Lymphocytic choriomeningitis virus (LCMV) is an old-world arenavirus capable of causing severe diseases in humans. Despite the extensive use of LCMV in studying immune responses to viral infection, very little is known about its entry pathway. As an enveloped virus, the main determinant of LCMV entry is the viral glycoprotein, which allows for the fusion of the viral membrane with that of the target cell, upon specific triggers. While the exact triggers for LCMV GP are currently unknown, low pH and interaction with a yet to be identified host-encoded receptor are likely involved in the activation of the GP fusion activity. This thesis finds that a triad of histidine residues on LCMV’s GP is absolutely critical for infection. Since mutation of the histidine triad had no effect on GP synthesis and did not completely abrogate its ability to bind to cells, our data suggest that the histidine triad are important for a step after virus internalization, potentially allowing low pH sensing. In addition, through the use of engineered soluble GPs, pulldown experiments, and mass spectrometry, various LCMV receptor candidates were identified. These candidates were further validated in order to identify crucial host proteins involved in LCMV entry. This study finds that LCMV GP interacts strongly with the Neuropilin proteins NRP1 and NRP2, and these cellular proteins may play a role in LCMV’s entry pathway during infection.

About 10% of the world population suffers from chronic virus infections such as infections with hepatitis B virus, hepatitis C virus, and human immunodeficiency virus. Chronic virus infection is associated with the loss of functional cytotoxic T cells. Recent studies uncovered several mechanisms that play a central role in T cell exhaustion and thus development of chronic infections. The role of the innate immune system in the development of chronic virus infections remains unclear. Here we show that type I interferons trigger not only inhibition of virus replication but also immunoregulatory functions. The antiviral effects of type I interferons in the liver were dependent on the presence of macrophages. Macrophage depletion led to excessive virus replication and the development of chronic infection. Moreover, specific deletion of the type I interferon receptor on macrophages led to a decreased innate immune response. The regulatory functions of type I interferons were carried out by natural killer cells. Activated natural killer cells inhibited the virus specific cytotoxic T cell response and therefore delayed virus control. Depletion of natural killer cells prevented development of chronic infections and immunopathology. Moreover, type I interferons acted directly on the cytotoxic T cell response. Treatment with interferon 3 lead to increased T cell function. This resulted in induction of autoimmune diabetes in transgenic mice. In conclusion, the nature of the innate immune response triggers the development of chronic virus infection. These results uncover new mechanisms that might provide the foundation for new therapeutic approaches for patients with chronic infections.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.

Viral hepatitis infection represents a significant epidemiological and economic burden on society. Following infection, some patients mount a blunted immune response to the virus, which ultimately can result in chronic infection. FGL2, a member of the fibrinogen-related protein superfamily, has been implicated in vitro in suppressing both innate and adaptive immune responses. In a murine model of acute viral hepatitis caused by Lymphocytic Choriomeningitis Virus strain WE, we demonstrate that FGL2 expressed by reticuloendothelial cells limits viral spread. When expressed by Treg cells FGL2 binds to FCγRIIB and prevents DC maturation and suppresses virus-specific T and B cell responses. We provide compelling evidence to suggest that hepatitis viruses utilize the FGL2-FCγRIIB pathway to evade immune detection. Inhibition of this pathway restores effective cellular and humoral antiviral immune responses towards hepatitis viruses.

No description available.

Professional antigen presenting cells (pAPCs) process and present antigens on their cell surface in association with MHC class I molecules through two general pathways: direct or cross-presentation. The process of antigen presentation by pAPCs to naïve T cells resulting in their proliferation and differentiation into activated cytotoxic lymphocytes (CTLs) is called T cell priming. In these studies, we examine the cross-presentation of antigens from two non-replicating viruses: inactivated Lymphocytic Choriomeningitis virus (LCMV), and recombinant baculovirus encoding the LCMV nucleoprotein (NP). Since effective activation of pAPCs is essential for efficient priming of CD8+ T cells and CTL activation, and because infection with inactivated viruses generally induces an extremely poor level of CTL activation, we examined the activation state of pAPCs by measuring their cytokine profiles following infection to help further delineate their involvement in the CTL response to inactivated viruses. Our results indicate a pro-inflammatory cytokine mRNA upregulation in pAPCs in response to the inactivated virus, similar to the cytokine profiles subsequent to live LCMV infection, but to a lesser extent. In these studies, we also examined CTL activation following infection with inactivated LCMV and bAc-NP. We have demonstrated that the presentation of antigens from inactivated LCMV and bAC-NP results in a low level of CTL activation in vivo, though there is an undetectable level of CTL activation in vitro, in comparison to activation following infection with the live virus. Ultimately, the characterization of the cytokine profiles of pAPCs and the CD8+ T cell profiles induced in response to inactivated LCMV or the baculovirus derived NP may lead to a better understanding of how cross-presentation of these viral antigens may occur. This information may be applied to enhance the process of pAPC activation and T cell priming, for the induction of more effective cellular immune responses and the generation of stronger protective immunity. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-09-02 15:30:13.883

Lymphocytic Choriomeningitis Virus (LCMV) is a common arenavirus and natural murine pathogen that causes congenital neurodevelopmental disease in humans. Exposure to the virus in utero often results in severe and permanent damage the fetal brain and eyes. While usually severe, symptoms vary from case to case. Little is known about the pathological mechanism of congenital LCMV disease. Animal models of congenital LCMV infections suggest that timing of infection during gestation may influence disease outcomes; however, time alone cannot explain all of the variation observed in humans. Another possibility is that individuals are infected with different strains of LCMV. The LCMV genome is composed of four highly conserved genes, yet even single amino acid mutations can cause the virus to exhibit very different properties in animal models. However, the role of viral strain in the context of neurodevelopment remains relatively unexplored. Here, using a rat model of congenital LCMV infection, we demonstrate that three related strains of LCMV produce different patterns of infection and disease states. Infection with the highly neurotropic E350 strain induces a disease comparable to that observed in many confirmed cases of human congenital LCMV infection. While most of these animals survive to adulthood, they suffer permanent motor and behavioral abnormalities. Postmortem analyses of infected brains suggest that this strain has a proclivity for infecting mitotically active regions of the brain, including the cerebellum, olfactory bulb, hippocampus and subventricular region. E350 is not known to induce immunosuppression and viral clearance is likely mediated by a robust T-cell response. Indeed, we find high numbers of inflammatory cells in the brains of E350 animals and elevated pro-inflammatory cytokines and chemokines. The immune response, though responsible for the clearance of the virus from the brain, is also implicated in severe and sometimes permanent brain damage. The Clone 13 strain, a strain typically associated with lymphatic tissue, readily infects the brains of developing rats. Many of these animals do not live to adulthood. Those that survive exhibit extreme stunted growth, but relatively normal neurodevelopment and little discernable neurological disease. By adulthood, the brains of these animals are comparable in size and structure to controls, despite reduced body mass. The Clone 13 infects the same brain regions as the E350, but is not cleared and remained at high titers for the duration of the study. Chronic infection is likely a consequence of the immunosuppressive effects of Clone 13 on the host immune system. The WE2.2 causes a severe disease with both neurological and systemic symptoms. These animals exhibit persistent seizure like discharges during peak infection and significant motor deficits. They all fail to thrive, losing weight shortly after infection and die invariably 9-11 days post-inoculation. The brains of WE2.2 animals exhibit widespread infection of neurons in the cerebellum, hippocampus, olfactory bulbs, and cortex. WE2.2 does not cause immunosuppression and high levels of inflammatory cells are observed in the brain. Cytokine and chemokine expression is complex, without discernible trends and variable by brain region. Finally, we looked at alpha-dystroglycan (α-DG) expression in the brain and compared it with infectivity among the strains. Alpha-dystroglycan is recognized as the principle receptor for LCMV and due to mutations in the viral glycoprotein, certain strains are more dependent on α-DG for infection. Several associations between α-DG expression and viral infectivity were observed; however α-DG expression alone could not explain all the differences in infection patterns.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.

Chronic viral infection is a significant burden on healthcare systems worldwide. Robust anti-viral immune responses are essential for viral clearance. Persistent viruses use a variety of mechanisms to evade immune surveillance including the upregulation of host immunesuppressive factors. Secreted fibrinogen-like protein 2 (FGL2) has been identified as an inhibitory effector molecule in suppressing immune responses in patients with chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) disease. In a murine model of chronic infection caused by Lymphocytic choriomeningitis virus (LCMV) clone 13, we demonstrate that mice deficient in Fgl2 have increased numbers of mature antigen-presenting cells (APC), improved virus-specific cytotoxic T cell immunity and enhanced viral clearance when compared to wild-type mice. These results highlight the importance of the FGL2 inhibitory pathway in immune evasion and provide a rationale to investigate the effects of blocking FGL2 as a novel immune therapeutic in patients suffering from persistent infections.

During viral infections, CD8+ T cells only respond to a select few epitopes derived from the respective foreign pathogen. These epitopes can be organized into a hierarchy, based on their ability to induce T cell priming. Such phenomenon is known as immunodominance. Cytotoxic T cells can be primed through the direct pathway, or the cross-priming pathway. The latter involves exogenously derived viral epitope presentation by uninfected professional antigen presenting cells. It has been previously reported that Lymphocytic Choriomeningitis nucleoprotein expressed in HEK cells (HEK-NP) could be cross presented to CD8+ T cells. In these studies we have used this same HEK-NP model to study the effects of LCMV-NP cross priming on the LCMV immunodominance hierarchy following viral challenge. Our results provide strong evidence that cross priming is an efficient route with which to induce cell-mediated immunity. We also highlight a regulatory role for cross priming in immunodominance by showing that a single dose of HEK-NP can completely shift the immunodominance hierarchy of a typical LCMV infection. Furthermore, we see that the induction of LCMV-NP cross priming boosts anti-viral immunity to subsequent LCMV infections. This work provides strong support for the physiological role that cross priming plays in normal cell-mediated immune responses. It may also provide relevant information to the realm of immunotherapy. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-10 14:33:18.115