Sean Murphy - US grants

DESCRIPTION (provided by applicant): Finding interventions that improve outcome from ischemic stroke has proved to be challenging and current treatment is limited to thrombolysis, aspirin, and management in a stroke unit. An ideal stroke therapeutic would minimize damage to mature neurons and white matter, and also maximize the generation of new neurons from endogenous progenitors. Recent findings of our own and of others, both in vitro and in animal models of stroke, suggest that histone deacetylase (HDAC) inhibitors meet these criteria. Drug administration protects isolated neurons from induced apoptotic cell death and white matter from oxygen-glucose deprivation, results in improved histologic and functional outcomes following cerebral ischemia, and appears to drive 'neuronal'differentiation of multipotent neural progenitor cells. Therefore, we hypothesize that HDAC inhibition in stroke may have acute effects (to ameliorate white matter excitotoxicity), and in the intermediate and longer term, added benefit by reducing apoptosis and encouraging regeneration. As a number of HDAC inhibitors are either already approved by the FDA (vorinostat), or well-advanced in clinical trials (MS-275) for other reasons, then re- purposing one or more of these drugs for stroke could be expedited. In addition, development of new and specific inhibitors is ongoing, especially against the Class I HDACs which are the focus of interest here. Studies described in Aim 1 are designed to identify the specific HDACs which account for the beneficial actions of these inhibitors, using in vitro and ex vivo preparations in which individual HDACs are knocked down with shRNA and transduced cells/tissues then subjected to oxygen-glucose deprivation. Having identified specific HDACs, we shall explore in Aim 2 the potential substrates and mechanisms responsible for the beneficial actions of HDAC inhibition. Finally, in the last aim we consider whether treatment of mice with MS-275, a Class I HDAC inhibitor, preserves anatomic integrity and promotes long term functional (motor, sensory, cognitive) recovery from transient cerebral ischemia (middle cerebral artery occlusion), and whether restoration of function correlates with the extent of 'neuronogenesis'. Two recently developed transgenic lines, p53+/+ and p53 -/- mitoCFP mice, will be employed so that we can monitor ischemic pathology in CFP+ fiber tracts, assess drug-associated changes in mitochondrial frequency and distribution within neuronal cell bodies and their processes, and also determine whether HDAC inhibition ultimately involves targets in addition to neuronal p53. PUBLIC HEALTH RELEVANCE: This proposal is submitted in response to PA-08-099 Mechanisms of functional recovery after stroke, "a funding opportunity to promote research to understand the processes of brain repair that lead to functional recovery in order to develop methods to optimize existing practices and to develop new approaches to improve post-stroke outcomes". Currently, there is no drug available to enhance neuroprotection and restore function following human stroke. Experimental animal studies suggest that broad inhibition of histone deacetylase (HDAC) activities in the brain following injury results in neuronal protection and also promotes the generation of new neurons. The purpose of the studies outlined in this proposal is to identify which HDACs are involved in order to select specific drugs to target these particular enzymes. Selected drugs will be tested to reveal what functional benefits they confer on the recovery of mice from stroke injury.

DESCRIPTION (provided by applicant): The success of malaria eradication efforts hinges on the development of a safe and efficacious malaria vaccine that induces complete protection against infection. To date, efforts to develop such a vaccine for worldwide use have been unsuccessful. However, vaccination with the subunit RTS,S/AS02 vaccine encoding the Plasmodium falciparum circumsporozoite protein significantly reduced deaths due to severe malaria in Africa children and demonstrated for the first time that development of an anti-infection malaria subunit vaccine is feasible. We know that live-attenuated Plasmodium parasites yield complete and long-lasting CD8+ cytotoxic T lymphocyte (CTL)-mediated protection in mice and humans, but such vaccine preparations are not easily deployed in the field. The dependence of protection on antigens targeted by CTLs indicates that the targets of such cells would make ideal subunit vaccine antigens. The search for such targets was previously impossible on a whole organism scale because attenuated parasite vaccines contain thousands of different immune targets leading to complex polyspecific CTL responses - until now there was no methodology capable of identifying discrete antigenic targets in the midst of such a complex response. To solve this problem, I will employ a high-throughput method to efficiently screen thousands of candidate targets against CTLs from malaria-exposed mice in order to decipher the key CTL responses that confer immunity against malaria. I hypothesize that protective CTL responses encompass a wide range of antigenic specificities and include parasite proteins expressed in both liver and erythrocyte stages. These target antigens can be rapidly identified using this high-throughput minigene-driven screening approach. In Specific Aim 1, I will combine this approach with different vaccination models in mice to assess the kinetics and diversity of CTLs and identify cognate target antigens associated with protection. I will also use complementary experimental and in silico approaches to guide development of a focused candidate target library. In Specific Aim 2, I will evaluate cross-priming of CTLs by malaria-infected erythrocytes and determine how such targets elicit cross-stage CTL immunity. Immune targets defined in this work can later be used to rationally test CTLs from malaria-exposed humans to define a polyspecific protective CTL repertoire in humans. The proposed approach will serve as a powerful paradigm for identifying protective CTL targets and can be generally applied to malaria and other infections. This application addresses PA-10-059 (K08 Career Development Award) and was crafted to ensure excellent mentorship, strong institutional support and successful collaborations. NARRATIVE Global eradication of malaria requires an efficacious anti-infection malaria vaccine. Development of such a vaccine requires the identification of discrete antigenic targets from among thousands of proteins expressed by malaria parasites. We propose here to study CTLs induced by different immunization approaches in mice and identify protective antigens by profiling CTL responses using high-throughput screening technologies and other cutting-edge approaches to accelerate malaria vaccine development.

The Animal Behavior Core (ABC) is comprised of two components: the Infant Primate Research Laboratory (IPRL) and the Mouse Behavior Laboratory (MBL). The overall objective of the ABC is to provide Research Affiliates with the infrastructure needed to perform the most advanced and innovative functional outcome measures when assessing the effectiveness of intrinsic (gene-based) and/or extrinsic (surgical, drug-based) interventions in animal models of neurodevelopmental disorders.

ABSTRACT Our laboratory?s overarching goals support NIAID?s mission to better understand, treat, and prevent infectious diseases by focusing on pre-erythrocytic malaria vaccine development through identification of protective cytotoxic T cell (CTL) antigens using novel biological and immunological methods. Subunit vaccines that efficiently stop the Plasmodium sporozoite (spz) or liver stage (LS) completely protect against malarial disease and will enable eradication efforts. There are currently no FDA-approved malaria subunit vaccines for use in humans although attenuated spz show sterile protection against challenge in multiple Phase 1/2 clinical trials. This project aims to identify parasite proteins exported from the parasite to the host for testing as novel vaccine subunits. Two proximity labeling methods will be used to identify parasite proteins exported to the parasitophorous vacuolar membrane and/or host cell cytoplasm during LS infection. Such exported proteins are more likely to be sampled by host antigen processing pathways for display on Class I MHC as CTL antigens. Since little is known about LS protein trafficking, this work may identify export motifs that would expand our basic knowledge of LS parasite biology. Selected proteins will be tested to validate their export in vitro and those that demonstrate true export will be tested in preliminary immunization/challenge studies. Exported proteins may be ideal CTL target antigens and could be incorporated into our ongoing efforts to develop effective multi-subunit CTL DNA vaccines.

ABSTRACT Our R01 project supports NIAID?s mission to better understand, treat, and prevent infectious diseases by focusing on pre-erythrocytic malaria vaccine development. Vaccines that efficiently stop the Plasmodium sporozoite (spz) or liver stage provide complete protection against malarial disease and will enable eradication efforts. There are currently no FDA-approved malaria vaccines for use in humans although repeated dosing with intravenously-administered attenuated spz has shown sterile protection against challenge in multiple Phase 1-2 clinical trials. Recently, CD8+ T cells that reside in the liver (liver resident memory T cells or Trm cells) have been identified as key cell types in protection against liver stage infection. Vaccine strategies that increase liver Trm cells and can be readily adapted to clinical use are therefore critically needed. Such vaccines could bolster CD8+ T cell immunity and may result in T cell-focused vaccines that achieve durable, high-grade protection for persons in endemic and non-endemic regions. Our laboratory has developed a two-dose vaccine that increases liver Trm cells and achieves sterile protection. This approach requires only a single dose of spz. This project aims to provide pre-clinical support for development of this two-dose ?prime-and-trap? vaccine. The University of Washington (UW) will collaborate with established partners at Sanaria Inc. In Aim 1, we will evaluate biological and technical questions about the proposed vaccine regimen, including dose dependence, effects of spz cryopreservation, adjuvant effects, interference from pre-existing antibodies, and use of multiple DNA plasmids. In Aim 2, we will investigate the magnitude and degree of antigen spreading following vaccination, a phenomenon that could enhance protection in the liver. In Aim 3, we will evaluate the prime-and-trap vaccine in the P. knowlesi non-human primate (NHP) immunization-challenge model and demonstrate Trm cell targeting in a P. falciparum NHP model. Tolerability and toxicology endpoints will be obtained in NHP studies in preparation for future clinical studies. In summary, this project will optimize and assess a two-dose prime-and-trap vaccine rationally designed to elicit complete protection against the Plasmodium liver stage.

ABSTRACT The world needs a highly effective pre-erythrocytic vaccine for malaria prevention. Immunocompetent animal models are needed to test candidate vaccines targeting the most lethal species: Plasmodium falciparum (Pf). However, Pf does not infect mice and does not cause blood-stage infections in the common rhesus non-human primate (NHP) model. Although rhesus do not support blood stage infections, our preliminary data shows that parasites actively proliferate in the preceding liver stage, making it possible to monitor liver burden in naïve vs. immunized animals by RT-PCR as an efficacy endpoint. This sort of endpoint is well accepted in rodent models, but has not been widely used in NHP studies because the growth of Pf in rhesus livers was unappreciated until now. We propose to establish this model and compare the liver burden in immunologically- naïve NHP compared to NHP vaccinated in a pilot study using our ?prime-and-trap? approach that generates protective liver resident memory CD8+ T cells. This model is enabled by high quality diagnostics, and our lab is considered a reference center for molecular detection of malaria parasites ? therefore we are well suited to evaluate the proposed model. The study will (a) provide additional data on use of Pf/rhesus as a useful NHP model for pre-erythrocytic malaria vaccines and (b) provide translational data on our prime-and-trap vaccine to advance this promising vaccine approach and accelerate funding for further translational studies.

ABSTRACT There are a significant number of low-density Plasmodium infections in endemic populations, and the proportion of this type of infection appears to increase with decreasing transmission intensity. However, most studies to date have assessed these infections at a single time point or at infrequent time points, which offers little to no information on the dynamics of these infections and their possible contribution to malaria transmission. Recent data suggest that asymptomatic parasite densities may be much more dynamic than previously known. The goal of this project is to demonstrate the feasibility and quality of daily dried blood spot (DBS) sampling in order to quantify parasite and gametocyte density kinetics of low-density asymptomatic Plasmodium falciparum infections to better understand their possible contribution to malaria transmission by studying the natural history of such infections in a population experiencing intermediate transmission. We will measure the proportion of subjects who successfully collect daily DBS over a 28-day period, and quantify the quality of the samples collected over time. Ultrasensitive quantitative reverse transcription PCR for P. falciparum 18S rRNA and gametocyte mRNA will be combined to cost-effectively identify infected participants and then deconvolute daily samples to illustrate the parasite density kinetics. If successful, the results from this study will offer a technique to further study the dynamics of low-density parasite carriage and be generalizable to other malaria-endemic countries confronting the public health problem of asymptomatic malaria for malaria control and elimination.

ABSTRACT Our U01 project supports NIAID?s mission to better understand, treat, and prevent infectious diseases by focusing on pre-erythrocytic malaria vaccine development. Vaccines that efficiently stop the Plasmodium sporozoite (spz) or liver stage can provide complete protection against malarial disease and will enable eradication efforts. There are currently no FDA-approved malaria vaccines for use in humans although repeated dosing with intravenously-administered attenuated spz has shown sterile protection against challenge in multiple Phase 1-2 clinical trials. Recently, CD8+ T cells that reside in the liver, namely liver resident memory T cells or TRM cells, have been identified as key cell types in protection against liver stage infection. Vaccine strategies that increase liver TRM cells and can be readily adapted to clinical use are therefore critically needed. Such vaccines could bolster CD8+ T cell immunity and may result in T cell-focused vaccines that achieve durable, high-grade protection for persons in endemic and non-endemic regions. Our laboratory has developed a two-dose vaccine that uses a DNA prime followed by an attenuated spz boost or ?trapping dose? that increases liver TRM cells and achieves sterile protection. This project aims to improve upon spz-based trapping by developing an orally-administered nanoparticle-based trapping vaccine. The University of Washington will collaborate with Johns Hopkins University to develop this more easily manufactured, more easily deliverable, and less expensive vaccine. In Project 1, we will define a threshold of Pf antigen-specific TRM cells needed to achieve protection using DNA prime/sporozoite trapping. In Project 2, we will optimize nanoparticles for liver- specific delivery and expression profile in hepatocytes using a variety of nanoparticle compositions, sizes, surface characteristics, and formulation strategies. In Project 3, we will evaluate the optimized nanoparticles in prime-and-trap vaccination in mice and non-human primates for safety, tolerability, immunogenicity, and efficacy. If successful, this project will deliver an optimized prime-and-oral trap vaccine rationally designed to elicit complete protection against the Plasmodium liver stage.