John Williams - US grants

Brain functions are executed by intricately coordinated networks of neurons, whose modes of operation are highly sensitive to a constellation of neuromodulators. More specifically, neuromodulators such as dopamine, norepinephrine, serotonin, and acetylcholine exert dramatic control over global brain processes such as arousal, attention, emotion, or cognitive perception. Altered neuromodulator signaling has been linked to neurological and psychiatric disorders such as Parkinson's disease, schizophrenia, depression and addiction. Similarly, opioid neuropeptides play important roles in the modulation of cognition and behavior. While the anatomical structures that produce neuromodulatory signals are well known, little is known about the spatial and temporal evolution of these signals in the innervated brain regions. This is because current measurement techniques, such as microdialysis or cyclic voltammetry, lack the spatial or temporal resolution (and often the molecular specificity) to resolve respective signals. This technical challenge has been a long-standing barrier to our understanding of how neuromodulation alters neural circuit function in order to influence behavior. To address this challenge, this project will develop, validate, and disseminate novel genetically encoded fluorescent proteins for large-scale optical measurement of monoamine neuromodulator and opioid neuropeptide signaling in behaving animals, by bringing together a multi-disciplinary team of investigators with unique and complementary expertise. These sensor proteins have the potential to revolutionize neuroscience in a way similar to genetically encoded indicators for calcium, glutamate, and voltage, which are now in widespread use. Combined with calcium and voltage imaging, neuromodulator sensors will reveal how these systems impinge on cellular and circuit function. In particular, proposed sensors will enable minimally invasive, high-resolution, long-term, and direct measurement of neuromodulator and neuropeptide signaling at synaptic, cellular, and system levels. Sensors for neuromodulatory signaling have remained elusive for a long time. Our team recently developed a first generation of genetically encoded indicators for serotonin (5-HT), norepinephrine (NE), and dopamine (DA) that can report nano- to micromolar concentration changes with high spatial and temporal resolution. Building on this work, the following specific aims are proposed: 1) Optimize and diversify genetically encoded sensors for the monoamines using computational modeling, directed evolution and high-throughput screening; 2) Develop and optimize genetically encoded sensors for opiate neuropeptides using novel protein scaffolds; and 3) Systematically validate the novel neuromodulator and neuropeptide sensors in acute brain slices and behaving animals. Together, this work will provide the neuroscience community with a wide range of well-characterized multi-color indicators for probing the functional role of neuromodulators and neuropeptides in regulating neural circuit function and behavior in both health and disease.

PROJECT SUMMARY The long-term goal of our research program is to understand mechanisms of human metapneumovirus (HMPV) immunity and pathogenesis and facilitate vaccine development. HMPV is a leading cause of severe lower respiratory infection (LRI) in children and adults worldwide. There is no licensed vaccine against HMPV. Recurrent infections with HMPV and other respiratory viruses such as respiratory syncytial virus (RSV) occur throughout life. Consequently, HMPV and RSV cause severe illness in older adults and persons with underlying conditions such as asthma, immune compromise, and chronic cardiopulmonary disease. Limited induction of CD8+ T cell (TCD8) memory by respiratory viruses may be a contributing factor to reinfection and presents an obstacle to the development of effective vaccines. RSV, influenza, and parainfluenza viruses have been shown to induce TCD8 with impaired cytolytic function and IFN? secretion, but the mechanism of this inhibition was unknown. Similar antigen unresponsiveness termed TCD8 exhaustion is associated with chronic viral infections and cancer. A key mediator of exhausted TCD8 is programmed cell death-1 (PD-1), a negative regulator of T cell activation implicated in maintaining peripheral tolerance and preventing autoimmunity. PD-1 and other inhibitory receptors including LAG-3 are therapeutic targets in chronic infections and cancer. A recognized adverse effect in these patients is respiratory inflammation, likely due to over-activated TCD8. We discovered that HMPV, influenza, and other respiratory viruses induce TCD8 impairment mediated initially by the PD-1 pathway. However, our preliminary data suggest that other inhibitory receptors, including LAG-3, are involved in maintaining lung TCD8 impairment in later stages of exhaustion. We hypothesize that respiratory viruses activate an orchestrated network of inhibitory pathways, which limit the acute TCD8 response and immunopathology, but also limit the induction of effective memory TCD8. These inhibitory pathways are likely natural immunoregulatory responses to limit lung inflammation, but which also limit effective host response and memory. Elucidating these mechanisms would increase understanding of poor immune memory against respiratory viruses and help guide vaccine development. In Specific Aim 1, we will test the hypothesis that PD-L1 has distinct functions on different cell types in the lung. In Aim 2, we will define the contribution of other inhibitory receptor pathways to late TCD8 impairment. In Specific Aim 3, we will test the hypothesis that lung TCD8 impairment programming can be bypassed using non-replicating virus-like particle (VLP) vaccines with local inhibitory receptor blockade. This work will elucidate mechanisms of TCD8 impairment and define the contribution of TCD8 immunoregulatory pathways in the memory response to vaccination and viral respiratory infections. The findings will guide HMPV vaccine development and identify biomarkers of effective TCD8 responses to vaccines. The results of these experiments will be applicable to other respiratory pathogens as well as relevant to respiratory complications of inhibitory receptor blockade therapy.

Summary Acute respiratory illness (ARI) and acute gastroenteritis (AGE) are leading causes of disease in children in the U.S. and globally. These infections contribute a substantial burden of morbidity, mortality, and direct health care costs, in addition to the indirect costs associated with parental leave from work. Many cases of ARI are caused by viruses, including influenza, respiratory syncytial virus, and others. Similarly, most AGE in the U.S. is associated with viruses, including rotavirus and others. There are few or no effective antivirals for these, and therefore vaccination is the most promising intervention. Licensed vaccines are available for influenza and rotavirus; candidate vaccines for other pathogens are in development. Active, prospective surveillance is necessary to establish ?real-world? vaccine effectiveness (VE). Moreover, population-based surveillance can discover the burden of potentially vaccine- preventable diseases and guide policymakers and industry. The infrastructure provided by the New Vaccine Surveillance Network (NVSN) will facilitate these goals, as well as allowing the description of the clinical features, natural history, and population dynamics of these illnesses. The Children?s Hospital of Pittsburgh (CHP) offers an ideal environment to conduct the proposed population-based research, as CHP is the only major provider of pediatric inpatient and Emergency Department (ED) care in Allegheny County and the region. We propose three Specific Aims. Aim 1: To evaluate the effectiveness and impact(s) of current or upcoming vaccines and other immunoprophylaxis strategies, and inform pediatric vaccine-related policies. Using test-negative case-control methods, we will calculate the annual VE of influenza and rotavirus vaccines against medically attended ARI and AGE. Aim 2: To actively assess the burden of AGE and ARI (including illness with laboratory-confirmed viral etiologies) in the pediatric population. We will perform laboratory confirmation of viral etiologies of ARI and AGE among ill subjects enrolled in the inpatient or ED, and healthy controls enrolled at well-child visits. Aim 3: To establish the natural history of disease for pediatric infectious diseases, transmission dynamics, vaccine impacts for targeted and vulnerable populations, and socioeconomic and microbiological environments potentially relevant to public health interventions. We will capture extensive clinical and demographic data on enrolled subjects and healthy controls. Subjects will be tested for additional ARI- and AGE-associated viruses. The completion of this project will provide new data regarding the VE of licensed vaccines; define the population-based burden of potentially vaccine-preventable diseases; and establish the natural history and disease association of multiple human viruses. These results will guide the development of new vaccines and antivirals, inform public health policies, and enhance the health outcomes of children in the U.S. and globally.

SUMMARY (For Components A, B, and C). Acute gastroenteritis (AGE) and acute respiratory illness (ARI) are leading causes of childhood disease, accounting for a large proportion of hospitalizations, Emergency Department (ED) visits, and outpatient visits annually in the US. These illnesses are caused by diverse pathogens, including influenza, respiratory syncytial virus, human metapneumovirus, parainfluenza viruses, rhinovirus, enterovirus (EV), coronavirus including SARS-CoV-2, adenovirus, rotavirus, norovirus, astrovirus, and sapovirus. Moreover, some of these viruses are associated with other emerging childhood syndromes, including acute flaccid myelitis (AFM) associated with EV, and multisystem inflammatory syndrome in children (MIS-C) associated with SARS-CoV-2. Thus, viral AGE and ARI are of major public health importance, and result in serious long-term consequences for some children. There are few or no effective antivirals for these viruses and vaccination is the most promising intervention. Our goals are to conduct active, prospective population-based surveillance for AGE and ARI; to define the burden of vaccine-preventable diseases; describe the clinical features, natural history, and population dynamics; and establish vaccine effectiveness (VE) of licensed and impending vaccines and monitor VE over time. The New Vaccine Surveillance Network (NVSN) will facilitate these goals. We propose four Specific Aims: Aim 1: to conduct prospective active surveillance for AGE due to norovirus, rotavirus, and other enteric viruses among children seeking healthcare in ED, inpatient, and outpatient settings. (Component A, Mandatory Component 1; Optional Component B) Aim 2: to conduct prospective active surveillance for ARI due to respiratory viruses in these settings. (Component A, Mandatory Component 1; Optional Component B) Aim 3: to conduct prospective active surveillance for AFM syndrome in these settings. (Component A, Mandatory Component 2) Aim 4: to conduct prospective active surveillance for MIS-C. (Optional Component C) The Pittsburgh site has extensive experience with pediatric clinical research, including as a top enrolling NVSN site in the current NVSN cycle. UPMC Children?s Hospital of Pittsburgh (CHP) has a catchment area of >5.5 million people and admits >95% of hospitalized children in the surrounding county of >1.2 million. Thus, the environment is excellent for population-based research. The experienced investigative team includes experts from pediatric infectious diseases, critical care, rheumatology, and cardiology. The data and samples collected in this project will facilitate the capacity to calculate VE for multiple licensed and pending vaccines. The results of this project will inform best practices for diagnosis and treatment, guide vaccine recommendations, and determine public health interventions to prevent viral illness-related medical visits among children.