediction approach identifies the breadth of murine TCD8+-cell responses to vaccinia virus. Nat Biotechnol 24:817-9. Tscharke DC, Woo W-P, Sakala IG, Sidney J, Sette A, Moss DJ, Bennink JR, Karupiah G, and Yewdell JW. (2006). Poxvirus CD8+ T-cell determinants and cross-reactivity in BALB/c mice. J. Virol. 80:6318-23. Clark RH, Kenyon JC, Bartlett NW, Tscharke DC , and Smith GL. (2006). Deletion of gene A41L enhances vaccinia virus immunogenicity and vaccine efficacy. J. Gen. Virol. 87:29-38. 2005 Tscharke D, and Suhrbier A. (2005). From mice to humans. Murine intelligence for human CD8+ T cell vaccine design. Expert Opin. Biol. Ther. 5:263-71. Smith CL, Mirza F, Pasquetto V, Tscharke DC, Palmowski MJ, Dunbar PR, Sette A, Harris AL, and Cerundolo V. (2005). Immunodominance of poxviral-specific CTL in a human trial of recombinant-modified vaccinia Ankara. J. Immunol. 175:8431-7. Tscharke DC , Karupiah G, Zhou J, Palmore T, Irvine KR, Haeryfar SMM, Williams S, Sidney J, Sette A, Bennink JR, and Yewdell JW. (2005). Identification of poxvirus CD8 + T cell determinants to enable rational design and characterization of smallpox vaccines. J. Exp. Med. 201:95-104. Haeryfar SMM, DiPaolo RJ, Tscharke DC, Bennink JR, and Yewdell JW. (2005). Regulatory T cells suppress CD8 + T cell responses induced by direct priming and cross-priming and moderate immunodominance disparities. J. Immunol. 174:3344-51. Pasquetto V, Bui H-H, Giannino R, Banh C, Mirza F, Sidney J, Oseroff C, Tscharke DC, Irvine K, Bennink JR, Peters B, Southwood S, Cerundolo V, Grey HM, Yewdell JW, and Sette A. (2005). HLA-A*0201, HLA-A*1101, and HLA-B*0702 transgenic mice recognize numerous poxvirus determinants from a wide variety of viral gene products. J. Immunol. 175:5504-15. Oseroff C, Kos F, Bui H-H, Peters B, Pasquetto V, Glenn J, Palmore T, Sidney J, Tscharke DC, Bennink JR, Southwood S, Grey HM, Yewdell JW, and Sette A. (2005). HLA class I-restricted responses to vaccinia recognize a broad array of proteins mainly involved in virulence and viral gene regulation. PNAS 102:13980-5. 2004 Norbury CC, Basta S, Donohue KB, Tscharke DC, Princiotta MF, Berglund P, Gibbs J, Bennink JR, and Yewdell JW. (2004). CD8 + T Cell Cross-Priming via Transfer of Proteasome Substrates. Science 304:1318-21. 2003 Tscharke DC , and Yewdell JW. (2003). T cells bite the hand that feeds them. Nat. Med. 9:647-8. Pires de Miranda M, Reading PC, Tscharke DC, Murphy BJ, and Smith GL. (2003). The vaccinia virus kelch-like protein C2L affects calcium-independent adhesion to the extracellular matrix and inflammation in a murine intradermal model. J. Gen. Virol. 84:2459-71. 2002 Price N, Tscharke DC, and Smith GL. (2002). The vaccinia virus B9R protein is a 6 kDa intracellular protein that is non-essential for virus replication and virulence. J. Gen. Virol. 83:873-8. Bartlett N, Symons JA, Tscharke DC, and Smith GL. (2002). The vaccinia virus N1L protein is an intracellular homodimer that promotes virulence. J. Gen. Virol. 83:1965-76. Symons JA, Tscharke DC, Price N, and Smith GL. (2002). A study of the vaccinia virus interferon- ? receptor and its contribution to virus virulence. J. Gen. Virol. 83:1953-64. Tscharke DC , Reading PC, and Smith GL. (2002). Dermal infection with vaccinia virus reveals roles for virus proteins not seen using other inoculation routes. J. Gen. Virol. 83:1977-86. Yewdell JW, and Tscharke DC . (2002). Inside the professionals. Nature 418:923-4. Tscharke DC , and Smith GL. (2002). Notes on transient host range selection for engineering vaccinia virus strain MVA. BioTechniques 33:186-8. Symons JA, Adams E, Tscharke DC , Reading PC, Waldmann H, and Smith GL. (2002). The vaccinia virus C12L protein inhibits mouse IL-18 and promotes virus virulence in the murine intranasal model. J. Gen. Virol. 83:2833-44. Selected earlier publications Tscharke DC, Wilkinson R, and Simmons A. (2000). Use of mRNA differential display to study the action of lymphocyte subsets in vivo and application to a murine model of herpes simplex virus infection. Immunol Lett 74:127-32. Tscharke DC , and Simmons A. (1999). Anti-CD8 treatment alters interleukin-4 but not interferon-gamma mRNA levels in murine sensory ganglia during herpes simplex virus infection. Arch. Virol. 144:2229-38. Tscharke DC , and Smith GL. (1999). A model for vaccinia virus pathogenesis and immunity based on intradermal injection of mouse ear pinnae. J. Gen. Virol. 80:2751-5. Pereira RA, Tscharke DC , and Simmons A. (1994). Upregulation of class I major histocompatibility complex gene expression in primary sensory neurons, satellite cells, and Schwann cells of mice in response to acute but not latent herpes simplex virus infection in vivo. J. Exp. Med. 180: 841-50. Simmons A, and Tscharke DC. (1992). Anti-CD8 impairs clearance of herpes simplex virus from the nervous system: implications for the fate of virally infected neurons. J. Exp. Med. 175: 1337-44.