John Schrader, MD., Ph,D.
Professor, Department of Medicine
Associate Professor of Pathology and Laboratory Medicine
Our research has two major themes both of which concern major classes of proteins secreted by the immune system, cytokines and antibodies.
Antibodies are unique proteins encoded by millions of genes which are made and mutated in our body. The slight differences in structure between antibodies enable them to bind specifically to different targets. The number of different antibodies in each of our personal “repertoire ” of antibodies is enormous, meaning that each of us is likely to make antibodies that can bind any toxin, virus or bacterium. When we encounter a virus for the first time, even a “new’ one like “bird flu”, we will make greatly amplify the production of that specific type of antibody, meaning that –if we recover from that initial infection- we will be immune to that virus. We are exploring the idea that our ability to make antibodies specific for some common viruses or bacteria may not be dependent on us being lucky enough to make by chance an antibody that protected against that microbe. Instead we suspect that our genes have hard-wired us to make antibodies that protect us against common microbes. To study human antibodies, we use a novel technique we have developed to copy human antibodies as “monoclonal” antibodies. “Monoclonal” antibodies are the fastest growing class of new drugs and have provided breakthrough treatments for arthritis, cancer and other diseases. Therefore we are also studying how human monoclonal antibodies can be exploited as research tools or novel therapeutic agents. Our research on antibodies is directly relevant to combating two important human pathogens, human cytolomegalovirus (HCMV), a virus that is an important cause of birth defects, and the pneumococcus, which is a common cause of death from pneumonia. We are also exploring the use of human monoclonal antibodies to treat arthritis or cancer.
Cytokines are hormone-like secreted proteins that are normally produced only when the body is invaded by microorganisms or suffers damage, for example as result of trauma. Cytokines orchestrate the immune response, mobilizing the white blood cells that attack invaders by increasing the production of blood cells, readying them for action and ensuring that they congregate at the site of infection or damage. We have studied the biochemical signals that cytokines make in cells. Our work on cytokines is relevant to diseases caused by dysregulation of the immune system like rheumatoid arthritis and asthma. Because cytokines stimulate stem cells to generate white blood cells, through a process that involves extensive cellular proliferation, our research is also relevant to leukemia. Indeed, because of the similarities in the molecular processes that control cells in all tissues, our work is also relevant to cancers like breast cancer.
Germline V-genes encode key structural features of protective antibodies against human cytomegalovirus (HCMV) and the pneumoccus. HCMV chronically infects most healthy humans, but can cause serious intra-uterine infections in the fetus and life-threatening illnesses in immunocompromised individuals. We used our novel method to generate a series of human monoclonal antibodies against HCMV that bound a critical site on HCMV and neutralized its ability to infect cells. Surprisingly, all known human antibodies against this part of HCMV, even those generated from different individuals, are encoded by genes derived from the same pair of germline IGHV and IGKV genes. Given that these particular germline genes are well-conserved and are present in all humans, we hypothesized that they had co-evolved with HCMV, to enable humans to generate germline-based, primary immunoglobulins antibodies that would bind HCMV and trigger subsequent somatic mutation and affinity maturation. This would increase the chance of generating high-affinity neutralizing antibodies against this potentially damaging virus. To test this idea, we recreated the hypothetical germline-based encoded antibodies, and confirmed that they indeed bound the HCMV epitope. Moreover, the genome of chimpanzees, which are infected with a co-specifically evolved relative of HCMV, contains IGHV and IGKV germline elements that are almost identical to those we identified in humans. We postulate that these genetic elements have been selected during primate evolution to ensure the reliable generation of antibodies that neutralize CMV, provide an innate foundation for the subsequent adaptive immune response. In collaboration with Dr Emil Pai of the University of Toronto, we have compared dimensional structures of these germline-based antibodies and their somatically mutated, high-affinity progeny. These studies indicate that many of the amino acids that make important contacts with the antigen are encoded by germline V-genes. Moreover, somatic hypermutation and affinity maturation did not result in new side-chain contacts, but instead stabilized these germline-encoded contacts. Thus, these germline V-genes provide a pre-adapted foundation for the generation of primary antibodies against HCMV. We have shown that the same pair of germline IGHV and IGKV genes is also used in primary immunoglobulins that bind a sero-type of pneumococcal polysaccharide. Dr Pai has obtained crystallographic data of complexes of a Fab fragment and a pneumococcal polysaccharide subunit that indicates that germline-encoded amino-acids contact the antigen. This suggests that this pair of IGHV and IGKV genes may have evolved under selective pressure from multiple pathogens.
Broadly cross-protective antibodies made by humans against influenza – the development of a universal influenza vaccine Our influenza research started in the spring of 2009, when we started in a collaboration with many people across Canada to generate human monoclonal antibodies against hemagglutinin (HA) of what was known then as “swine flu” from people that had recovered from the pandemic H1N1 influenza virus. These monoclonal antibodies could be used to potentially treat people seriously ill with the infection. In August of 2009 we noticed that many of the antibodies from people recovering from swine flu had the genetic characteristics of artificial antibodies against the HA stem of avian H5N1 influenza. These artificial antibodies, because they targeted the conserved HA stem, cross-protected against many varieties of influenza viruses. We tried our antibodies against the H5 HA and it bound to the HA stem. So we knew in the summer of 2009 that broadly cross-protective antibodies against many types of influenza could be made by humans. For 50 years people thought that humans could only make strain-specific protective antibodies against the mutable HA head. We also noticed that the antibodies that bound to the H5 HA stem also bound well to different batches of the seasonal influenza vaccine. That meant the standard flu vaccine contained the constant part of the flu virus – the HA stem. We predicted that the pandemic H1N1 influenza vaccine would induce antibodies against the HA stem and protect against many different influenza viruses, as the pandemic H1N1 influenza vaccine was produced by the same methods as the seasonal influenza vaccine. When the pandemic H1N1 influenza vaccine become available in November 2009, we analysed the antibodies from vaccinated humans. We found that vaccinated humans produced many antibodies against the HA stem and that these antibodies could protect mice against lethal infections with H5N1. We proved that these antibodies block the function of the HA stem and prevented the entry of the virus into the cell. The influenza virus cannot afford to vary the HA stem because, mutations in the stem weakens the virus and it cannot penetrate and enter the cell.
What was the difference between pandemic influenza and seasonal influenza? Why, when you were vaccinated with the seasonal influenza vaccine, did you not make broadly cross-protective antibodies against many varieties of influenza viruses and why did not long-lasting immunity normally develop to influenza? We suspected that the different thing about pandemic influenza was that humans had no contact with a closely related HA head. When the human immune system is repeatedly stimulated with variants of the head of HA by seasonal influenza every year – either by infection or vaccination – the memory B cells that make antibodies against the HA head, get stimulated, receive T cell help and enter germinal centres to undergo affinity maturation to make antibodies that bind tightly to the new variant of the HA head. However, because the process of affinity maturation takes time, if you have been infected rather than vaccinated, you still get ill. In seasonal influenza, the few memory B cells that make antibodies to the conserved part of the HA stem get out-competed for T cell help by the memory B cells that target the HA head. But if we vaccinate with an influenza virus that was circulating not in humans but in animals – like the 2009 pandemic H1N1 influenza that was circulating in swine – and had a very different head on the HA, the memory B cells that make antibodies against the seasonal influenza HA head would not get stimulated and enter the germinal centres. Thus the few memory B cells that make antibodies to the conserved part of the HA stem could access T cell help and enter affinity maturation. So we are developing a universal flu vaccine based on conventional vaccines based on a mixture flu viruses that were circulating not in humans, but in animals like ducks.
M-Ras Our work on the cytokines and the regulation of hemopoietic cells uncovered a novel protein, M-Ras, which is activated by many cytokines and is involved in some of their effects on cellular proliferation and survival. M-Ras is a new member of the Ras family of proteins that function as molecular switches that control many important biological processes. We identified M-Ras through bioinformatics approaches and went on to identify its binding partners using yeast-two hybrid screens or affinity-directed mass spectrometry. Activation of M-Ras-mediated signaling pathways in normal bone-marrow stem cells immortalized them and transformed them into cancer stem cells that give rise to leukemias. While expression of activated p21 Ras also resulted in the generation of myeloid leukemias, these resembled dendritic cells. These results raise the intriguing possibility that differences in the signals generated by the two Ras proteins determine the direction of differentiation taken by stem cells. We are investigating these ideas using transgenic mice and M-Ras knockout mice. We are also following up on clues that M-Ras may play a critical role in breast cancer and other human cancers. Expression of activated mutants of M-Ras in a mammary epithelial cell line resulted in epithelial- mesenchymal transition and tumorigenicity in vivo.
Ras and Innate Immunity We are investigating the role of Ras proteins in regulating inflammation, focusing on their role downstream of receptors that sense products of microbes or released from tissues by damage. Activation of Ras is likely to be important in regulating both gene expression and morphological changes in inflammatory cells.
RNA-binding Proteins and Immunity While studying the M-Ras pathway, we serendipitously discovered a novel RNA-binding protein that increased in levels when resting T- or B-lymphocytes were activated. We used mass spectrometry to identify the protein, which we call Caprin-1. Caprin-1 is also expressed in all dividing cells as well as in the brain. We have shown using gene-targeting that Caprin-1 is essential for normal cellular proliferation and used proteomic approaches to identify its binding partners. We showed that Caprin-1 heterodimerizes with an RNA-binding protein that has been associated with the Ras pathway, called G3BP-1. We have shown that Caprin-1 also selectively binds mRNA that are involved in cellular proliferation and other processes. We are investigating the physiology of Caprin1 in the immune system.
Wang Y , Thomson CA , Allan LL , Jackson LM , Olson M , Hercus TR , Nero TL , Turner A , Parker MW , Lopez AL , Waddell TK , Anderson GP , Hamilton JA , Schrader JW. (2013). Characterization of pathogenic human monoclonal autoantibodies against GM-CSF. Proceedings of the National Academy of Sciences of the United States of America. 110(19): 7832-7
Thomson CA, Wang Y, Jackson LM, Olson M, Wang W, Liavonchanka A, Keleta L, Silva V, Diederich S, Jones RB, Gubbay J, Pasick J, Petric M, Jean F, Allen VG, Brown EG, Rini JM, Schrader JW. Pandemic H1N1 Influenza Infection and Vaccination in Humans Induces Cross-Protective Antibodies that Target the Hemagglutinin Stem. Frontiers in Immunology, 2012 May; doi: 10.3389/fimmu.2012.00087
Thomson CA, Little KQ, Reason DC, Schrader JW. Somatic Diversity in CDR3 Loops Allows Single V-Genes To Encode Innate Immunological Memories for Multiple Pathogens. J Immunol. 2011 186:2291-2298
Lorén CE, Schrader JW, Ahlgren U, Gunhaga L. FGF signals induce Caprin2 expression in the vertebrate lens. Differentiation. 2009 77:386-94
Kolobova E, Efimov A, Kaerina I, Rishi AK, Schrader JW, Ham A, Larocca MC, Goldenring JR. Microtubule-dependent association of AKAP350A and CCAR1 with RNA stress granules. Exp Cell Res. 2009 Feb 1;315(3):542-55
Thomson, C, Bryson, S., McLean, G, Creagh, L., Pai, E., Schrader, JW. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus. EMBO Journal 27(19):2592-602 (2008)
Schrader JW and McLean GR. Location, location, timing: analysis of cytomegalovirus epitopes for neutralizing antibodies. Immunology Letters.112:58-60 (2007).
Solomon S, Xu Y, Wang B, David MD, Schubert P, Kennedy D, Schrader JW. Distinct structural features of caprin-1 mediate its interaction with G3BP-1 and its induction of phosphorylation of eukaryotic translation initiation factor 2alpha, entry to cytoplasmic stress granules, and selective interaction with a subset of mRNAs. Mol Cell Biol. ;27:2324-42 (2007)
Guo, X, Stratton L, and Schrader JW. Expression of activated M-Ras in hematopoietic stem cells initiates leukemogenic transformation, immortalization and preferential differentiation to mast cell. Oncogene. 13;25(30):4241-4. (2006)
David, M., Cochrane, C.L., Duncan, S.K., and Schrader, J.W. Pure lipopolysaccharide or synthetic Lipid A induces activation of p21Ras in primary macrophages through a pathway dependent on Src family kinases and PI3K. J Immunol 175(12):8236-41 (2005)
Wang, B., David, M, and Schrader, J.W. Absence of Caprin-1 results in defects in cellular proliferation. J Immunol 175(7): 4274-82 (2005)
McLean, G.R., Olsen, O.A., Watt, I.N., Rathanaswami, P., Leslie, K.B., Babcook, J.S., Schrader, JW Recognition of HCMV by Human Primary Immunoglobulins Identifies an Innate Foundation to an Adaptive Immune Response. J Immunol174(8): 4768-78 (2005)
Ward KR, Zhang KX, Somasiri AM, Roskelley CD and Schrader JW Expression of activated M-Ras in a murine mammary epithelial cell line induces epithelial-mesenchymal transition and tumorigenesis. Oncogene. 23(54): 8858 (2004)
Ehrhardt, A, David, M, Ehrhardt, GRA, and Schrader, JW. Distinct mechanisms determine the patterns of differential activation of H-Ras, N-Ras, K-Ras 4B, and M-Ras by receptors for growth factors or antigen. Molecular and Cellular Biology. 24:6311-23 (2004)
Bin Wang PhD, Research Associate
Yanni Wang, Research Assistant/Technician