Michael S. Diamond - US grants

DESCRIPTION (adapted from application abstract): Dr. Diamond received his Ph.D. in 1992 (Cell/Dev Biology) and M.D. degree from Harvard University, where he worked with Timothy Springer, who describes him as `an extraordinary level of graduate student that one may see only several times in a lifetime.` The candidate has nineteen publications, eight of these as first author, the first four research done in college and the next fifteen from his Ph.D. work in immunology, focusing on the role of CD11b/CD18 on leukocytes. After graduating from the MD/Ph.D. program he initially began a post-doc to continue his study of integrins in the Drosophila genetic system, but then decided to continue clinical training in order to be able to combine clinical work with basic investigation. Having now completed a residency and in the midst of an infectious diseases fellowship, he requests funding to obtain new training in viral pathogenesis and molecular epidemiology so that he can apply his skills in a system that is new to him. Dr. Diamond is at the very highest level of prior research training and experience that can be considered appropriate for the K08 award. The career development plan is outlined for five years, with courses in epidemiology and biostatistics during phase I, followed by the period of independent research in phase II. The proposed research addresses the role of interferon in infection by dengue virus (DV), both to investigate how IFN modulates DV infection, and how DV alters the IFN response. The candidate proposes to use this award as a vehicle for learning a new area of research that will allow him to add virology and molecular pathogenesis to his knowledge base and to apply his prior research and clinical skills in a novel system.

DESCRIPTION (provided by applicant): Using both cell culture and in vivo models of infection, our collaborative group investigates the molecular pathogenesis of positive strand RNA viruses of global importance. West Nile virus (WNV) is an emerging mosquito-borne human and animal pathogen that has caused outbreaks of fatal encephalitis in Europe, Asia, the Middle East, and most recently, the United States. At present, there is no available antiviral therapy or vaccine. This proposal is directed at the discovery of agents that inhibit replication of WNV with the long-term goal of developing antiviral agents to treat WNV-associated disease. Several independent approaches are used to identify inhibitory agents against WNV and the degree of their antiviral effects. One goal of this grant is to develop nucleic-acid specific antagonists against WNV by generating small interfering double-stranded RNA based on the recently described phenomenon of RNA interference (Specific Aims 1 and 2). These antagonists will be tested in cells and in vivo in a murine model of West Nile encephalitis. A second goal is to use genetic screens to identify the viral determinants that mediate sensitivity and resistance to RNA interference (Specific Aim 3). The third goal is to utilize subgenomic WNV replicons as a screening tool to identify small molecule inhibitors of WNV replication (Specific Aim 4). The identification of sequence-specific and small molecule antagonists of WNV and the characterization of pharmacologic enhancers and/or suppressors of RNA interference will provide a platform for the development of novel therapeutic agents that may be used against WNV as well as other positive strand RNA viruses.

[unreadable] DESCRIPTION (provided by applicant): West Nile Virus (WNV) is an emerging mosquito-borne human and animal pathogen (NIAID Category B) that has caused outbreaks of fatal encephalitis in Europe, Asia, the Middle East, and most recently, the United States. Despite the increasing numbers of cases and deaths attributed to WNV, no effective therapy or vaccine is available for humans. Recently, using a mouse model of WNV encephalitis, we have demonstrated that a deficiency of antibody leads to disseminated infection, and passive administration of immune serum or purified polyclonal antibody protects antibody-deficient and wild type mice from WNV-induced mortality. Based on these observations, the proposed research plans to generate a new panel of monoclonal antibodies (mAbs) against WNV E and NS1 proteins, test their therapeutic efficacy in our mouse model of WNV encephalitis, and humanize the best candidates for possible clinical intervention. In Specific Aim 1, a panel of mAbs against the E and NSl proteins of WNV will be generated and characterized for neutralizing activity and effector function, and for localization to discrete structural domains on each protein. In Specific Aim 2, the mAbs with the greatest inhibitory and effector activity in vitro will be evaluated for therapeutic efficacy in the mouse model of WNV infection. Because the RNA-dependent RNA polymerase of WNV has a high error rate and thus, a potential to rapidly alter immunodominant residues, trials will be also conducted with combinations of mAbs. A novel ranking system has been developed to streamline the selection process of mAbs with the greatest potential inhibitory activity. In Specific Aim 3, the six anti-E and anti-NS1 mAbs with the greatest inhibitory activity will be humanized using chimerization and complementarily determining region (CDR) grafting techniques and re-evaluated in vitro and in vivo for anti-WNV activity. By studying the mechanisms of antibody-mediated protection, we hope to rationally design immunotherapeutic agents against WNV. These studies are an essential first step in the generation of humanized mAbs that have utility as therapeutic agents against WNV in humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dengue virus (DENV) causes dengue fever, the most prevalent arthropod-borne viral illness in humans (NIAID Category A pathogen). Globally, the four serotypes of DENV cause an estimated 100 million new cases of dengue fever and 250,000 cases of dengue hemorrhagic fever (DHF) per year. Currently, no specific therapy is available, and vaccines are still in early stages of development. Given that the most advanced tetravalent live attenuated DENV vaccine candidate showed a poor 30% overall efficacy rate in a recently published phase 2b clinical trial, there remains a pressing need for new approaches for safe, effective vaccines. In the first cycle of this R01 we developed a large panel of ~500 novel monoclonal antibodies (MAbs) against all four DENV serotypes and analyzed the structural, biophysical, and cellular mechanisms of Ab-mediated neutralization of several of them. These studies defined novel epitopes on DENV E proteins recognized by inhibitory antibodies (Abs), many of which are not solvent-accessible according to existing atomic models of the virus particle. Our studies revealed that the structure of DENV was more complex than anticipated and is likely a heterogeneous and structurally dynamic ensemble of different states, each of which may interact differentially with Ab. In this renewal application, we propose to define the spectrum of structural states sampled by the DENV virion, determine the structural basis of differential neutralization of individual DENV serotype by cross-reactive, fusion-loop specific Abs, and assess the role of anti-prM Abs in recognition and neutralization of DENV. This information will be translated into developing a novel inactivated vaccine strategy that traps DENV particles in structural states that preferentially elicit highly neutralizing Abs.

Animals; Antibodies; Antiviral Agents; Antiviral Drugs; Antivirals; Asia; B blood cells; B-Cell Subsets; B-Cells; B-Lymphocyte Subsets; B-Lymphocytes; Bursa-Dependent Lymphocytes; Bursa-Equivalent Lymphocyte; Central Nervous System; Complement; Complement Proteins; Complement Receptor; Culicidae; Diamond; Disease; Disease Outbreaks; Disorder; Egypt 101 virus; Encephalitis; Epidemiology; Europe; Gamma Globulin, 19S; Human; Human, General; IFN; IgM; Immune response; Immune system; Immunity; Immunoglobulin M; Immunomodulation; Infection; Inflammation, Brain; Interferons; Man (Taxonomy); Man, Modern; Mediating; Middle East; Mosquitoes; Nervous System, CNS; Neuraxis; Outbreaks; Pathogenesis; Phase; Population; Proteins; Research; Role; Therapeutic Intervention; United States; Viral Diseases; Viral Pathogenesis; Virus; Virus Diseases; Viruses, General; WNV; West Nile; West Nile virus; base; body system, allergic/immunologic; disease/disorder; gene product; host response; immune modulation; immunologic reactivity control; immunoregulation; immunoresponse; in vivo; intervention therapy; mouse model; novel; organ system, allergic/immunologic; pathogen; prevent; preventing; response; social role; viral infection; virus infection

[unreadable] DESCRIPTION (provided by applicant): Dengue virus (DV) is a single-stranded RNA flavivirus that causes dengue fever, the most prevalent arthropod-borne viral illness in humans (NIAID Category A pathogen). Globally, the four serotypes of DV cause an estimated 100 million new cases of dengue fever and 250,000 cases of dengue hemorrhagic fever (DHF) per year, primarily in tropical and subtropical areas. Currently, no specific therapy is available for human use. Given its global burden, there is a pressing need for the development of safe therapeutics against DV. Recently, we defined a dominant neutralizing epitope on domain III of the envelope (E) protein o West Nile virus (WNV), a related flavivirus, and humanized one monoclonal antibody (Hu-E16) with clinical potential as a post-infection therapeutic. Hu-E16, which is now entering human clinical trials, binds to a conserved WNV-specific epitope, and neutralizes at a post-attachment step of infection. While in theory, antibody-based therapeutics could have analogous activity against DV, the risk for antibody-dependent enhancement of infection (ADE) with adverse outcome has limited this approach. For this proposal, we will use an existing academic-industry collaborative partnership to generate an antiviral biological product consisting of humanized monoclonal antibodies (mAbs) that potently neutralize all serotypes of DV without he risk of ADE. In Specific Aim 1, we will generate mouse mAbs against the E protein of all four DV serotypes and test their relative neutralizing potential in vitro and in vivo. In Specific Aim 2, we will identify recognition determinants on DV E proteins for the strongly inhibitory mAbs using high-throughput yeast surface display epitope mapping. Using this information, we will create DV variant strains that are resistant to mAb neutralization and test them for virulence. In Specific Aim 3, we will humanize candidate inhibitory mAbs that strongly block infection against each DV serotype. These will be tested for neutralizing activity in cell culture and in mice. In Specific Aim 4, we will engineer safe DV immunotherapeutics by altering the effector functions of candidate humanized mAbs. Cell lines producing IgG subclass variants will be developed that strongly neutralize yet abolish Fc receptor binding and prevent ADE. Overall, the generation of humanized mAbs against all four DV serotypes with no possibility of ADE will foster the development of potent and safe immunotherapeutics against DV infection. [unreadable] [unreadable] [unreadable] [unreadable]

West Nile Virus (WNV) is a NIAID category B mosquito-borne flavivirus that causes fatal outbreaks of epidemic encephalitis in Europe, Asia, the Middle East, and North America, including the United States. Using genetically deficient mice, we have recently demonstrated that a deficiency of the complement components C1q or C4 leads to a disseminated, fatal WNV infection. Based on these observations, the proposed research plans to directly determine how activation of the classical and lectin complement pathways inhibits WNV infection. These results may be directly applicable to other related NIAID Category A and B flavivirus human pathogens. In Specific Aim 1, we will define the protective anti-WNV function of the lectin pathway of complement activation using mannan-binding lectin serine peptidase 2 (MASP-2)-/- and mannose binding lectin (MBL)-/- mice. In Specific Aim 2, the role and mechanism of C1q modulation of antibody neutralization will be examined. This information will be directly applied to generating novel recombinant antibody therapeutic reagents with increased complement-dependent effector functions. In Specific Aim 3, the mechanisms by which complement primes the amplitude and specificity of memory B cell responses against WNV will be studied. The identification of specific pathways for controlling early dissemination of WNV and for triggering long-term immune protection will enhance our understanding of WNV pathogenesis and provide novel strategies for prophylaxis and/or therapeutic intervention.

DESCRIPTION (provided by applicant): Currently, there is no specific antiviral therapy available for the treatment of flavivirus infections, including West Nile Virus (WNV), a Biodefense Category B agent. Therefore, the development of novel classes of antiviral agents is of high priority for public health and defense against a possible bioterrorism attack. For this initiative, our goal is to develop small molecule inhibitors of WNV. In our preliminary experiments, we used a WNV replicon-based cell .culture high throughput screening platform to identify several candidate lead compounds and confirmed their inhibitory activity in WNV and other flavivirus infection assays in cell culture. Our collaborative group will bring the potential anti-viral lead compounds we discovered in our screens through a preclinical drug development program that includes lead optimization using structure-activity relationships (SAP) to enhance compound potency and drug-like properties. Preclinical toxicology and pharmacokinetic (PK) studies will be performed to help predict how the antiviral compounds distribute and behave in vivo and will include an analysis of cytotoxicity, solubility, metabolic stability, cell permeability, and plasma protein binding. As a cost-saving measure, we will perform preliminary preclinical toxicology studies in-house, for early identification of compounds that merit further consideration in advanced in vitro and In vivo toxicokinetics preclinical studies in small animals. Compounds with acceptable toxicity and PK profiles will be tested in efficacy studies using a well-characterized mouse model of WNV infection. The ability of our small molecule anti-WNV compounds to synergize with a neutralizing humanized monoclonal antibody to generate a more potent combination therapeutic regimen will also be studied. For the lead compounds with the greatest WNV anti-viral potential in vivo, virological studies will be performed to define the mechanism of action. It is expected that these studies will result in the production of an antiviral drug for future clinical trials to determine safety and efficacy against WNV, and possibly other flavivirus infections. Project Narrative There are currently no effective therapies against the Category B agent WNV to address the immediate health needs of the public in case of a bioterrorism incident. In this project, we are proposing to use the combined expertise of Apath.LLC and the Diamond laboratory at Washington University in St. Louis to develop anti-WNV compounds we discovered in our screening program into an antiviral drug to be tested in future clinical trials.

DESCRIPTION (provided by applicant): Chikungunya virus (CHIKV) is a mosquito-transmitted, single-stranded RNA alphavirus of the Togaviridae family that causes explosive epidemics of a severe febrile illness characterized by debilitating polyarthalgias in humans. CHIKV (NIAID Category C pathogen) caused an estimated 1.4 million new cases in India alone in 2006, and has the potential to spread globally because of the distribution and abundance of its primary mosquito vector, Aedes aegypti. During recent outbreaks, more severe forms of CHIKV infection were observed including encephalopathy and hemorrhagic fever, suggesting the emergence of more virulent strains. Currently, no specific treatment or vaccine is available. Given its global burden, the increased travel into CHIKV-endemic areas, and the worldwide spread of its vector, there is a pressing need for the development of prophylactic and therapeutic agents against CHIKV. In preliminary experiments, we have generated strongly neutralizing monoclonal antibodies (MAbs) against CHIKV that prevent infection in mice. Here, an established academic-industry collaborative partnership between the Diamond laboratory and MacroGenics will develop humanized antibody therapeutics that protect against virtually all strains of CHIKV. In Aim 1, panels of mouse MAbs against the E1 and E2 envelope proteins of CHIKV will be generated and screened functionally. The most potent neutralizing MAbs that recognize an array of genetically diverse CHIKV strains will be evaluated for protection in mice. In Aim 2, mechanistic correlates of antibody protection will be defined by characterizing the cellular stages of antibody blockade. Additionally, we will identify CHIKV recognition determinants using high-throughput yeast surface display epitope-mapping technology. In Aim 3, we will engineer humanized MAbs that block infection of CHIKV. These will be tested for neutralizing activity in cell culture and in mice. Humanized MAbs will be tested for protective efficacy, durability, and therapeutic effect in vivo. High-expressing cell lines producing the best candidates will be established. Overall, the generation of strongly protective humanized MAbs against CHIKV will foster the clinical development of immunotherapeutic agents against CHIKV infection.

DESCRIPTION (provided by applicant): Members of Flavivirus genus are the most important arthropod-borne viruses causing disease in humans. This genus includes viruses (West Nile virus (WNV), Japanese encephalitis virus (JEV) and Dengue virus (DENV)) that are re-emerging and becoming endemic in new areas of the world. Flaviviruses account for ~100 millions infections per year, with billions at risk and no specific therapy available. Although interferon (IFN) responses control the cell and tissue tropism of WNV and other flaviviruses, the specific effector molecules that restrict infection remain poorly characterized. The studies in this collaborative and inter-disciplinary project between the Diamond and Chanda laboratories will use genetic screens to identify novel interferon stimulated genes (ISG) that modulate flavivirus infection in specific cell types ex vivo and in vivo. Loss-of-function high-throughput genetic screens will be performed with a custom- generated GFP-marked shRNA library targeting ~700 mouse and human ISGs, to identify novel ISG that restrict infection of virulent and attenuated strains of WNV. Using stable cell lines that have targeted reductions or ectopic expression of candidate ISGs, we will define mechanistically how novel ISG effector molecules restrict specific steps in the viral lifecycle and influence infection outcome. In preliminary studies, we have identified several candidate inhibitory ISG (Ifi27, IFIT2, and IFITM3) that attenuate WNV infection, and already acquired or generated knockout mice. Using these mice, we will evaluate the function of these particular ISGs in restricting WNV replication in a cell-specific manner and in vivo. We hypothesize that specific ISGs have antiviral properties that differentially control WNV infection and spread, and that these demonstrate tissue and cell-type specificity. Overall, these experiments will more clearly define the interface between IFN control and flavivirus pathogenesis and possibly, guide strategies that modulate immunity to infection by this family of viruses.

DESCRIPTION (provided by applicant): ABSTRACT West Nile virus (WNV) is an NIAID Category B infectious zoonotic agent that causes severe neurological disease in humans and other animals. The virus and host processes that control WNV infection and immunity are not fully understood. Our preliminary studies indicate that susceptibility to WNV infection is in part regulated by innate immune defenses triggered by pathogen-associated molecular pattern (PAMP) recognition of WNV RNA by the RIG-I-like receptors (RLRs), RIG-I and MDA5. These defenses are mediated by the type I interferon (IFN) antiviral response that includes a novel IKK? signaling pathway of STAT1-serine 708 phosphorylation and the actions of specific antiviral effector interferon-stimulated genes (ISGs). Our preliminary studies also have revealed that pathogenic WNV disrupts IKK? signaling to attenuate IFN actions and evade antiviral immunity. These observations identify STAT1 serine-708 phosphorylation control as a virulence determinant of WNV infection. The proposed studies will investigate the hypothesis that WNV infection outcome is regulated by virus/host interactions that modulate RLR signaling and specific IFN-? innate immune defenses in distinct cell types. Our studies will include Aims to: (1) Define the dual role of RIG-I and MDA5 in restricting WNV infection [and modulating adaptive immunity], (2) Identify the PAMPs within the WNV RNA that are recognized by RIG-I and MDA5, (3) Define the IFN-?-mediated antiviral pathways that specifically restrict WNV infection in neurons, which are key targets of in vivo infection, and (4) Determine the mechanism of immune escape from the IKK??pathway.

PROJECT SUMMARY (See instructions): Members of the Flavivirus genus are the most important arthropod-borne viruses causing disease in humans, many of which are characterized as NIH Category A, B, and C organisms. This genus includes viruses [West Nile virus (WNV), Japanese encephalitis virus (JEV) and Dengue virus (DENV)] that are endemic and continue to spread in many areas of the world. Flaviviruses account for -100 millions infections per year, with billions at risk and no specific therapy available. Although much work has focused on understanding the mechanisms of flavivirus replication in cultured cells, and on defining virulence in vivo, less remains known about host responses to strains of different virulence, and the genes and cell types that modulate infection in vivo. Here, we propose to identify and define the mechanism of action of novel host genes that modulate WNV infection. A systems biology approach to studying complex host-pathogen interactions associated with infection by virulent and attenuated WNV strains will provide new insight into host response mechanisms. With the support of several Cores (computational; proteomics, metabolomics, and lipidomics; data management and resources dissemination), we will acquire a global picture of the host response to infection by WNV strains of distinct pathogenic potential. Such an analysis has never been performed for any flavivirus. This approach to studying WNV pathogenesis will interact directly with work in Project 1 on influenza A and Ebola viruses to identify novel common genes, networks, and pathways that restrict infection with the viruses studied here. Target genes identified by systems biology will be validated in cells using ectopic expression and gene silencing, and these phenotypes will help prioritizing the generation of new KO mice for evaluation of the function of target genes in vivo in the context of WNV infection. Overall, our studies will provide new insight into the cellular processes that restrict infection by WNV and likely other viruses, and thus may promote novel strategies for development of therapeutic agents that contain virus spread and disease. Moreover, it may have implications for understanding the genetic variation in humans, which could explain susceptibility to particular viral infections.

The primary goal of Project 2 is to determine how innate immune responses impact on the entry, infection, and replication of encephalitic flaviviruses within the central nervous system (CNS). Members of the Flavivirus genus include neurotropic viruses (e.g., West Nile (WNV), Japanese encephalitis (JEV), and tickborne encephalitis viruses) that continue to spread and cause human disease in new areas of the world. Although much work has focused on understanding flavivirus replication in cells, and on defining the virulence features of strains in vivo, less is known as to how the host innate immune mediators limit entry into the CNS and controls infection of neuronal target cells. Outside the CNS, innate immune cells recognize viral pathogen associated molecular patterns (PAMPs) and respond to infection with the orchestrated release of proinflammatory cytokines, which trigger cell-intrinsic antiviral responses and the induction of adaptive immunity. Because the CNS lacks secondary lymphoid tissues and cannot initiate adaptive immune responses, it relies, in part on innate responses of resident neural cells to limit viral invasion and the extent of infection. In preliminary studies, we have determined that types I (IF Nab) and 111 (lL-28) interferons (IFNs), modulate the ability of WNV to enter and infect the CNS. Thus, systemic and local IFN responses, which occur during WNV infection, induce brain microvascular endothelial cells (BMECs) to regulate blood-brain barrier (BBB) permeability and restrict viral entry. Once encephalitic flaviviruses enter the brain, several factors likely contribute to the regional heterogeneity and cellular distribution of infection that is observed in mouse models and human autopsy specimens. We hypothesize that innate immune responses in the CNS after flavivirus infection exhibit cell- and region-specific effects that restrict viral entry, infection, and injury of neurons. We also hypothesize that non-neuronal cells in the CNS (e.g., astrocytes, oligodendrocytes, and microglia) provide critical innate cues and produce inflammatory mediators that instruct neurons in developing specific innate immune programs to control virus infection. Insight into the cell-intrinsic and cell extrinsic processes by which the host controls flavivirus infection and minimizes neuronal injury is essential for developing strategies to contain virus spread, persistence, and disease.

? DESCRIPTION (provided by applicant): Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that cycles directly between humans and mosquitoes and causes a debilitating febrile illness in humans on a global scale. Autochthonous transmission in the New World now has been described, with outbreaks in at least 19 countries in the Americas and over a quarter million cases in just 6 months. CHIKV disease cases have been reported to ArboNET in travelers returning to the US from the Caribbean in 29 states, as of July 1. The potential for epidemics in North and South America is high due to the ubiquitous distribution of one of its primary vectors, Aedes albopictus. Despite the possibility for infecting and causing disease in millions, specific treatments or vaccines for CHIKV are not available. A primary goal of this project is to define the molecular, genetic, immunologic, and structural characteristics of ultra-potent neutralizing human mAbs with broad activity against all genotypes of CHIKV. Additional goals include defining the mechanistic correlates of protection by these ultra-potent neutralizing mAbs. In these studies, we will elucidate how antiviral Abs with exceptional inhibitory activity exert their action in cell culture and in vivo. The approach will include high efficiency isolationof human mAbs, coupled with innovative antibody gene repertoire studies based on nextgen sequencing. Several hypotheses will be tested, including the concept that ultra-potent neutralizing activity results from features of both the antibodies (extensive mutations due to persistent CHIKV infection) and the antigen (binding to quaternary epitopes on multiple adjacent envelope proteins and blocking structural transitions critical for virus entry or release). Althoug our focus is to understand how and why ultra-potent human mAbs inhibit CHIKV, the studies likely will be relevant to general principles of antibody neutralization of many different viruses. Beyond defining the molecular and structural basis of Ab neutralization of CHIKV, these studies will generate a group of fully human mAbs that can prevent and treat CHIKV infection and persistence in mice, which could be developed in the future as a possible combination immunotherapeutic for humans. Studies in this project, while targeted against CHIKV, likely will inform future Ab-based and/or vaccine efforts against other alphaviruses that cause human disease. We have assembled a unique group of investigators, including a human Ab expert, a molecular virologist with experience in Ab-virus interactions, and two accomplished structural biologists with specific expertise in alphaviruses, including CHIKV, to pursue these studies.

? DESCRIPTION (provided by applicant): Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes massive epidemics of a debilitating musculoskeletal inflammatory disease. There are currently no approved CHIKV-specific vaccines or antiviral agents. CHIKV initiates infection after the E2 glycoprotein binds to glycosaminoglycans (GAGs) on the surface of host cells and promotes internalization by clathrin-dependent uptake into the endocytic pathway. Both attenuated and virulent CHIKV strains bind GAGs, and GAG-binding efficiency influences virulence, including in a mouse model of CHIKV disease. However, the molecular basis of CHIKV-GAG interactions is unknown, which precludes a complete understanding of the function of GAGs in CHIKV pathogenesis. Moreover, the host factors that promote CHIKV replication, the precise cellular targets for CHIKV infection in the host, and links between host CHIKV replication factors and disease pathogenesis also remain unclear. The proposed research combines the expertise of the laboratories of Terence Dermody, Michael Diamond, and Thomas Morrison in virus-receptor interactions, RNA virus replication, viral immunology, and mouse models of viral disease to enhance knowledge of CHIKV replication and pathogenesis. Three integrated and interactive specific aims are proposed. In Specific Aim 1, the mechanisms and pathological significance of CHIKV binding to GAGs will be determined. Specific GAG subtypes bound by CHIKV will be defined using glycan-array screening and genetically altered cell lines with defects in GAG biosynthesis, and sequences in E2 required for binding to GAGs will be defined using structure-guided mutagenesis. The function of GAG binding in acute and chronic CHIKV disease will be determined using mutant CHIKV strains with reduced or abolished GAG-binding capacity and GAG-knockout mice. In Specific Aim 2, the function in CHIKV infection of COPI coatomer subunits and regulatory factors, which were recently identified in a small-interfering RNA screen, will be elucidated. Cells with diminished COPI transport activity will be infected with CHIKV and tested for formation of viral replication compartments, synthesis of viral RNA, and assembly and release of viral progeny. The function of COPI coatomer ARCN1 and regulatory factor GBF1 in CHIKV pathogenesis will be determined using newly established gene-targeted mice. In Specific Aim 3, cell types in the mammalian host that contribute to CHIKV pathogenesis and persistence will be identified. CHIKV strains engineered to contain tissue-specific microRNA seed sequences will be tested for acute and chronic disease in mice and elaboration of chemokines and cytokines, infiltration of musculoskeletal tissues with inflammatory leukocytes, and development of humoral immune responses. Overall, studies in this collaborative proposal will enhance our understanding of mechanisms by which CHIKV binds to GAGs, determine the function of COPI transport in CHIKV infection and pathogenesis, and define specific cells in the host targeted by CHIKV to produce disease. Knowledge gained from the proposed research may illuminate new targets for anti-CHIKV therapeutics.

Aging is associated with dysregulation of innate and adaptive immunity, which impairs an individual's ability to control infection by newly encountered pathogens. Aging also impacts neuroprotective mechanisms within the central nervous system (CNS), including the function of the blood-brain barrier (BBB), which limits the entry and replication of neutrotropic viruses. West Nile virus (WNV) is the leading cause of arthropod-transmitted viral infections in the United States. While most WNV infections are asymptomatic, individuals with symptomatic disease present either with a flu-like febrile illness that can progress to severe neuroinvasive diseases including meningitis, encephalitis, or flaccid paralysis. Severe WNV infection, especially fatal forms, predominate in the elderly. It is unclear whether this is due to effects of aging on viral invasion and/or virologic control within peripheral tissues or the CNS. To study many different aspects of immune regulation in adult mice, the Diamond and Klein laboratories have established a murine model of infection with a virulent WNV strain. However, interactions between antiviral immunity, neuroinflammation, and aging within the CNS have not been investigated. In this proposal, we will examine the impact of age on innate and adaptive immune mechanisms that control viral entry and clearance specifically within the CNS. The UH2 phase we will establish breeding cohorts and determine the feasibility of using 21 month-old aged animals in WNV infection studies. In the UH3 phase, we will explore how changes in skin immunity, BBB integrity, and CNS inflammation during aging influence neuroinvasion and disease progression associated with WNV infection. 

Project Summary: Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that has become a global public health threat. ZIKV infection has now been causally linked to cases of Guillain-Barre syndrome in adults and microcephaly, intrauterine growth restriction (IUGR), and spontaneous abortion in the setting of maternal infection during pregnancy. Given the recognition of this causal relationship, it is imperative to determine the mechanism(s) of maternal-fetal transmission. Insights into such mechanisms could improve our ability to reduce the burden of the effects of Zika virus infection during pregnancy. The goal of this collaborative and interactive project is to define how host defense responses can control or possibly contribute to ZIKV pathogenesis during and after pregnancy. The premise is based on the extensive newly published data showing that ZIKV infection of pregnant mice results in infection of the placenta, in utero transmission, and injury to the developing fetus. O ur central hypotheses are that maternal-fetal transmission occurs because ZIKV overcomes placental trophoblast host defenses (autophagy) (Aim 1), usurps TAM receptors to enter placental trophoblasts and endothelial cells (Aim 2); and this entire process is enhanced in flavivirus-immune individuals or animals who have sufficient levels of cross-reactive, non-neutralizing anti-ZIKV antibodies that promote ADE (Aim 3). Collectively, our efforts will be highly significant for understanding ZIKV pathogenesis. This work will provide a foundation for clinical studies that define the risks of maternal to child transmission of ZIKV, generate possible new therapeutic approaches, and determine the interaction between ZIKV and natural immunity to other flaviviruses.

PROJECT SUMMARY/ABSTRACT The receptor tyrosine kinases of the TAM family ? Tyro3, Axl, and Mer ? are essential regulators of infection by enveloped viruses, and Axl and Mer are especially important to arbovirus infection of cells of the central nervous system (CNS). Infection of the CNS by encephalitic arboviruses, including West Nile virus (WNV), often has devastating consequences, both acutely and after recovery, and deciphering the molecular mechanisms through which TAM receptors control virus entry, propagation, and clearance is therefore a key objective. Genetic, molecular biologic, cell biologic, and behavioral assays will be used to elucidate these mechanisms. In Aim 1, a set of new conditional mouse mutants and cell-specific Cre drivers will be used to investigate the CNS cell types through which TAM receptors control infection by WNV and two other neurotropic enveloped arboviruses - La Crosse encephalitis virus and Venezuelan equine encephalitis virus. These studies will elucidate the specific roles played by Axl and Mer in brain microvascular endothelial cells (BMECs), microglia, astrocytes, and pericytes in neuroinvasion and CNS pathogenesis by these viruses. In Aim 2, a new mouse model of learning impairment after recovery from CNS infection by WNV will be used to probe the role that Axl and Mer in microglia and astrocytes play in spatial learning and memory after infection. These experiments also will assess the role that cell-specific TAM signaling plays in synapse elimination and neurogenesis subsequent to WNV infection of the brain. In Aim 3, molecular genetics and cell-based signaling assays will be used to elucidate the molecular architecture of TAM receptor-Interferon receptor (IFNAR) interaction, which is crucial to the phenomena of Aims 1 and 2, in BMECs, microglia, macrophages, and dendritic cells. These studies will identify the signaling pathways activated by cooperative TAM receptor/INFAR signaling in these cells, and assess the ability of these interacting receptor systems to reorganize actin cytoskeletons. Together, the experiments of this proposal will guide the formulation of novel strategies for inhibiting virus entry into the CNS, attenuating virus infection of neural cells, and promoting the repair and recovery of infected neural tissues.

Abstract Chikungunya virus (CHIKV) is an emerging mosquito-transmitted alphavirus that causes an abrupt onset of fever with severe joint and muscle pain. In a significant number of patients chronic and debilitating arthritis can persists for months to years. CHIKV pathogenesis reflects an interplay between events controlling viral replication and the host inflammatory immune response. Key mediators of the host response to viral infection are type I interferons (IFNs), which collectively is comprised of 13 IFN-alpha (?) proteins and single IFN-beta (?), kappa (?), epsilon (?), and omega (?) proteins. The type I IFN response is known to control CHIKV infection, as mice lacking the type I IFN receptor (IFNAR) die soon after infection. However, the role of individual IFN subtypes is poorly understood. In preliminary data, we have found that IFN-?-/- and IRF7-/- (a surrogate for IFN-?) are more susceptible to CHIKV induced disease, whereas IFN-?-/- mice are resistant, providing evidence for functional differences between the IFN subtypes in vivo. In this proposal, we will define the mechanistic basis for how different type I IFN subtypes modulate CHIKV pathogenesis by exploring their ability to control viral replication and spread, function as immunomodulatory proteins, or temper the host response. In Aim 1 we will define the role that IFN-? plays during acute and chronic CHIKV infection by exploring its ability to limit viral replication and dissemination and determining whether IFN-? regulates the cell tropism of viral persistence. In Aim 2 we will determine the function of IFN-? during acute and chronic disease and evaluate its impact on modulating the cellular and adaptive immune response during acute and chronic infection. In Aim 3 we will investigate the novel actions of IFN-? by evaluating its impact on CHIKV pathogenesis in vivo and defining functional differences in signaling through IFNAR between IFN-?, IFN-?, and IFN-?. Overall, our studies will provide new insight into the effects of type I IFN immunity on CHIKV induced disease, which may promote the development of novel therapeutic strategies.

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that has become a global public health threat. The World Health Organization declared ZIKV and its suspected link to birth defects an international public health emergency on February 1, 2016. Epidemics of ZIKV infection have been reported in Mexico, and Central and South America and linked to cases of Guillain-Barre syndrome in adults and microcephaly in newborn infants in the setting of maternal infection during pregnancy. Despite the potential for infecting and causing disease in millions, specific diagnostics, treatments, or vaccines for ZIKV are not available. The primary goal of this collaborative and interactive project is to define the molecular, genetic, immunologic, characteristics of newly- isolated neutralizing human mAbs with broad specificity against all strains of ZIKV. A second goal is to define the mechanistic correlates of protection by neutralizing mAbs. A third goal is to determine whether cross-reactive anti-DENV human mAbs that bind to ZIKV are protective/therapeutic or pathogenic in a newly developed mouse model of ZIKV. We hypothesize that potently inhibitory mAbs recognize epitopes associated with key ZIKV structural transitions with high affinity and block one or more keys step during entry (e.g., attachment, entry, or fusion). Our approach will include high-efficiency isolation of human mAbs and with detailed functional and structural analyses to define how and why human mAbs inhibit ZIKV. We also will explore the significance of Fc?R binding and determine whether ZIKV is similar or different than DENV in the context of antibody-mediated immune enhancement of disease. In addition to fundamental studies of ZIKV pathogenesis and immunity, these studies also will result in the generation of a group of fully human mAbs that can be tested in preclinical models and could be developed rapidly as therapeutic or prophylactic biologic drugs for humans. Studies in this project also will inform ongoing diagnostic and future vaccine efforts against ZIKV, as they will define the principal major antigenic sites epitopes associated with potent type-specific antibody-mediated virus neutralization and protection. The collaborative multidisciplinary group assembled to conduct studies in this multi-PI application already collaborates productively, with a strong track record in studies of dengue, chikungunya and other arthropod-borne viruses, and has a clear division of labor for the studies proposed in this application.

Flaviviruses are a genus of related positive-stranded enveloped RNA viruses that significantly impact human health, including dengue (DENV), Zika (ZIKV). West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses. The discovery of host factors critical for viral infection reveals new aspects of cell biology, intricate virus-host relationships, and potential targets for antiviral therapeutics. After entering cells and fusing in the acidified endosome, the flavivirus RNA genome penetrates into the cytoplasm and is then translated into a polyprotein and processed at the endoplasmic reticulum (ER). In addition to utilizing many functions of the ER for protein production, flaviviruses extensively remodel ER membranes to create a niche for RNA- dependent RNA replication. The ER also is the site for flavivirus assembly, which enables the production and secretion of new infectious viruses. Thus, the ER serves as a central point for orchestrating many of the essential steps in the flavivirus infection life cycle. Despite this, little is known about the host factors and molecular mechanisms at the ER that are required for optimal translation and processing of the viral proteins or for the assembly of the replication niche. We recently have performed several genetic screens to identify important components in this process. Our genome-wide RNAi screen with WNV in insect cells validated 18 genes associated with ER biology that promote infection. Our CRISPR/Cas9 gene-editing screen in human cells with WNV also identified 12 ER-associated genes. These screens converged on ER-resident proteins as being critical for WNV infection and included genes associated with ER-translocation and signal peptide processing, ER-associated degradation (ERAD), protein glycosylation, protein folding and lipid metabolism. Indeed, infection of WNV, ZIKV, JEV, DENV, and YFV all required specific subunit components of the host signal peptidase complex (SPCS) for processing of the viral polyprotein, the production of viral glycoproteins and thus generation of nascent virions. The objective of this proposal is to define the molecular mechanisms by which flaviviruses use specific ER-associated host proteins to promote viral translation, polyprotein processing, RNA replication, and/or assembly. Aim 1 will define the mechanism by which the ER translocon promotes polyprotein translation and processing while Aim 2 will dissect the role of ER-associated decay (ERAD) in promoting flavivirus replication. Our long-term goal is to determine the mechanisms by which flaviviruses exploit the ER for their replication, as this will reveal both fundamental aspects of virology as well as new avenues for antiviral therapeutics.

PROJET SUMMARY/ABSTRACT Globally, the four serotypes of DENV cause an estimated 390 million new cases of dengue fever and 500,000 cases of dengue hemorrhagic fever (DHF) per year. Zika virus (ZIKV) is a related emerging virus linked to Guillain-Barre syndrome in adults, and microcephaly, congenital malformations, and spontaneous abortion of the fetus during pregnancy. Currently, no antiviral therapy is available for either virus. In the first two cycles of this collaborative and inter-disciplinary R01, we developed panels of monoclonal antibodies (Abs) against the E proteins of all four DENV serotypes and analyzed their structural, biophysical, and cellular mechanisms of neutralization. These studies defined novel epitopes on DENV E proteins recognized by inhibitory Abs, some of which are not solvent-accessible according to existing atomic models of the virus particle. We also defined the dynamic state of DENV and related flaviviruses, determined the atomic structure of mature and immature ZIKV, characterized atomic interactions between DENV and ZIKV virions, E protein, and new highly neutralizing mouse and human Abs, and identified how different maturation states influence epitope accessibility and Ab neutralization. Our studies revealed that flavivirus structure is more complex than anticipated and is likely a heterogeneous and dynamic ensemble of different states, each of which may interact differentially with Abs. In this renewal, our collaborative and inter-disciplinary team hopes to gain insight into questions about the antigenicity of flavivirus virions and subviral particles (SVPs) as well as the cross-reactivity that limits vaccine and diagnostic development and may affect pathogenesis. Our group will define new states of DENV and ZIKV particle structure and determine how these states influence epitope exposure and interaction with specific neutralizing Abs available in our laboratories. We also will compare the atomic structure of SVPs with infectious virions, determine how these differences affect binding and induction of neutralization Abs, and characterize further the structural basis of cross-reactivity of DENV and ZIKV. Our studies may facilitate the generation of novel antigens and immunogens with improved capacity to detect and elicit specific protective Ab responses against DENV, ZIKV, and likely other emerging flaviviruses.

The overarching goal of this application is to identify human placental innate immune pathways and factors that alter maternal-fetal sensitivity to teratogenic virus infections. The hematogenous spread of viruses from the maternal circulation to the fetus can induce devastating consequences in the developing embryo, compromise maternal health, and jeopardize pregnancy outcome. The placenta is a primary immunological and physical barrier to the spread of viruses from both the maternal circulation and the vaginal and cervical mucosa. However, despite the importance of this barrier, relatively little is known regarding the innate immune pathways by which the placenta senses and responds to viral infections. The proposed research by the Coyne and Diamond laboratories combines expertise in virology, immunology, and placental biology to identify placental-derived innate immune pathways that bolster antiviral defenses in a placental cell-type specific manner. We have previously identified pathways employed by placental trophoblasts to restrict viral infections. These include the constitutive release of antiviral type III interferons (IFNs), which protect both maternal- and fetal- derived cells from viral infections. These previous studies suggest that trophoblasts form an innate IFN-mediated barrier to the vertical transmission of viruses and that viruses associated with fetal disease must bypass these trophoblast intrinsic pathways to be trans-placentally transmitted. In this application, we will define the innate immune antiviral pathways by which fetal-derived components of the placenta, including chorionic villi and the amnion and chorion, sense and respond to infection by known teratogenic viruses, including Zika virus (ZIKV), Rubella virus (RuV), and herpesvirus-2 (HSV-2). These studies will utilize the individual and complementary expertise of the Coyne and Diamond laboratories, who specialize in virology (CC and MD), immunology (CC and MD), placental biology (CC), and in vivo modeling of maternal-fetal transmission (MD). In addition, we will define the mechanism(s) by which disparate IFN types (type I and III) impact placental antiviral signaling and placental damage. In deciphering the underlying mechanisms that constitute placental-derived antiviral innate immune pathways, we may illuminate the basis of placental sensitivity or resistance to viruses and identify cell populations that may be particularly sensitive to viral infections during pregnancy. These studies could inform the development of innovative therapeutics designed to mitigate and/or prevent viral infections or inflammation-induced injury, thus reducing the burden of infection related feto-maternal morbidity and mortality.

PROJECT SUMMARY/ABSTRACT Zika virus (ZIKV) is a flavivirus that has recently emerged from Uganda, into Asia, across the Pacific and now into the Americas. ZIKV infection is transmitted to humans by the bite of an infected mosquito. Person to person sexual transmission of ZIKV has also been documented. ZIKV infection poses a major threat to unborn children because it efficiently crosses the placental barrier to mediate maternal to fetal transmission in utero. Fetal infection can lead to varied pathologies including microcephaly and fetal death. Little is known of how placental cells respond to infection to control ZIKV tropism and to mount innate immune defenses to protect against ZIKV spread and fetal infection. Our preliminary studies show that ZIKV triggers placental innate immune activation through RIG-I sensing of viral pathogen associated molecular patterns (PAMPS) but that it directs a broad blockade to cytokine signaling through signal transducer and activator of transcription (STAT) proteins in the infected cell. This broad STAT suppression attenuates interferon (IFN) innate immune defenses to support virus replication and spread to the fetus. This proposal will investigate the central hypotheses that the outcome of ZIKV infection and disease is linked with regulation of innate immune defenses and control of JAK-STAT signaling, and that viral evasion of host defenses enables maternal-fetal ZIKV transmission and fetal disease. We will conduct three Aims: 1) Determine the molecular mechanisms by which ZIKV induces innate immune responses in key cell types of the maternal-fetal interface throughout human pregnancy; 2) Determine the molecular mechanisms of broad JAK-STAT regulation by ZIKV, and 3) Define the role of innate immune activation and STAT regulation of antiviral defenses to control ZIKV infection of human placental cells ex vivo, and determine how viral innate immune evasion impacts fetal disease in the STAT2-KI model of maternal/fetal transmission.

PROJECT SUMMARY B cell responses are essential for clearance of viral infections. The mechanisms by which chronic viruses subvert B cell immunity are poorly understood. Chikungunya virus (CHIKV) is a mosquito-transmitted RNA alphavirus that causes explosive epidemics of debilitating polyarthralgia. Joint swelling, joint stiffness, and tenosynovitis can last for months to years and are associated with persistent CHIKV infection. The mechanistic basis for how CHIKV evades B cell immunity to establish chronic infection is unknown. We found that an attenuated CHIKV strain, but not the parental CHIKV strain (which differs by 5 amino acids), is cleared from joints of WT mice. Persistent infection of the attenuated strain was restored in mice unable to produce virus-specific antibody (Ab). Mapping studies revealed that a single arginine or glycine residue at position 82 in the CHIKV E2 glycoprotein dictates viral clearance or persistence, respectively. In comparison with acutely cleared CHIKV, persistent CHIKV infection was associated with altered B cell responses, a failure to develop germinal centers in the draining lymph node (DLN), poorer quality virus-specific neutralizing Abs, and distinct viral localization and inflammatory responses in lymphoid tissue. Remarkably, depletion of inflammatory myeloid cells improved B cell responses to pathogenic CHIKV. The goal of this highly interactive project between the Morrison and Diamond laboratories is to define mechanistically how pathogenic CHIKV strains evade B cell immunity and the contribution of inflammatory monocytes to this process. In Specific Aim 1, the mechanisms by which a specific CHIKV E2-82 residue dictates inflammatory responses in the DLN will be determined. Viruses differing only at E2-82, and new mice with genetic deficiencies in glycosaminoglycans (GAGs), will be used to determine how E2-GAG interactions dictate CHIKV localization in the DLN. LN macrophage depletion and single-cell RNAseq will define the role of LN myeloid cells in inflammatory and B cell responses to persistent and acutely cleared CHIKV. In Specific Aim 2, how type I IFN signaling modulates B cell responses during acutely cleared and persistent CHIKV infection will be defined. Type I IFN reporter mice, flow cytometry, and IHC will be used to define the cells that produce type I IFN in lymphoid tissue. Ab-mediated blockade of IFNAR1 or distinct type I IFN subtypes (pan-? versus ?), and mice with B cell-specific defects in type I IFN signaling will be used to determine the effects of type I IFN on B cell, Th, and Tfh cell responses. In Specific Aim 3, how inflammatory myeloid cells antagonize B cell responses during persistent CHIKV infection will be elucidated. The impact of inflammatory myeloid cells on the avidity, neutralization capacity, and epitope repertoire of the polyclonal anti- CHIKV Ab response will be defined. Using new Nos2F/F or Nox2F/F mice, and a panel of Cre driver strains, we will determine mechanisms by which specific subsets of myeloid cells inhibit optimal B cell responses. This work will elucidate new mechanisms by which viruses subvert B cell immunity and establish persistence. The knowledge gained may aid the development of vaccines or therapies against CHIKV or other chronic viral infections.

Project Summary/ Abstract Alphaviruses are mosquito-transmitted, positive-strand enveloped RNA viruses of the Togaviridae family that cause global disease in humans. At present, no antiviral agents or licensed vaccines exist for the treatment or prevention of any alphavirus infections. We recently used a genome-wide CRISPR/Cas9-based screen to identify the cell surface molecule Mxra8 as a novel entry receptor for multiple emerging alphaviruses that cause arthritis and musculoskeletal disease including chikungunya (CHIKV), Ross River (RRV), Mayaro, (MAYV), and O'nyong nyong (ONNV) viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced alphavirus infection of cells, and reciprocally, ectopic expression resulted in increased infection. Mxra8 bound directly to CHIKV particles and enhanced virus attachment and internalization into cells. We hypothesize that engagement of Mxra8 by different alphaviruses will explain how infection, tissue targeting, and disease pathogenesis occurs. The primary goals of this collaborative proposal between the Diamond, Fremont, and Smit laboratories are to define the precise mechanism(s) by which Mxra8 facilitates alphavirus entry into cells, to gain high- resolution structural insight as to how Mxra8 engages the alphavirus virion, and to determine the detailed role of Mxra8 in the pathogenesis of acute and chronic infection of multiple arthritogenic alphaviruses in vivo using novel murine models. The experiments in this proposal will define fundamental aspects of alphavirus biology that enhance our understanding of infection and cell tropism. This information may facilitate the development of small molecules or biologicals that disrupt Mxra8 interaction with alphavirus spike proteins, which could form the basis of future therapeutics that ameliorate disease of multiple emerging alphaviruses.

Project Summary / Abstract Dengue virus (DENV) causes dengue fever, the most prevalent arthropod-borne viral illness in humans. The existing four serotypes of DENV (DENV1, DENV2, DENV3, and DENV4) cause an estimated 390 million infections and at least 500,000 cases of Severe Dengue per year, which is a vascular leakage syndrome that can result in hypotension, shock, and death. Currently, no specific therapy is available, and only one vaccine is approved for use in humans (Dengvaxia®). This vaccine lacks efficacy in naïve individuals and appears to promote disease in some recipients upon subsequent natural infection. This phenotype may be due in part to suboptimal serotype responses and the phenomenon of antibody-dependent enhancement (ADE) of DENV infection, where cross-reactive yet sub-neutralizing levels of antibody can promote virus entry and infection in myeloid cells expressing activating Fc? receptors. Accordingly, all of the current DENV vaccines in trials have a goal of developing balanced and potently inhibitory tetravalent responses against the four serotypes of DENV. Recently, a new strain of DENV (DKE-121) was isolated in Malaysia from an individual with Severe Dengue, with up to 38, 37, 38, and 12% amino acid difference between DENV1, DENV2, DENV3, and DENV4 in the envelope protein. This strain may represent a new DENV serotype (DENV5). The potential emergence of a new DENV serotype could have major implications for existing tetravalent DENV vaccine platforms, which in theory, might lack coverage. Given the possible significance of such a strain (and serotype), in this exploratory R21 application, we propose to evaluate in detail the serological relatedness of DKE-121 to other DENV serotypes using a panel of existing and newly-generated sera and monoclonal antibodies. In parallel, we will develop a new mouse model of DKE-121 infection, which will be of utility to the field for assessing antibody activity, antibody cross-reactivity, and pathogenesis in vivo. We hypothesize that the genetic differences between DKE-121 strain and existing DENV serotypes will result in sufficient loss of serum cross-neutralization to classify this strain as a new DENV serotype, which could have significant consequences for existing DENV vaccine platforms and trials if this or related strains emerged.

ABSTRACT This proposal aims to define a new mechanism of antiviral activity by an Interferon stimulated gene (ISG) Oligoadenylate Synthetase 1 (OAS1). The host innate immune response is initiated by the sensing of non-self viral nucleic acid, and largely mediated through type I and III interferons (IFN). IFNs induce expression of hundreds of ISGs, many of which inhibit virus replication in infected cells, protect uninfected neighboring cells, and shape the adaptive immune response to clear virus infection. OASs are a family of ISG that belongs to an evolutionarily ancient family of nucleotidyl-transferases (NTase). The canonical antiviral mechanism of OAS proteins involves the enzymatic synthesis of 2'-5'-oligoadenylates, causing downstream activation of RNase L and leading to the inhibition of protein synthesis. However, the mechanisms of antiviral activity of multiple enzymatically active and inactive OAS isoforms present in human and mouse genomes are not yet clear. We have found that a specific OAS1 isoform enhances the translation of multiple antiviral proteins, including cGAS and IRF1. This OAS1 isoform (OAS1 P46) enhances translation independent of its enzymatic activity and RNase L. Our preliminary results suggest that OAS1 P46 enhances the translation of specific proteins through binding respective mRNAs. In humans, OAS1 P46 is generated due to an alternative splicing event at the C- terminal of the OAS1 gene. A naturally occurring polymorphism (rs10774671, A/G) at this alternative splice site regulates P46 expression, and has been associated with disease severity to multiple virus infections. Using primary human hematopoietic cells, we demonstrate that rs10774671 G allele coding for P46 also enhances IRF1 protein expression in T cells. We also provide multiple evidences suggesting the functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no NTase activity), which similarly affects WNV susceptibility in vivo. Based on these observations, we hypothesize that specific isoforms of OAS1 modulate innate immune responses against viruses through unique NTase activity-independent mechanisms. The goal of this proposal is to determine how OAS1 enhances specific protein translation, and the consequences of this newly identified function of OAS1 on the antiviral innate and adaptive immunity. Our three independent Aims are to: (1) Determine the molecular mechanism of OAS1-mediated translational regulation through biochemical and cell biology approaches; (2) Define the cellular targets mediating the antiviral effect of OAS1 using several gene-deficient cells; and (3) Define the in vivo role of Oas1b during virus infection using a newly generated Oas1b knock-in mouse. Upon completion of this study, we should establish a new paradigm of antiviral activity by OAS proteins that may lead to therapeutic strategies modulating this OAS1-cGAS-type I IFN axis.

Project Summary Alphaviruses are mosquito-transmitted, positive-strand enveloped RNA viruses of the Togaviridae family that cause global disease in humans. At present, no antiviral agents or licensed vaccines exist for the treatment or prevention of any alphavirus infections. We recently used a genome-wide CRISPR/Cas9-based screen to identify the cell surface molecule LDLRAD3 as a novel, highly conserved entry receptor for Venezuelan equine encephalitis virus (VEEV), an emerging pathogen capable of causing fatal neuroinvasive disease in humans and other vertebrate animals. Gene editing of mouse or human LDLRAD3 resulted in reduced VEEV infection of neuronal cells, and reciprocally, ectopic expression of LDLRAD3 resulted in increased infection. LDLRAD3 bound directly to VEEV virions and enhanced virus attachment and internalization into cells. Genetic studies indicated that domain 1 (D1) of LDLRAD3 is necessary and sufficient to support VEEV infection. We hypothesize that engagement of LDLRAD3 by VEEV will explain how infection, tissue targeting, and disease pathogenesis occurs. The primary goals of this collaborative, interactive project between the Diamond, Fremont, and Whelan laboratories are to define the precise mechanism(s) by which LDLRAD3 facilitates alphavirus entry into cells, to gain high-resolution structural insight as to how LDLRAD3 engages the spike proteins on the virion, and to determine the cell-type specific role of LDLRAD3 in VEEV pathogenesis in vivo. The experiments in this proposal will define fundamental aspects of VEEV biology that enhance our understanding of infection and cell tropism. This information may facilitate the development of small molecules or biologicals that disrupt LDLRAD3 interaction with VEEV spike proteins, which could form the basis of future therapeutics that ameliorate disease of this emerging and highly pathogenic alphavirus.

ABSTRACT This proposal aims to define a new mechanism of antiviral activity by an Interferon stimulated gene (ISG) Oligoadenylate Synthetase 1 (OAS1). The host innate immune response is initiated by the sensing of non-self viral nucleic acid, and largely mediated through type I and III interferons (IFN). IFNs induce expression of hundreds of ISGs, many of which inhibit virus replication in infected cells, protect uninfected neighboring cells, and shape the adaptive immune response to clear virus infection. OASs are a family of ISG that belongs to an evolutionarily ancient family of nucleotidyl-transferases (NTase). The canonical antiviral mechanism of OAS proteins involves the enzymatic synthesis of 2'-5'-oligoadenylates, causing downstream activation of RNase L and leading to the inhibition of protein synthesis. However, the mechanisms of antiviral activity of multiple enzymatically active and inactive OAS isoforms present in human and mouse genomes are not yet clear. We have found that a specific OAS1 isoform enhances the translation of multiple antiviral proteins, including cGAS and IRF1. This OAS1 isoform (OAS1 P46) enhances translation independent of its enzymatic activity and RNase L. Our preliminary results suggest that OAS1 P46 enhances the translation of specific proteins through binding respective mRNAs. In humans, OAS1 P46 is generated due to an alternative splicing event at the C- terminal of the OAS1 gene. A naturally occurring polymorphism (rs10774671, A/G) at this alternative splice site regulates P46 expression, and has been associated with disease severity to multiple virus infections. Using primary human hematopoietic cells, we demonstrate that rs10774671 G allele coding for P46 also enhances IRF1 protein expression in T cells. We also provide multiple evidences suggesting the functional equivalence between OAS1 P46 and a mouse ortholog, Oas1b (no NTase activity), which similarly affects WNV susceptibility in vivo. Based on these observations, we hypothesize that specific isoforms of OAS1 modulate innate immune responses against viruses through unique NTase activity-independent mechanisms. The goal of this proposal is to determine how OAS1 enhances specific protein translation, and the consequences of this newly identified function of OAS1 on the antiviral innate and adaptive immunity. Our three independent Aims are to: (1) Determine the molecular mechanism of OAS1-mediated translational regulation through biochemical and cell biology approaches; (2) Define the cellular targets mediating the antiviral effect of OAS1 using several gene-deficient cells; and (3) Define the in vivo role of Oas1b during virus infection using a newly generated Oas1b knock-in mouse. Upon completion of this study, we should establish a new paradigm of antiviral activity by OAS proteins that may lead to therapeutic strategies modulating this OAS1-cGAS-type I IFN axis.

Project Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that was first isolated in Wuhan China in December, 2019. SARS-CoV-2 is the cause of coronavirus disease 2019 (COVID-19), which is now a pandemic and has caused more than 1.3 million confirmed cases and 72,000 deaths, with an estimated case fatality rate of 4%, with substantially higher death rates (~15%) in the elderly or immunocompromised. Virtually all countries and territories have reported cases, with major epidemics in China, Italy, Spain, France, Germany, Iran, and the United States. SARS-CoV-2 is thought to be of zoonotic origin, most likely bats, and is about 75% identical to the original SARS-CoV. Most cases are spread by direct human-to- human transmission, with community transmission in asymptomatic individuals described. Currently, no countermeasures are licensed for human use. The development, characterization, and ultimately deployment of an antibody-based treatment against SARS-CoV-2 could prevent substantial morbidity and mortality, and possibly mitigate its epidemic spread. This interactive multi-PI proposal leverages complementary expertise in the Diamond, Crowe, and Baric laboratories to rapidly develop highly neutralizing and therapeutic human monoclonal antibodies (mAbs) against SARS-CoV-2 for immediate use in humans. To achieve this goal, we will generate and interrogate human mAbs against SARS-CoV-2 that are obtained from multiple convalescent subjects. We will identify potently neutralizing mAbs and optimize them for affinity by selecting naturally occurring somatic variants identified by repertoire sequencing and sibling analysis and Fc effector functions. Protective activity of top candidate coronavirus mAbs will be tested in newly-generated and optimized mouse models of SARS-CoV-2 infection, including those expressing human ACE2 receptors (hACE2). To define correlates of protection, we will use chimeric viruses, shotgun mutagenesis, and neutralization escape to identify the epitopes of our most protective mAbs. Our team has extensive experience in the generation, characterization and optimization of antibodies, CoV biology, and animal models of disease and protection. A therapy composed of one to three highly neutralizing mAbs may provide an immediate countermeasure against the pandemic spread of SARS-CoV-2 and help establish correlates of structural and functional humoral protection that ultimately inform vaccine efforts.