Paul Bates - US grants

This project will contribute to our nation's capacity for increasing the number of
people with disabilities employed in the science, technology, engineering, and
mathematics (STEM) work force by demonstrating the effectiveness of an innovative
combination of person centered career planning activities, ongoing mentoring, and hands
on experiences in the sciences. This combination of activities is designed to 1.) increase
recognition of the interests and needs of students with disabilities in the STEM career
areas, 2.) create a more supportive academic and professional climate for persons with
disabilities, 3.) promote accessibility and appropriateness of instructional materials and
educational technologies at multiple levels (high school, community college, and
university), and 4.) increase the availability of academic enrichment experiences such as
mentoring and hands-on experiences in the STEM areas.

To meet these broad goals, the SIU SY-STEM project will:
*Identify students' preferences and interests for specific STEM-related career
areas.
*Increase receptivity of educators (secondary and post-secondary math and science
educators) and of guidance counselors for including of students with disabilities
in STEM-related curriculum experiences.
*Impact adaptations, accommodations, and instructional strategies used by
educators in STEM classes and labs at multiple levels.
*Foster academic and professional development in STEM related career areas by
increased access to career exploration experiences and mentoring.

A fundamental premise of this project is that the best way to impact employment of
persons with disabilities in the sciences is by simultaneously increasing the numbers of
people in the pipeline leading to these careers and enhancing the capacity of secondary
and post-secondary training institutions to successfully include students with disabilities.
Twenty-five teams of students, parents, educators, and guidance counselors from 25 high
schools in southern Illinois will participate in intensive one-day workshops on person
centered career planning. From these 25 student teams, six students will be selected to
participate in an intensive, on-campus Summer Institute at Southern Illinois University.
This Institute will expose students to a variety of careers in the sciences and engage them
in a series of rotations in various lab sciences. University students with disabilities will be
recruited to work in close affiliation with a graduate student mentor and faculty mentors
to provide ongoing support for the workshop and Institute participants.

The SIU SY-STEM project will impact involvement of persons with disabilities in
STEM areas of study at multiple levels, including the personal and professional
development of high school and college students with disabilities, and changes in the
educational resources, willingness, and capacity to support these students in high school,
community college, and university programs. Although this project is designed as a
regional demonstration involving high schools, consortia of community colleges, and
Southern Illinois University, it is the intention of the applicant to sustain this initiative by
building broader, more comprehensive regional/national networks and alliances. To that
end, the project includes a comprehensive evaluation component and widespread
dissemination plan.

Recent evidence demonstrates an essential role for cellular cathepsins in viral glycoprotein processing and cellular entry for the highly pathogenic viruses Ebola, SARS coronavirus and Nipah/Hendra. Cathepsin L appears to be important for Nipah and SARS coronavirus virus glycoprotein activation while Ebola requires both cathepsins L and B for viral entry. Inhibitors of these enzymes effectively block viral entry and replication in cell culture. Studies in mice with broad spectrum cathepsin inhibitors as well as specific inhibitors and genetic knockouts all suggest that cathepsin activity can be impaired in vivo without significant detrimental effects. In preliminary studies we have used a novel methodology to screen chemical libraries on microarrays and have identified two previously unrecognized cathepsin L inhibitors. One of these inhibitors, PC-185, is highly specific for cathepsin L and blocks Ebola entry with an IC50 of ~193nM. This proposal will build upon these significant preliminary findings and will develop cathepsin inhibitors as therapeutics for Ebola. For this application, we propose the following aims: Specific Aim 1) Utilize high throughput screening of diverse libraries to identify additional new cathepsin inhibitors. Analyze structureactivity relationships (SAR), optimize lead compounds, and develop second-generation agents and screening libraries. Specific Aim 2) Test the candidate compounds for inhibition of viral entry using a rapid, quantitative and safe assay employing viral pseudotypes carrying the glycoproteins of Ebola virus. A novel virus-like particle system that we developed to study Ebola entry will be employed to confirm the ability of lead compounds to block filamentous Ebola infection. Additionally, we will send promising candidates to collaborators at USAMRIID for efficacy testing against Ebola infection. Effective candidate compounds will be cross screened against SARS-CoV and Hendra infections. Specific Aim 3) Determine the mechanism(s) by which viruses can escape cathepsin inhibition by selection of escape mutants using a novel VSV vector that relies upon Ebola GP for replication (VSV-GP).

DESCRIPTION (provided by applicant): This proposal requests support for renewal of the long-standing pre-and postdoctoral Training in Virology T32 at the University of Pennsylvania. Over the past 20 years hundreds of PhD and postdoctoral trainees have been mentored in viral research by our faculty. This training program currently includes 21 laboratories directed by well- established and well-funded investigators at the University of Pennsylvania, the Wistar Institute, and CHOP selected from over 40 NIH-funded laboratories studying viruses on the contiguous campus that houses these institutions. Addition of new trainers including George Shaw, Matt Weitzman, Sara Cherry, Kyong-Mi Chang and Carolina Lopez has strengthened an already outstanding cadre of mentors. The primary goal of the Virology Program is to identify, mentor and develop the careers of future leaders dedicated to biomedical research in the field of virology. Toward this end, we crafted a well-organized training program that includes outstanding mentors, an exceptional pool of candidates, outstanding institutional support and facilities, and training activities comprised of formal coursework in virology and immunology, an invited scientist speaker series, a student and postdoc research in progress seminar series, and a career development/Alumni day. New initiatives include Individualized Development Plans for all trainees, a UPenn Virology LinkedIn group to track and network with former trainees, a well described set of metrics to gauge success, and fellowship preparation instruction for incoming PhD students. Penn continues to provide direct and tangible institutional commitment to training in the biomedical sciences by supporting predoctoral trainees for their first 21 months of graduate school and funding the Biomedical Postdoctoral Programs office. Over the past five year funding period, this Virology T32 directly supported a total of 27 trainees including 19 predoctoral trainees (12 PhD, 6 MD/PhD, 1 VMD/PhD, 9 men, 10 women, 1 under-represented minority) and eight postdoctoral fellows (2 men, 6 women, 1 under-represented minority). These 27 trainees worked in the labs of 15 different trainers. Success of this program is exemplified by the former trainees who developed into independent scientists studying viral biology including Carolyn Coyne, Blossom Damnia, Anthony Nicola, Andy Pekosz, and Matt Weitzman among others. In following the review recommendations, we propose supporting four pre-doctoral and three postdoctoral trainees per year.

Zika virus (ZIKV) is an emergent viruses of the family Flaviviridae that is spreading explosively through South & Central America. Of concern, the virus, which usually causes only mild symptoms, has been linked to a reported increase in the number of cases of babies born in Brazil with microcephaly and may also be associated with an increase in Guillain-Barré syndrome. Currently there are no therapeutics or licensed vaccines to treat or prevent ZIKV infection. Indeed ZIKV is understudied and very little is known about the basic biology of how ZIKV interacts with human cells. The aim of this research proposal is to 1) identify key human factors exploited by this virus during infection and to 2) delineate innate host cellular responses to Zika infection with the expectation that a better understanding of how this virus interacts with host cells may aid in the broad goal of identifying potential therapeutic targets. This proposal will utilize two independent, complimentary forward genetic screens that have not previously been applied to flaviviruses. It builds on our experience using a human haploid cell screen that has identified several human genes required by pathogenic hantaviruses. In specific aim 1 a library of insertionally-mutagenized haploid cells will be selected using lethal challenge by ZIKV. Deep sequencing will be used to map the locations of mutagenic insertion sites within the human haploid library prior to and following selection with ZIKV. By statistically ranking the number of independent insertions into genes within these two populations, aim 1 will define genes important for ZIKV infection. The importance of these genes will be validated by creating expression knockdowns and knockouts of these genes to retest infectivity with ZIKV. Aim 2 will identify cellular genes that when activated can restrict ZIKV infection using a modified CRISPR/Cas9 system. To accomplish this goal we will use RNA-guided DNA binding of a cleavage-defective Cas9 protein and sgRNAs that are fused to strong transcriptional activators. A library of >70,000 sgRNAs that target every isoform of every human gene will be transduced into cells. Illumina sequencing of the sgRNAs in the cell population before and after lethal ZIKV challenge will be used to identify sgRNAs (and the corresponding genes) that are enriched in cells that resist or restrict infection. Bioinformatic tools will be employed to define pathways or cellular processes restricting ZIKV infection. Together, these aims serve to initiate a research program that will yield important basic scientific data on this emergent virus.

Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) is a pathogenic, tick-transmitted bunyavirus that can cause a severe febrile hemorrhagic-like disease with case fatality rates of up to 30%. Discovered during a 2009 outbreak of febrile illness in China, the geographic distribution of SFTSV extends into Korea and Japan with recent reports of infection in Vietnam and Russia. The tick vector for SFTSV is widespread throughout Asia. Numerous domestic and wild animals are naturally infected by SFTSV suggesting a large reservoir with potential spillover to humans. There are currently no vaccines or therapeutics for SFTSV. Because of its epidemic threat the WHO included SFTSV in its 2017 recommendation ?A research and development Blueprint for action to prevent epidemics? and identified SFTSV as one of 11 pathogens most likely to cause severe outbreaks in the near future and proposed development of vaccines. Here we will explore two complementary and potentially synergistic strategies for an SFTSV vaccine: a recombinant viral vector and nucleoside-modified mRNA encoding the SFTSV viral glycoproteins. Vesicular stomatitis virus (VSV) is a cytopathic virus that has been developed as a vaccine vector due to its ability to rapidly induce strong, protective antibody and T cell responses to encoded foreign antigens after a single dose. Using a VSV vector expressing the SFTSV viral glycoproteins (similar to the currently employed VSV-Ebola vaccine), we demonstrate single dose induction of a neutralizing antibody response and protection from SFTSV challenge in an IFNAR1 knockout mouse model. Separately, we show that vaccination of wt mice with a single dose of nucleoside-modified mRNA lipid nanoparticles (mRNA-LNP) encoding the SFTSV glycoproteins elicits high levels of SFTSV neutralizing antibodies that are capable of conferring partial SFTSV protection when transferred into the IFNAR1 KO model. Based upon these strong preliminary findings we propose to characterize antibody and T-cell responses in rVSV and mRNA vaccinated mice when these vaccines are used alone or in a prime-boost regimen. These studies are significant as there is limited knowledge regarding vaccines for this highly pathogenic virus (a single report) and use of rVSV and mRNA in a prime-boost vaccination has not been reported. Finally, current small animal models of SFTSV infection are limited to animals with type I IFN responses knocked out. Because these animals lack an important innate immune response mechanism that supports amplification of cellular and humoral immune responses, we will develop an immune competent mouse vaccination model using transient monoclonal antibody blockade of IFNAR1 during SFTSV challenge.

Vesicular stomatitis virus (VSV) is a cytopathic virus that has been developed as a vaccine vector due to its ability to induce strong, protective antibody and T cell responses to encoded foreign antigens after a single dose. VSV efficiently incorporates glycoproteins ( GP) from a different virus on virion surface allowing production of replication-competent recombinant vectors in which the cognate VSV G gene is replaced by a foreign GP gene. Recombinant VSVs (rVSVs) expressing foreign GPs have been studied as vaccine vectors for a number of pathogens including the recent successful deployment of a rVSV-Ebola vaccine. Despite this success, studies in animal models demonstrate significant pathogenic effects when some rVSV are injected intracerebrally or when used to infect animals with defective interferon responses. Concerns raised by these studies support development of more attenuated rVSV vectors with better safety profiles. Specific Aim 1 will utilize a novel strategy for attenuating rVSV vaccine vectors by joining adjacent VSV transcripts using a P2A ribosomal skipping sequence between adjacent genes in the VSV genome. rVSV with single or multiple fused transcripts will be tested for pathogenesis in immunocompromised mouse models or in the CNS. In Specific Aim 2 these next generation, low pathogenicity vectors expressing the glycoproteins of Severe Fever with Thrombocytopenia Syndrome virus (SFTSV) will be analyzed for their ability to induce neutralizing responses. SFTSV is a pathogenic, tick-transmitted bunyaviruses that causes a severe febrile hemorrhagic-like disease with case fatality rates of up to 30%. Initially discovered during a 2009 outbreak of febrile illness in China, the geographic distribution of SFTSV extends into Korea and Japan. There are currently no vaccine or therapeutics for SFTSV. Because of its potential threat the WHO included SFTSV in its 2017 recommendation ?A research and development Blueprint for action to prevent epidemics? and identified SFTSV as one of 11 pathogens most likely to cause severe outbreaks in the near future and proposed development of vaccines. Included in this revised application are preliminary data demonstrating induction of strong neutralizing antibody responses that correlate with protection from SFTSV challenge in mice vaccinated with a 1st generation rVSV-SFTSV. Additionally, vaccination of IFNAR-/- mice with the 1st generation rVSV-SFTSV demonstrated significant pathology (weight loss) supporting the premise for Aim 1. This short IDEA proposal is designed to produce attenuated rVSV vectors useful for vaccine development for many pathogens and will generate proof-of-principle data that will permit further development of a vaccine for SFTSV.