J. Lindsay Whitton - US grants

This competing continuation application has three overall goals: First, to extend the reach of vaccination, by increasing the number of effective epitopes. Second, to understand and manipulate epitope dominance and subdominance, permitting us to maximize vaccine-induced immunity. Third, to clarify the effects of serial microbial infections upon vaccine-induced immunological memory. These three goals are divided into five specific aims: Aim 1. To identify subdominant epitopes and to carefully map the kinetics of the CTL responses to all epitopes, dominant and subdominant, during acute infection and in the memory phase. Aim 2. To extend the reach of vaccination by expanding the number of virus sequences open to immune surveillance. Can we convert a very poor epitope into a strong one? Aim 3. To design vaccines which circumvent immunodominance, and to evaluate the protective benefits. Current vaccines are limited by immunodominance; they induce responses only to the few dominant epitopes they contain. Can we design new vaccines which circumvent this problem, permitting the concurrent induction of responses to all encoded epitopes? Aim 4. To determine the role of lysis of antigen presenting cells in control of immunodominance. Is the phenomenon of immunodominance controlled by lysis of APCs by the "strongest" CTL responses? Aim 5. How stable is vaccine-induced memory for CTL, and what are the effects of subsequent microbial infections? If memory is diminished by subsequent infection, as has been suggested, this could have profound implications.

Viral infections continue, particularly since the advent of HIV, to exact a massive toll of human morbidity and mortality. This is despite the use of antiviral vaccines, which nevertheless represent one of the most remarkable achievements in medical care over the past three decades, having eradicated smallpox, and diminished the load imposed by many other pathogenic viruses. HIV is the major concern at present, but other agents too must be considered threatening, particularly in underdeveloped countries: e.g. measles still kills around 2 million people annually, polio still paralyses, and influenza is a constant Date Released: threat. To meet these challenges, a full understanding of the immunological basis of vaccine-induced protection must be obtained. In the first period of funding Dr. Whitton's group has demonstrated a protective role for vaccine-induced CD8+ cytotoxic T lymphocytes: a vaccine has been designed which induces these cells without inducing antiviral antibodies, and which confers complete protection against normally-lethal virus challenge. This competing proposal has three aims: first, to further dissect the factors which determine whether a given virus sequence will induce CD8+ CTL responses. The effects of amino-acid residues flanking the epitopes will be determined, and the ability to link multiple short epitopes in a "string-of-beads" vaccine (which "concentrates" the immunologically- critical sequences, thus increasing the effective capacity of many delivery vectors) will be assessed. MHC class I and II, and B cell, epitopes will be included in such a string-of-beads, and the immune responses measured. Second, the role of CD4+ T cells will be measured, exploiting Dr. Whitton's recent collaborative finding that mice unable to mount normal CD8+ CTL responses instead mount a significant CD4+ CTL response. Are CD4+ CTL instrumental in controlling LCMV infection Le are they a somewhat-effective "backup" system? If so, what mechanism underlies this? Thirdly, a comparative evaluation will be undertaken of several antigen delivery systems, to determine which one provides the most balanced and effective antiviral response. Vaccinia virus will be used (continuing and expanding upon the applicants previous studies); in addition, retroviruses and retrovirus-transformed cells will be evaluated (how well do these agents, already approved for human gene therapy trials, induce immunity?); and purified plasmid DNA will be directly injected intramuscularly (this is known to induce responses, but the biological efficacy has never been determined). Furthermore, the effect of administration of pre-formed antigen will be measured, using synthetic peptides (what are the optimal sizes and sequences for induction of effective responses?) and/or immuno-stimulatory complexes (ISCOMs). Immune responses will be evaluated by several criteria.

virus diseases; nervous system infection; cytotoxic T lymphocyte; aging; ribozymes; virus replication; viral vaccines; amyloid proteins; disease /disorder prevention /control; Alzheimer's disease; genetic disorder; antisense nucleic acid; nonhuman therapy evaluation; MHC class I antigen; tissue /cell culture; genetically modified animals; laboratory mouse; histopathology;

DESCRIPTION (provided by applicant): Coxsackievirus B3 causes myocarditis, pancreatitis and meningo-encephalitis but, despite the resulting human morbidity and mortality, neither a treatment, nor a vaccine, is available. My lab has shown that the metabolic status of the host cell plays a key role in determining the outcome of CVB3 infection and, in the previous period of support, we identified stem cells as early targets of CVB3 infection. In this renewal application, I propose 4 Specific Aims, focusing on the following topics: 1. Bone marrow is the main repository of stem cells in the adult, and we show herein that CVB naturally infects ~1% of bone marrow cells in vivo. We shall identify and characterize the bone marrow cells that become infected;and will evaluate the biological implications of this infection. 2. We shall determine the role of cellular activation in regulating CVB3 infection in the heart. We shall use a variety of methods to ask: are proliferating cells targeted? Are myocardial stem cells a preferred site of infection? Does prior myocardial damage alter viral replication in the heart, and does this exacerbate the viral myocarditis? 3. The innate immune response to picornaviruses in general, and to enteroviruses in particular, is poorly understood. We shall investigate the innate responses to CVB3 infection in lymphoid tissues (spleen &lymph nodes). What responses are mounted? Which of the many innate molecular sensors are involved? How does activation of the innate system affect the outcome of subsequent CVB3 infection? 4. Many virus infections induce very strong T cell responses, but CVB3 appears not to do so;neither CD4+ nor CD8+ T cells are strongly activated during wtCVB3 infection. We shall use novel methods to map, kinetically and anatomically, the presentation of CVB3-encoded MHC class I &class II epitopes, and will ask how the innate responses to CVB3 infection affect the subsequent development of adaptive T cell immunity. PUBLIC HEALTH RELEVANCE: Coxsackieviruses infect millions of people each year in the USA. In most cases, the infections cause little harm, but in some cases - especially in very young children - the diseases can be serious, and sometimes fatal. This research will help us understand how these viruses cause disease, and will provide clues about how to prevent or treat these dangerous infections.

DNA immunization is a potentially important approach to vaccination. It has been shown to work in many animal models, producing immune responses which can counter viruses, bacteria, and tumors. However the mechanisms which underpin this approach remain poorly defined. This proposal is aimed towards analyzing these mechanisms, and using the accrued knowledge to manipulate and optimize DNA vaccines. The proposal has four specific aims. Aim 1. To identify the cells which take up and express DNA following intramuscular injection, and which present antigen to T cells. It is known that DNA is expressed in muscle cells; are APCs also transfected? We shall use cloned CTL as probes to identify the cells actually presenting antigen following DNA immunization. Aim 2. To precisely identify which cells induce immunity, and to determine whether muscle cells are important. That cells can be recognized by T cells does not imply that they can induce responses. Can we use cell sorting & transfer to identify the cells responsible for induction of immunity? Aim 3. To evaluate the role of antigen release in DNA immunization, and to identify the underlying mechanisms. Does induction of antibody & CD4+ T cells require antigen release into the humoral phase? If so, what mechanisms underlie this release? Does T cell mediated lysis play a role, and if so, what are the roles of the perforin & fas pathways? Does T cell lysis subsequently limit the immune response? Aim 4. To use the accumulated knowledge to optimize DNA immunization. The knowledge from aims 1-3 will be drawn together to optimize induction of antibodies, CD4+ T cells, and CD8+ T cells each of which probably will have unique requirements for optimal responses.

prions; virus diseases; nervous system infection; aging; cytotoxic T lymphocyte; ribozymes; viral vaccines; nonhuman therapy evaluation; central nervous system disorders; immune tolerance /unresponsiveness; disease /disorder prevention /control; Alzheimer's disease; antiviral agents; pathologic process; gene expression; virus genetics; spongiform encephalopathy; neurons; virus replication; lymphocytic choriomeningitis virus; tissue /cell culture; genetically modified animals; laboratory mouse; western blottings; histopathology;

DNA immunization is a potentially important approach to vaccination. It has been shown to work in many animal models, producing immune responses which can counter viruses, bacteria, and tumors. However the mechanisms which underpin this approach remain poorly defined. This proposal is aimed towards analyzing these mechanisms, and using the accrued knowledge to manipulate and optimize DNA vaccines. The proposal has four specific aims. Aim 1. To identify the cells which take up and express DNA following intramuscular injection, and which present antigen to T cells. It is known that DNA is expressed in muscle cells; are APCs also transfected? We shall use cloned CTL as probes to identify the cells actually presenting antigen following DNA immunization. Aim 2. To precisely identify which cells induce immunity, and to determine whether muscle cells are important. That cells can be recognized by T cells does not imply that they can induce responses. Can we use cell sorting & transfer to identify the cells responsible for induction of immunity? Aim 3. To evaluate the role of antigen release in DNA immunization, and to identify the underlying mechanisms. Does induction of antibody & CD4+ T cells require antigen release into the humoral phase? If so, what mechanisms underlie this release? Does T cell mediated lysis play a role, and if so, what are the roles of the perforin & fas pathways? Does T cell lysis subsequently limit the immune response? Aim 4. To use the accumulated knowledge to optimize DNA immunization. The knowledge from aims 1-3 will be drawn together to optimize induction of antibodies, CD4+ T cells, and CD8+ T cells each of which probably will have unique requirements for optimal responses.

DESCRIPTION (provided by applicant): In this application, I propose to analyze virus-specific CD4+ T cells in two animal models of virus infection, selected because they represent the two ends of the immunological spectrum; one induces strong CD8+ T cell responses [lymphocytic choriomeningitis virus (LCMV)], while the other induces strong CD4+ T cell/antibody responses [coxsackievirus B3 (CVB3)]. We have two general goals: first, to measure CD4+ T cell function at the cellular and organismal levels (Aims 1 & 2 respectively); and, second, to identify the factors which appear to restrict the expansion of antigen-specific CD4+ T cells (Aim 3). All of our analyses will be done using normal T cells, evaluated directly ex vivo.Aim 1: We shall examine the antigen-responsiveness of primary and memory CD4+ T cells, and determine whether or not they undergo functional avidity maturation during the course of viral infection.Aim 2: We shall investigate the role of vaccine-induced CD4+ T cells in modifying the immune responses to subsequent viral infection; and we will measure the protective benefits of these cells. These experiments will exploit our experience with LCMV (a virus controlled by CD8 +T cells) and with CVB3 (which is controlled by antibodies). We shall generate a stable of recombinant CVB expressing a variety of CD8 and CD4 epitopes.Aim 3. Immunodominance is an important immunological phenomenon, which profoundly affects CD8+ T cell responses. Our preliminary data indicate that it also affects CD4+ T cell responses; and that CD8+ T cells actively suppress CD4+ T cell expansion. This may explain why, during LCMV infection, CD8+ cells outnumber CD4+ cells by 20:1. We shall investigate this further, and will determine the underlying mechanism. This may allow us to circumvent the problem, thus permitting the design of vaccines, which could induce elevated levels of CD4+ T cell memory.

CD8 + T cells, which are strongly induced by most viral infections, are conceptually attractive as effector cells in autoimmunity, because most cell types express MHC class I and are, therefore, open to CD8 + T cell surveillance. Many studies of autoimmune disease have focused on CD4 + T cells, but emerging data indicate that CD8 + T cells may play a critical role. In this component of the program grant, I focus on CD8 + T cells and, in particular, on their ability to undergo bystander activation during viral infections. We postulate two general types of virus-induced bystander activation, both of which will be investigated herein. First, we have evidence that, during virus infection, a substantial proportion of naive CD8 + T cells can undergo some degree of TcR-independent bystander activation. In Specific Aim 1, we shall: (i) evaluate how effectively naive, primary, and memory CD8 + T cells are activated in vivo in a TcR-independent manner; (ii) determine whether naive cells, activated by these bystander effects, mature into "bystander memory" cells; (iii) evaluate the roles of several cytokines in TcR-independent bystander activation. Second I propose a novel mechanism to explain TcR-dependent bystander activation. I suggest that virus-induced IFNgamma, up-regulates expression of the immunoproteasome and that this, in turn, leads to the presentation of new self epitopes, against which autoreactive CD8 + T cell responses may be mounted. Little is known about how, where & when the immunoproteasome is induced during virus infection, and this is the focus of Specific Aim 2. In Specific Aim 3, we shall evaluate the role of the immunoproteasome in type 1 diabetes. These studies will be carried out together with Dr. von Herrath (PI of this program grant, and of Project I). We shall determine immunoproteasome expression during the development of T1D, and assess whether T1D develops in the absence of the immunoproteasome. Finally, in Specific Aim 4, in collaboration with Dr. Fujinami (Project II), we shall study the role of the immunoproteasome in CNS autoimmune disease, including a new animal model of multiple sclerosis, in which CD8 + T cells appear to be the culprits.

DESCRIPTION (provided by applicant): CD8+ memory T cells are one of the cornerstones of successful vaccination, and much is known about their generation, i.e. the pathways that determine na¿ve CD8+ T cell activation, expansion & contraction, and the factors that affect the establishment of CD8+ memory T cell subsets. In contrast, we know relatively little about CD8+ memory T cell recall responses, upon which vaccine efficacy relies. Memory cells deal with individual virus-infected cells in vivo and, to do so effectively, they must respond to secondary infection (i.e., begin their recall response) by imposing their effector functions upon an infected cell before virus progeny has been released; for many virus infections, this means that - if they are to be maximally protective - memory T cells must act within hours of infection. Hence, my lab recently has begun to investigate the very early (d24 hours p.i.) recall responses of CD8+ memory T cells; and, so far as is possible, we measure the responses in vivo. Unpublished data (presented herein) show that: (i) CD8+ memory T cells initiate effector responses to virus infection in vivo within 3-6 hours (long before a single round of virus replication has been completed); and (ii) very surprisingly, in vivo cytokine synthesis (IFN¿, TNF & IL-2) is largely terminated soon thereafter (by 24 hours p.i.). This termination of effector function occurs despite the presence of immunostimulatory viral antigen, suggesting that there is active down-regulation of effector function by CD8+ memory T cells that have responded to infection. These events occur before the memory T cells have multiplied. These early recall responses, and the molecular mechanisms that control them, will be explored in four Specific Aims. 1. To investigate the in vivo regulation of CD8+ memory T cell early recall responses. Our observations regarding the rapid on/off expression of effector functions by CD8+ memory T cells will be expanded. 2. To identify and manipulate the molecular pathway(s) underpinning the early recall response of CD8+ memory T cells. The molecular mechanisms regulating the initiation, and the termination, of effector function(s) will be identified using several approaches. 3. To evaluate the role of dendritic cells in regulating these very early memory T cell responses. The majority of studies of DC / T cell interactions have (understandably) focused on priming of na¿ve T cells. Here, we shall evaluate the role of DCs in the rapid in vivo responses of CD8+ memory T cells. 4. To generate and test a mouse line that would allow us, for the first time, to evaluate the importance of various proteins in regulating the recall responses of CD8+ memory T cells. We shall generate a transgenic mouse line which will allow the investigator to deletion a gene from CD8+ T cells at the time of his/her choosing. An infected mouse would mount a completely normal primary T cell response, and would establish a normal memory cell pool; only then would we delete the gene of interest.

DESCRIPTION (provided by applicant): The purpose of this training grant is to train predoctoral and postdoctoral fellows in a multidisciplinary approach to study the molecular basis of viral pathogenesis. The program brings together 19 accomplished investigators representing a broad range of virologic, immunologic, and structural and molecular genetics expertise. A common theme among these laboratories is the goal of elucidating molecular mechanisms of viral pathogenesis. Recent experience with the AIDS epidemic as well as with outbreaks of emerging viral diseases, virally induced autoimmune and neurodegenerative diseases, and the threat posed by bioterrorism underscores the need to train competent investigators in viral pathogenesis. The goals of this program are as follows: 1) to provide trainees with a solid experimental background in contemporary virology; 2) to acquaint fellows with structural, immunologic, cell biological molecular biological and chemical approaches used to investigate viral pathogenesis over a broad range of model systems; 3) to ensure that trainees have in addition to their specific experimental problem, an appreciation of the full range of critical issues and concepts of viral pathogenesis both from the point of view of virus and host. Applicants will be selected on the basis of their academic and research track records, letters of recommendation from their mentors and associates, and their commitment to a career in basic research. Specific efforts will be undertaken to identify and attract qualified candidates from underrepresented minorities. Scientific development of the postdoctoral fellows will be guided by: 1) carrying out an original basic research project in the laboratory of a member of the training grant faculty; 2) regular presentation and critique of data in laboratory meetings and group retreats; 3) immersion in the rich intellectual environment of The Scripps Research Institute (TSRI) with participation intensive seminar series and journal clubs sponsored by the pathogenesis, immunology, and molecular and cellular biology affinity groups: and 4) accessibility of highly sophisticated instrumentation and laboratory facilities. In addition, fellows are provided access to core and elective course options offered by the TSRI graduate program. It is our experience that such a training program produces well-rounded scientists who are able to contribute effectively to the field of viral molecular pathogenesis in an academic or research environment.

[unreadable] DESCRIPTION (provided by applicant): Protective immunity, whether induced by infection or by vaccination, relies on the generation of memory B & T cells in sufficient quantity, and of sufficient quality. In the case of CD8+ T cells - to be studied herein - these memory cells usually represent the few survivors of a dramatic and rapid cull, T cell contraction, in which 90-95% of virus specific CD8+ T cells (in our model, ~50 million cells) are removed over a period of 1-2 weeks. The process of T cell contraction remains poorly-understood, and the overall goal of this proposal is to better characterize the causes and consequences of T cell contraction. The application has the following 4 Specific Aims: 1. To investigate the role of direct IFN3 signaling in regulating CD8+ T cell contraction. IFN3 is a key cytokine. Its receptor is expressed on almost all somatic cells, allowing it to act both as an antiviral effector molecule, and as an immunomodulatory molecule. We have developed a novel approach that allows us to evaluate the direct effects of IFN3 on T cells during virus infection, and we have found that - contrary to much of the published literature - the effects of IFN3 are strongly positive; T cells that are unable to receive IFN3 signals during virus infection are 100-fold less likely to enter the memory pool. In this aim, we propose a detailed analysis of these direct effects. 2. To determine how indirect effects of IFN3 affect T cell contraction. The widespread expression of the IFN3 receptor means that IFN3 also can affect T cell biology indirectly, via a multitude of paths. Some of these indirect effects will be evaluated. 3. To evaluate antigen persistence after acute virus infection is cleared, determine its effects on the quality and quantity of T cells, and analyze the role of IFN3 in these effects. Antigen contact plays an important part in regulating T cell function, and we have developed a new approach that allows us to identify T cells that have recently encountered authentic viral antigen in vivo. We shall ask how recent antigen contact correlates with a variety of T cell phenotypes (proliferative status, expression of cytokine receptors, etc.). 4. To manipulate T cell contraction, and determine the consequences on the quantity and quality of memory cells. Most studies suggest that T cell contraction is relatively random, but I propose that it may be selective, possibly serving to separate the wheat from the chaff. To investigate this, various approaches will be taken to modify the contraction phase, and the quantity and quality of the surviving T cells will be determined. PUBLIC HEALTH RELEVENCE:T cells are a vital part of the immune response. They help us recover from many viral and bacterial infections, and they are important in vaccine-mediated immunity to infection and disease. When a microbe enters our body, we produce many millions of these T cells to fight the infection but, after the infection has been cleared, the vast majority of the T cells die. In this grant, I investigate how and why the T cells die. I also will attempt to reduce the amount of T cell death, in the hope that the increased number of surviving T cells will give us increased protection against infection and disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Coxsackieviruses are the commonest infectious cause of acute myocarditis, a disease that causes substantial human morbidity and mortality. Even after the virus has (apparently) been cleared, low- grade inflammation may continue, eventually leading to the serious outcome of dilated cardiomyopathy. There is general agreement that the innate and adaptive host immune responses contribute both to virus control, and to (immunopathological) heart disease. However, our knowledge of coxsackievirus-induced immune responses is rudimentary. In this proposal, using existing and new mouse models of coxsackievirus B3 (CVB3) infection, we shall evaluate the intracardiac effects of type I interferons (T1IFN) on acute and persistent CVB3 infection, and we shall use a unique set of reagents, developed in my laboratory, to assess CVB-specific T cell responses in the hearts of infected mice. There are 3 Specific Aims : 1. The effects of T1IFN in the heart are controversial. Some published work suggests that these innate cytokines may clear virus from the heart, but other work has suggested that they play no role whatsoever within the infected heart. We shall cross two existing mouse strains to develop a new mouse model in which the expression of T1IFN receptor on cardiomyocytes can be ablated at will. These mice will allow us to resolve the above controversy, and to determine if T1IFN is responsible for constraining viral spread in the acutely-infected heart. In addition, by removing this receptor during persistent infection, we shall be able to determine if T1IFN is required for the maintenance of the persistent state. 2. Despite many years of study, our understanding of CVB-specific T cell responses remains minimal. To address this deficiency, my lab has generated a variety of unique reagents including recombinant CVB that expressed well-characterized CD4+ and CD8+ T cell epitopes, and genetically-marked T cells specific for those epitopes. We shall exploit our reagents to investigate the kinetics of CVB- specific CD4+ & CD8+ T cell infiltration in the heart over the course of acute myocarditis, and will determine the extent to which this T cell infiltration is regulated by T1IFN. 3. We also shall use the above reagents to map CVB3 antigen expression within the heart during acute and persistent infection. In which heart cells are antigens expressed? How quickly do antigen- specific CD4+ and CD8+ T cells home to the infected heart at various times after infection? For how long after viral clearance do epitopes remain detectable in the heart? How do virus-specific CD4+ and CD8+ T cells contribute to virus clearance, and to the development of immunopathological heart disease?

DESCRIPTION (provided by applicant): The immune system mounts strong CD8+ T cell responses to almost all acute virus infections. However, one virus genus - the enteroviruses - is a stark exception. These viruses can replicate to extremely high titers in vivo, and they induce CD4+ T cells and antibodies, yet these viruses almost completely avoid triggering na¿ve CD8+ T cells. Herein, using the CVB3 mouse model, we shall investigate the mechanism(s) by which enteroviruses achieve this remarkable feat. Antiviral CD8+ T cell responses are initiated when na¿ve CD8+ T cells are activated (or primed) by contact with an MHC class I / epitope peptide complex on the surface of dendritic cells (DCs). Some viruses can infect DCs, in which case their proteins, being synthesized endogenously (i.e., within the DC) will enter the cell's MHC class I pathway, ultimately triggering CD8+ T cells; this process is called direct priming. However, there are at least two situations in which direct priming cannot occur. First, some viruses that infect DCs also encode proteins that very effectively inhibit MHC class I presentation, rendering the infected DC incapable of antigen presentation. Second, many viruses do not infect DCs. Nevertheless, in both of these cases, the host still mounts strong CD8+ T cell responses to most viruses. It is now known that viral proteins that have been released from infected cells can be taken up by a subset of uninfected DCs, allowing immunogenic proteins to be separated from MHC-inhibitory proteins; these DCs can present viral epitopes on MHC class I. This process is called cross-presentation and, if it results in the triggering of naive CD8+ T cells, it is termed cross-priming. This explains how the immune system can mount strong CD8+ T cell responses to almost all acute virus infections. Why can't it do so for CVB3? In Aim 1, I will investigate the possibility that CVB3 specifically inhibits the cross-priming pathway, preventing uninfected host DCs from capturing viral proteins. In addition, I have conceived of another explanation: that immunological information may be transferred not as protein, but as mRNA, and that the unique capacity of enteroviruses to evade CD8+ T cells results from the unusual coding strategy of these viruses. This mRNA transfer idea may be important beyond enteroviruses, because it also can explain the absence of CD8+ T cell responses to extracellular bacteria. Aims 2 & 3 test this new, and potentially-important, hypothesis. Aim 1. To assess the effect of CVB3 infection on cross-presentation / cross-priming. Aim 2. To ask if mRNA coding strategy explains how enteroviruses can almost completely evade naive CD8+ T cells, while most viruses induce strong CD8+ T cell responses. Aim 3. To determine if mRNA regulatory sequences explain why extracellular bacteria fail to induce strong CD8+ T cell responses.

DESCRIPTION (provided by applicant): In vivo injection of mRNA triggers immune responses to the encoded proteins, and confers protection against disease. For this and other reasons, outlined below, RNA vaccines have great clinical potential, so it is important that we understand how they induce immunity. I hypothesize that RNA vaccines may be exploiting a cell type, and biological pathway, that have evolved specifically to capture, internalize, and translate mRNA. I propose that this pathway facilitates the transfer of immunological information into uninfected dendritic cells (DCs), thereby playing a key part in regulating CD8+ T cell responses to many viral and bacterial infections. Almost all acute virus infections induce strong CD8+ T cell responses, which are initiated when na?ve CD8+ T cells are activated by contact with an MHC class I / epitope peptide complex on the surface of DCs that express appropriate costimulatory molecules. However, many viruses do not infect DCs; and some viruses that infect DCs also encode proteins that quite effectively inhibit MHC class I presentation. These facts posed a puzzle: how could epitopes encoded by these viruses be effectively presented by DCs? The answer came with the identification of cross-presentation which, if it results in the triggering of naive CD8+ T cells, causes cross-priming. However, two in vivo observations show that cross-presentation/cross-priming (hereinafter, CP) is not always highly-efficient. First, enteroviruses replicate to very high titers and induce CD4+ T cells and antibodies, yet (unique among acute virus infections) they completely avoid triggering na?ve CD8+ T cells. Second, extracellular bacterial infections, in which microbial protein is hugely abundant, do not induce strong CD8+ T cell responses. For reasons described below, I hypothesize that both observations can be explained by proposing that some transfer of immunological information into uninfected DCs may rely on mRNA (rather than protein). So, this proposal has two goals: First, to evaluate how naked RNA induces immunity. Second, to test the hypothesis that, during most microbial infections, mRNA is transferred to uninfected DCs; if it is translated therein, the encoded protein will reach the class I MHC pathway, inducing strong CD8+ T cell responses; I have named this mechanism TATOR (transfer and translation of RNA). Conversely, if the mRNA cannot be translated, the organism is undetectable by CD8+ T cells. Thus, the specific characteristics of their mRNAs renders enteroviruses and extracellular bacteria invisible to CD8+ T cells. Aim 1. To identify and characterize the DC subset that is involved in RNA-triggered immunity. Aim 2. To determine if mRNA regulatory sequences explain why extracellular bacteria fail to induce strong CD8+ T cell responses.

DESCRIPTION (provided by applicant): Coxsackievirus B3 (CVB3), an enterovirus in the picornavirus family, is a frequent infectious cause of pancreatitis, a common, serious, and costly disease. Some forms of pancreatitis are known to involve the autophagy pathway; e.g., toxins that block late steps in the pathway can trigger the disease, perhaps because the blockade causes activation of intracellular trypsinogen. Paradoxically, complete inactivation of autophagy protects against those types of pancreatitis. Therefore, at least those forms of pancreatitis require that the pathway be both (i) active and (ii) blocked at a late stage. Autophagy is upregulated during, and often combats, many viral and bacterial infections. Predictably, evolution has led to several viruses developing mechanisms by which to evade the inhibitory effects of the pathway, and studies have suggested that some viruses - including CVB3 - have gone one step further, actively exploiting autophagy to enhance their replication. My lab has shown that, in acinar cells in vivo, CVB3 interrupts a late stage of the autophagy pathway. We therefore hypothesized that there might be a link between this effect of CVB3 on the autophagy pathway, and the virus' ability to cause pancreatitis. We wished to test this idea in vivo, (i.e., n the living animal, not only in tissue culture cells). To achieve this goal, we have recently developed conditional KO mice (Atg5f/f/Cre+ mice) in which a key protein in the autophagy pathway, Atg5, has been deleted only in pancreatic acinar cells. Using these mice, we have shown that CVB3 benefits from an intact autophagy pathway without which CVB3 replication in the pancreas is severely curtailed. Furthermore, virus-induced pancreatitis is dramatically reduced in the Atg5f/f/Cre+ mice. Thus, like some other types of pancreatitis, CVB pancreatitis requires that autophagy be active (as it is in wt mice), but dysfunctional (because the virus blocks the pathway). Furthermore, our unpublished data show that CVB3 infection induces autophagy-dependent trypsin activity inside acinar cells. These and other data lead me to propose that there is a common cause for all pancreatitides, viral and non- viral; I propose that they result from a late blockade of the autophagy pathway, which leads to cleavage of intracellular trypsinogen, unleashing trypsin activity inside the cell. These issues will be investigated in Aims 1 & 2. I further hypothesize that the trypsin-initiated damage can be exacerbated by T1IFN signaling into acinar cells; this will be investigated in Aim 3. If these concepts are validated, autophagy and T1IFNs would become key therapeutic targets for all forms of this currently-untreatable disease. There are three Specific Aims. Aim 1. To better define interactions among CVB3, autophagy, and trypsinogen in acinar cells in vivo. Aim 2. To evaluate how T1IFN signaling directly into acinar cells affects viral pancreatitis. Aim 3. To evaluate the role of T1IFNs in recruiting immune cells to the virus-infected pancreas.

? DESCRIPTION (provided by applicant): Viral myocarditis is a relatively common, sometimes severe, and potentially fatal disorder, and one of the most frequent causes is the enterovirus coxsackievirus B3 (CVB3). CVB3 infection of the heart is highly focal, even in mice lacking an intact adaptive immune system, suggesting that innate immunity may hold the virus in check. Type I interferons (T1IFNs) are an obvious candidate, and are known to positively affect the outcome of enteroviral myocarditis. However, there is much that we do not know about how the effects of T1IFNs are mediated. For example, one very well-respected group has argued that T1IFNs may not even act within the heart, and that their cardioprotective effects may be indirect, resulting from the suppression of viral replication in other organs. We have generated mice in which the T1IFN receptor (T1IFNR) can be ablated in vivo specifically from cardiomyocytes, and our unpublished data show unequivocally that T1IFNs do, in fact, act directly on cardiomyocytes during CVB3 infection, and play a substantial role in viral titers and CVB-induced disease. But many questions remain. How do they do this - what genes are activated by T1IFNs in infected (and in uninfected) cardiomyocytes? We shall identify the cardiomyocyte genes that are directly responsive to T1IFN signals in vivo. CVB3 also causes chronic myocarditis and dilated cardiomyopathy, which are related to RNA persistence in the heart, and our inducible gene knockout model confers a key experimental advantage in this regard; we can establish persistent CVB3 infection in genetically-intact mice that do not develop disease, and afterwards ablate T1IFN signaling into cardiomyocytes, to ask: do these mice now develop disease, i.e. might resistance/susceptibility to chronic myocarditis / DCM be related to T1IFN signaling in cardiomyocytes? Our preliminary data indicate that the effects of T1IFN during CVB3 infection of the heart may be biphasic, and we hypothesize that the two temporal phases are defined by differing cellular sources of the T1IFNs. So, Aims 2 and 3 extend our focus to address the production of T1IFNs. What cells serve as the source of the T1IFNs? Plasmacytoid DCs (pDCs) are obvious candidates, but recent work has raised questions about their true in vivo role during viral infection, and also has shown that TLR3-dependent production of T1IFNs may be more important in controlling virus infection. The proposal has three Specific Aims Aim 1. To investigate the in vivo consequences of type 1 IFN signaling in cardiomyocytes during acute and persistent CVB3 infection. Aim 2. To evaluate the role of pDCs during CVB3 infection Aim 3. To determine why TLR3 plays a key role in CVB3-induced myocarditis.