Renee C. Ireton

Genetic Diversity in the Collaborative Cross Model Recapitulates Human West Nile Virus Disease Outcomes

ABSTRACT West Nile virus (WNV) is an emerging neuroinvasive flavivirus that now causes significant morbidity and mortality worldwide. The innate and adaptive immune responses to WNV infection have been well studied in C57BL/6J inbred mice, but this model lacks the variations in susceptibility, immunity, and outcome to WNV infection that are observed in humans, thus limiting its usefulness to understand the mechanisms of WNV infection and immunity dynamics. To build a model of WNV infection that captures human infection outcomes, we have used the Collaborative Cross (CC) mouse model. We show that this model, which recapitulates the genetic diversity of the human population, demonstrates diversity in susceptibility and outcomes of WNV infection observed in humans. Using multiple F1 crosses of CC mice, we identified a wide range of susceptibilities to infection, as demonstrated through differences in survival, clinical disease score, viral titer, and innate and adaptive immune responses in both peripheral tissues and the central nervous system. Additionally, we examined the Oas1b alleles in the CC mice and confirmed the previous finding that Oas1b plays a role in susceptibility to WNV; however, even within a given Oas1b allele status, we identified a wide range of strain-specific WNV-associated phenotypes. These results confirmed that the CC model is effective for identifying a repertoire of host genes involved in WNV resistance and susceptibility. The CC effectively models a wide range of WNV clinical, virologic, and immune phenotypes, thus overcoming the limitations of the traditional C57BL/6J model, allowing genetic and mechanistic studies of WNV infection and immunity in differently susceptible populations. IMPORTANCE Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans. Mouse models of West Nile virus infection have revealed important details regarding the innate and adaptive immune responses to this emerging viral infection. However, traditional mouse models lack the genetic diversity present in human populations and therefore limit our ability to study various disease outcomes and immunologic mechanisms subsequent to West Nile virus infection. In this study, we used the Collaborative Cross mouse model to more effectively model the wide range of clinical, virologic, and immune phenotypes present upon West Nile virus infection in humans.

RIG-I like receptors in antiviral immunity and therapeutic applications.

The RNA helicase family of RIG-I-like receptors (RLRs) is a key component of host defense mechanisms responsible for detecting viruses and triggering innate immune signaling cascades to control viral replication and dissemination. As cytoplasm-based sensors, RLRs recognize foreign RNA in the cell and activate a cascade of antiviral responses including the induction of type I interferons, inflammasome activation, and expression of proinflammatory cytokines and chemokines. This review provides a brief overview of RLR function, ligand interactions, and downstream signaling events with an expanded discussion on the therapeutic potential of targeting RLRs for immune stimulation and treatment of virus infection.

Targeting Innate Immunity for Antiviral Therapy through Small Molecule Agonists of the RLR Pathway

ABSTRACT The cellular response to virus infection is initiated when pathogen recognition receptors (PRR) engage viral pathogen-associated molecular patterns (PAMPs). This process results in induction of downstream signaling pathways that activate the transcription factor interferon regulatory factor 3 (IRF3). IRF3 plays a critical role in antiviral immunity to drive the expression of innate immune response genes, including those encoding antiviral factors, type 1 interferon, and immune modulatory cytokines, that act in concert to restrict virus replication. Thus, small molecule agonists that can promote IRF3 activation and induce innate immune gene expression could serve as antivirals to induce tissue-wide innate immunity for effective control of virus infection. We identified small molecule compounds that activate IRF3 to differentially induce discrete subsets of antiviral genes. We tested a lead compound and derivatives for the ability to suppress infections caused by a broad range of RNA viruses. Compound administration significantly decreased the viral RNA load in cultured cells that were infected with viruses of the family Flaviviridae, including West Nile virus, dengue virus, and hepatitis C virus, as well as viruses of the families Filoviridae (Ebola virus), Orthomyxoviridae (influenza A virus), Arenaviridae (Lassa virus), and Paramyxoviridae (respiratory syncytial virus, Nipah virus) to suppress infectious virus production. Knockdown studies mapped this response to the RIG-I-like receptor pathway. This work identifies a novel class of host-directed immune modulatory molecules that activate IRF3 to promote host antiviral responses to broadly suppress infections caused by RNA viruses of distinct genera. IMPORTANCE Incidences of emerging and reemerging RNA viruses highlight a desperate need for broad-spectrum antiviral agents that can effectively control infections caused by viruses of distinct genera. We identified small molecule compounds that can selectively activate IRF3 for the purpose of identifying drug-like molecules that can be developed for the treatment of viral infections. Here, we report the discovery of a hydroxyquinoline family of small molecules that can activate IRF3 to promote cellular antiviral responses. These molecules can prophylactically or therapeutically control infection in cell culture by pathogenic RNA viruses, including West Nile virus, dengue virus, hepatitis C virus, influenza A virus, respiratory syncytial virus, Nipah virus, Lassa virus, and Ebola virus. Our study thus identifies a class of small molecules with a novel mechanism to enhance host immune responses for antiviral activity against a variety of RNA viruses that pose a significant health care burden and/or that are known to cause infections with high case fatality rates.

A Mouse Model of Chronic West Nile Virus Disease

Infection with West Nile virus (WNV) leads to a range of disease outcomes, including chronic infection, though lack of a robust mouse model of chronic WNV infection has precluded identification of the immune events contributing to persistent infection. Using the Collaborative Cross, a population of recombinant inbred mouse strains with high levels of standing genetic variation, we have identified a mouse model of persistent WNV disease, with persistence of viral loads within the brain. Compared to lines exhibiting no disease or marked disease, the F1 cross CC(032x013)F1 displays a strong immunoregulatory signature upon infection that correlates with restraint of the WNV-directed cytolytic response. We hypothesize that this regulatory T cell response sufficiently restrains the immune response such that a chronic infection can be maintained in the CNS. Use of this new mouse model of chronic neuroinvasive virus will be critical in developing improved strategies to prevent prolonged disease in humans.

Oas1b-dependent immune transcriptional profiles of west nile virus infection in the collaborative cross

The oligoadenylate-synthetase (Oas) gene locus provides innate immune resistance to virus infection. In mouse models, variation in the Oas1b gene influences host susceptibility to flavivirus infection. However, the impact of Oas variation on overall innate immune programming and global gene expression among tissues and in different genetic backgrounds has not been defined. We examined how Oas1b acts in spleen and brain tissue to limit West Nile virus (WNV) susceptibility and disease across a range of genetic backgrounds. The laboratory founder strains of the mouse Collaborative Cross (CC) (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/HlLtJ) all encode a truncated, defective Oas1b, whereas the three wild-derived inbred founder strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ) encode a full-length OAS1B protein. We assessed disease profiles and transcriptional signatures of F1 hybrids derived from these founder strains. F1 hybrids included wild-type Oas1b (F/F), homozygous null Oas1b (N/N), and heterozygous offspring of both parental combinations (F/N and N/F). These mice were challenged with WNV, and brain and spleen samples were harvested for global gene expression analysis. We found that the Oas1b haplotype played a role in WNV susceptibility and disease metrics, but the presence of a functional Oas1b allele in heterozygous offspring did not absolutely predict protection against disease. Our results indicate that Oas1b status as wild-type or truncated, and overall Oas1b gene dosage, link with novel innate immune gene signatures that impact specific biological pathways for the control of flavivirus infection and immunity through both Oas1b-dependent and independent processes.

A small-molecule IRF3 agonist functions as an influenza vaccine adjuvant by modulating the antiviral immune response

Vaccine adjuvants are essential to drive a protective immune response in cases where vaccine antigens are weakly immunogenic, where vaccine antigen is limited, or where an increase in potency is needed for a specific population, such as the elderly. To discover novel vaccine adjuvants, we used a high-throughput screen (HTS) designed to identify small-molecule agonists of the RIG-I-like receptor (RLR) pathway leading to interferon regulatory factor 3 (IRF3) activation. RLRs are a group of cytosolic pattern-recognition receptors that are essential for the recognition of viral nucleic acids during infection. Upon binding of viral nucleic acid ligands, the RLRs become activated and signal to transcription factors, including IRF3, to initiate an innate immune transcriptional program to control virus infection. Among our HTS hits were a series of benzothiazole compounds from which we designed the lead analog, KIN1148. KIN1148 induced dose-dependent IRF3 nuclear translocation and specific activation of IRF3-responsive promoters. Prime-boost immunization of mice with a suboptimal dose of a monovalent pandemic influenza split virus H1N1 A/California/07/2009 vaccine plus KIN1148 protected against a lethal challenge with mouse-adapted influenza virus (A/California/04/2009) and induced an influenza virus-specific IL-10 and Th2 response by T cells derived from lung and lung-draining lymph nodes. Prime-boost immunization with vaccine plus KIN1148, but not prime immunization alone, induced antibodies capable of inhibiting influenza virus hemagglutinin and neutralizing viral infectivity. Nevertheless, a single immunization with vaccine plus KIN1148 provided increased protection over vaccine alone and reduced viral load in the lungs after challenge. These findings suggest that protection was at least partially mediated by a cellular immune component and that the induction of Th2 and immunoregulatory cytokines by a KIN1148-adjuvanted vaccine may be particularly beneficial for ameliorating the immunopathogenesis that is associated with influenza viruses.

Transcriptional profiles of WNV neurovirulence in a genetically diverse Collaborative Cross population.

West Nile Virus (WNV) is a mosquito-transmitted virus from the Flaviviridae family that causes fever in 1 in 5 infected people. WNV can also become neuro-invasive and cross the blood-brain barrier leading to severe neurological symptoms in a subset of WNV infected individuals [1]. WNV neuro-invasion is believed to be influenced by a number of factors including host genetics. In order to explore these effects and recapitulate the complex immune genetic differences among individuals, we studied gene expression following WNV infection in the Collaborative Cross (CC) model. The CC is a mouse genetics resource composed of > 70 independently bred, octo-parental recombinant inbred mouse lines [2]. To identify the individual host gene expression signatures influencing protection or susceptibility to WNV disease and WNV neuroinvasion, we used the nanostring nsolver platform to quantify gene expression in brain tissue isolated from WNV-infected CC mice at days 4, 7 and 12 post-infection [3]. This nanostring technology provided a high throughput, non-amplification based mRNA quantitation method to detect immune genes involved in neuro-invasion. Data was deposited into the Gene Expression Omnibus (GEO) under accession GSE85999.

Immune correlates of protection from West Nile virus neuroinvasion and disease

BACKGROUND A challenge to the design of improved therapeutic agents and prevention strategies for neuroinvasive infection and associated disease is the lack of known natural immune correlates of protection. A relevant model to study such correlates is offered by the Collaborative Cross (CC), a panel of recombinant inbred mouse strains that exhibit a range of disease manifestations upon infection. METHODS We performed an extensive screen of CC-F1 lines infected with West Nile virus (WNV), including comprehensive immunophenotyping, to identify groups of lines that exhibited viral neuroinvasion or neuroinvasion with disease and lines that remained free of WNV neuroinvasion and disease. RESULTS Our data reveal that protection from neuroinvasion and disease is multifactorial and that several immune outcomes can contribute. Immune correlates identified include decreased suppressive activity of regulatory T cells at steady state, which correlates with peripheral restriction of the virus. Further, a rapid contraction of WNV-specific CD8+ T cells in the brain correlated with protection from disease. CONCLUSIONS These immune correlates of protection illustrate additional networks and pathways of the WNV immune response that cannot be observed in the C57BL/6 mouse model. Additionally, correlates of protection exhibited before infection, at baseline, provide insight into phenotypic differences in the human population that may predict clinical outcomes upon infection.

Interferon-stimulated genes: new platforms and computational approaches

Interferon-stimulated genes (ISGs) are the effectors of interferon (IFN) actions and play major roles in innate immune defense against microbial infection. During virus infection, ISGs impart antiviral actions to control virus replication and spread but can also contribute to disease pathology if their expression is unchecked. Antiviral ISGs have been identified by a variety of biochemical, genetic, and virologic methods. New computational approaches are expanding and redefining ISGs as responders to a variety of stimuli beyond IFNs, including virus infection, stress, and other events that induce cytokines. These studies reveal that the expression of ISG subsets link to interferon regulatory factors (IRF)s, NF-kB, and other transcription factors that impart gene expression in specific cell types independently of IFNs, including stem cells and other cell types where ISGs are constitutively expressed. Here, we provide a broad overview of ISGs, define virus-induced genes (VSG)s, and discuss the application of computational approaches and bioinformatics platforms to evaluate the functional role of ISGs in epigenetics, immune programming, and vaccine responses.

RNA PAMPs as Molecular Tools for Evaluating RIG-I Function in Innate Immunity

Pathogen recognition receptors (PRR)s and their cognate pathogen-associated molecular pattern (PAMP) represent the basis of innate immune activation and immune response induction driven by the host-pathogen interaction that occurs during microbial infection in humans and other animals. For RNA virus infection such as hepatitis C virus (HCV) and others, specific motifs within viral RNA mark it as nonself and visible to the host as a PAMP through interaction with RIG-I-like receptors including retinoic inducible gene-I (RIG-I). Here, we present methods for producing and using HCV PAMP RNA as a molecular tool to study RIG-I and its signaling pathway, both in vitro and in vivo, in innate immune regulation.