# data[].attributes.title
Mechanisms of non-HIV Collapsing Glomerulopathy in Hispanic Patients

# data[].attributes.funder_divisions[]
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)

# data[].attributes.abstract
Non-HIV collapsing glomerulopathy (CG) is a severe form of glomerular disease seen in all parts of the world. Major etiological factors include circulating proteins that cause recurrent CG, infections like SARS-CoV-2 and Parvovirus B19, drugs like pamidronate, and background genomic changes potentially present in any of the preceding categories. Whereas variants of the APOL1 gene have been implicated in select populations with West African heritage, a genomic basis in other patient populations has not been elucidated. Using data from two decades of investigations into CG centered around Hispanic patients from Mexico City and more recent genomic data from Peruvian Hispanic patients, several novel mechanistic rat and mouse models of CG were developed. Critical components of the CG upstream pathways are the podocyte expressed transcriptional factor ZHX2 and glomerular integrins, including α3β1 in the podocyte, αvβ5 in the glomerular endothelium, and αvβ3 at both locations. Finally, recombinant mutated human Angiopoietin-like 4 protein 8520 is known to have an integrin stabilizing effect specific to Integrins β1 and β5, and could be potentially used to treat CG in the future. The overall premise of this application is that high podocyte ZHX2 expression and low glomerular endothelial Integrin β5 expression predispose to the development of CG. In Aim 1, changes in glomeruli with high podocyte ZHX2 expression will be investigated using transgenic rat models. Disease mechanism during the development of CG and just prior to the collapse of capillary loops will be elucidated. In Aim 2, changes in glomeruli with low endothelial Integrin β5 expression will be investigated using knockout mouse models. Disease mechanism during the development of CG and just prior to the collapse of capillary loops will be elucidated. In Aim 3, rat and mouse models of CG will be treated with protein 8520 to test for prevention or improvement in CG, and to potentially halt the disease process before the development of collapse.

# data[].attributes.title
The multifaceted pathways of astrovirus entry and egress

# data[].attributes.funder_divisions[]
National Institute of General Medical Sciences (NIGMS)

# data[].attributes.abstract
Astroviruses are a major cause of pediatric diarrhea worldwide. Despite causing one of the most common early childhood infections, astroviruses are one of the least studied enteric RNA viruses. We previously discovered that the virus infects small intestinal goblet cells, specialized epithelial cells that secrete mucus. Few studies have investigated viral infection in goblet cells due to the lack of cell-specific models. Because the mechanisms by which viruses replicate inside of goblet cells are completely unknown, my lab is interested in addressing 1) how do astroviruses enter cells with highly dynamic apical membranes? and 2) what role does mucus secretion play in viral egress? We have established new in vitro models and tools to address these questions and have built a strong and collaborative investigative team with complementary expertise that will ensure the success of these projects. To evaluate receptor-mediated and fluid-phase endocytosis entry pathways into goblet cells, we will use a combination of CRISPR-Cas9 engineering, biochemical analysis, and high-resolution microscopy. We will use a similar suite of techniques as well as cryo-electron microscopy to define the egress pathway of astrovirus from goblet cells via mucus secretion. In addition to murine and human astroviruses, other respiratory and enteric viruses have also been shown to target goblet cells for infection. Thus, our work aims to initially provide foundational knowledge on the basic biology of astroviruses before shedding light on key host pathways in goblet cells that are co-opted by viruses from other families, including influenza and SARS-CoV2. Completion of these studies will provide the first major insights into the virus-host interactions at the apical membrane surface of intestinal goblet cells, which will pave the way for the future development of targeted drug treatments for the numerous viruses that target this unique cell population.

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Coronaviruses (CoV) are associated with severe diseases as demonstrated by the 2003 severe acute respiratory syndrome (SARS)-CoV1 epidemic and the SARS-CoV2 pandemic. One of the critical steps of infection involves viral mRNA mediated recoding of gene expression; a -1 frameshifting event that occurs during translation. It is this elegant mechanism that allows the ribosome to bypass a stop codon and synthesize viral enzymatic proteins. Furthermore, the frequency by which this event occurs is important for eﬃcient viral infectivity and is regulated by domains in the translating mRNA (in the case of the SARS-CoV, this domain is a pseudoknot). Although structural studies of frameshifting have received considerable aOention and various structures have been proposed and solved, information on exactly which structure causes the frameshifting is lacking. Our preliminary studies indicate that CoV gene expression is regulated by a dynamic, proton-driven equilibrium between an active, and two inactive pseudoknot conformations that allows for strict control over the protein ratios. This proposal aims to gain a complete structural and mechanistic understanding of the frameshifting frequency in CoV by combining structural studies with biochemical and in vivo experiments. Our aims will be: (#1) to understand the basis for how the frameshifting frequency is maintained by engineering structure-guided mutants to test our equilibrium model, (#2) to determine the structures of the pseudoknot signal in both conﬁgurations: permissive and nonpermissive for frameshifting, and (#3) to determine the structure of ribosomes as they encounter the permissive conformation of the pseudoknot.

# data[].attributes.title
Discovery and evolution of mammalian Serpins as host-encoded viral protease inhibitors

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Viral proteases are essential for the replication of many RNA and DNA viruses. Through sequence specific recognition of cleavage sites, these proteases process viral polyproteins into their functional components, while also cleaving host proteins to facilitate viral replication. Interestingly, organisms from every domain of life encode proteins known as SERPINs (SERine Protease INhibitors) that serve as natural inhibitors of serine and cysteine proteases, the types of proteases encoded by many viruses. SERPINs contain a conserved structural core and a disordered reactive center loop (RCL) that ‘baits’ a protease into cleaving it by mimicking the sequence specificity of the protease to be inhibited. Following protease-mediated cleavage of the RCL, the SERPIN undergoes a large conformation change that covalently traps and inactivates the protease, thus connecting sequence-specific recognition of the SERPIN RCL to protease inhibition. While SERPINs have been largely characterized as inhibitors of a wide range of cellular proteases, we hypothesize in this grant that SERPINs have also evolved, in a species-specific manner, to directly inhibit viral protease activity and thereby inhibit viral replication. Supporting this hypothesis, we have found that several mammalian SERPINs contain RCLs that are rapidly evolving under positive selection, consistent with SERPINs being engaged in evolutionary arms races with pathogen-encoded proteases. Moreover, we have identified primate SERPINs that have independently evolved sequences that mimic the cleavage site preferences of proteases from picornaviruses and coronaviruses, and find that these SERPINs can inhibit viral protease activity during infection. These data lead us to propose a model in which SERPINs, with their protease baiting RCLs, represent a modular platform for evolution of host-specific antiviral activity through viral protease inhibition. Based on this model, we now propose to use evolutionary biology, protease biochemistry, and virology to discover and characterize the natural variation, evolvability, protease specificity, and antiviral activity of viral protease inhibitors among mammalian SERPINs. In Aim 1, we will test the antiviral potency and specificity of primate SERPIN-mediated viral protease inhibition, while also computationally and functionally searching for additional mammalian SERPINs that can inhibit proteases from a wide range of RNA viruses. In Aim 2, we will probe the modularity and evolvability of SERPINs to determine the degree to which the SERPIN ‘core’ and RCL sequence impact function, and how sequence evolution of each can mediate selective viral protease inhibition. By identifying SERPINs as novel, rapidly evolving, host-encoded viral protease inhibitors, our work will reveal the impact of SERPIN and viral protease evolution on this new host-virus evolutionary conflict and on species-specific barriers to virus replication, and unveil the evolutionary potential of SERPINs to inhibit a wide range of viral proteases.

# data[].attributes.title
MARVELOUS: Maternal RSV Vaccination- Evaluating Optimal Immune Responses

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
The recent approval of the Respiratory Syncytial Virus (RSV) vaccine for administration in pregnancy presents a novel opportunity to define immune responses of the maternal-fetal dyad, and how that response crosses the placenta and mammary tissue. Recent work indicates that timing of maternal RSV vaccination alters placental antibody transfer to the fetus. To maximize infant protection after maternal RSV vaccination, key gaps in knowledge include: 1. The extent to which maternal vaccination elicits direct fetal antigen-agnostic and antigen- specific cellular responses to augment infant protection from RSV and other infections. 2. How gestational age at vaccination alters maternal antibody response, subsequent placental and breastmilk antibody transfer, and persistence of immunity in the infant. The proposed studies will test the central hypothesis that timing of maternal vaccination, antibody Fc-receptor binding properties, glycosylation profiles, neutralizing antibody levels and non-neutralizing antibody functions are all key determinants of placental and breastmilk antibody transfer to the neonate. These antibody features will work in concert with fetal innate and adaptive immune responses to maternal vaccination, driving protection of the infant through 6 months of age. In a cohort of 400 pregnant women and their infants, this study will examine fetal cellular responses to maternal RSV vaccination, both RSV-agnostic and RSV-specific, by evaluating fetal immune cells isolated from placental villi and cord blood (Aim 1). It will comprehensively profile placentally- and breastmilk-transferred antibodies after RSV vaccination in pregnancy, evaluating IgG subclass, Fc-receptor binding, glycosylation profile, and neutralizing capacity of antibodies using in vivo and in vitro assays (Aim 2). It will then evaluate how antibody properties and timing of maternal vaccination impact the durability of antibody-mediated and cellular immunity in infant blood and breastmilk, through 6 months of age (Aim 3). Machine learning approaches will be used to estimate the magnitude and specific features of protective immune responses induced by maternal vaccination, not only for RSV, but also for influenza, Tdap, and COVID-19. These methods will generate a comprehensive model of durable infant protection from maternal vaccination spanning multiple pathogens. Defining these immune principles across the maternal-fetal dyad will generate key biological insights necessary to optimize neonatal and infant protection.

# data[].attributes.title
Regulatory T cell memory in human tissues

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
T cell memory is stored across heterogeneous subsets with diverse functions in both tissues and circulation. While most studies have focused on the pro-inflammatory and cytotoxic functions of memory T cells, regulatory T cells (Tregs) serve an equally important immunomodulatory role in memory responses, particularly in tissues. While specific roles for Tregs in establishing tolerance and promoting tissue homeostasis have been elucidated in mouse models, the role of human Tregs in healthy immune responses and protective immunity in vivo has been difficult to assess. Moreover, the identity and function of human Tregs in diverse tissues remains unknown. We have established an organ donor tissue resource for human immunology that has allowed us to profile antigen-specific T cells across human tissues. Through these efforts, we found that antigen-specific Tregs are substantially enriched among memory T cells that respond to antigens from multiple viruses, including SARS-CoV-2, influenza, and EBV, and are particularly enriched in lymph nodes, spleen and lungs compared to blood, bone marrow and other sites. In addition, we found that memory Tregs induce an activation program that is distinct from effector memory T cells (TEM) involving CCL17 as a novel Treg-derived cytokine not produced by TEM cells or any other T cell subset. Moreover, tissue memory Tregs exhibit clonal overlap with TEM cells within and between sites. These findings raise the possibility that memory Tregs are generated along with TEM during priming and that they share a common pre-cursor with TEM. In the proposed studies, we will pursue three aims: 1) Determine the role of antigen and tissue in memory Treg induction; 2) Define the clonal and migratory relationships (i.e. tissue distributions) between memory Treg and other memory subsets; 3) Elucidate the functional and spatial interactions of tissue Tregs with immune and structural cells in the lymph node. We will combine state-of-the-art technologies for single-cell and spatial profiling with our unique human tissue resource to elucidate mechanisms for the generation, function, and maintenance of memory Tregs in human tissues. The results from this study will be important for designing strategies to promote immunoregulation and tissue repair for protective immunity and can inform Treg-directed therapies for autoimmunity and transplantation.

# data[].attributes.keywords[]
Cellular Indexing of Transcriptomes and Epitopes by Sequencing

# data[].attributes.title
Statistical Methods for Assessing Immune Correlates of Risk and Protection Using Flexible Two-Phase Sampling Designs that Enrich Longitudinal Samples

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Immune correlates of protection (CoP) are biomarkers that predict vaccine-induced protection against dis- eases and play a crucial role in the design and development of effective vaccines. The U.S. government (USG)-led initiative to identify CoPs for COVID vaccines highlighted the importance of neutralizing antibody titers as surrogate endpoints, significantly impacting vaccine recommendations and approvals. To effec- tively measure these immune biomarkers, researchers utilize two-phase designs, such as case-cohort or case-control studies, to boost statistical power and enhance representation. This proposal aims to develop novel two-phase sampling designs that allow enrichment of longitudinal immune response marker mea- surements in immune correlates studies. The proposal also aims to develop advanced statistical methods for datasets collected under sampling designs that would introduce bias if analyzed using conventional in- verse probability-weighted methods. Aim 1 focuses on the analysis of the immune response biomarkers measured at the peak immunogenicity time point, while Aim 2 delves into the study of the decaying im- mune response biomarkers. The final product will feature a user-friendly software implementation of the proposed methods, along with its application to analyze COVID correlates datasets from past and ongoing USG-sponsored vaccine efficacy trials.

# data[].attributes.title
Rapid point-of-care diagnosis of symptomatic and asymptomatic herpes infection

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Herpes simplex virus (HSV) is highly contagious and can be transmitted via physical contact. HSV can be diagnosed by detecting the presence of the virus in lesions or the antibodies in the blood. Yet, viral shedding can happen from asymptomatic infections, highlighting the need for early and accurate detection of HSV to prevent transmission. The most common ways to detect HSV are nucleic acid testing of an active infection via qPCR or serological testing of antibody levels in patient serum. However, qPCR is only accurate if a person is symptomatic and in asymptomatic people both the FDA and the CDC recommend against serological testing due to issues with sensitivity. Additionally, current testing for CNS complications arising from HSV infections requires highly invasive cerebral spinal fluid (CSF) sampling to diagnose. Thus, rapid, accessible, sensitive, and accurate point- of-care tests are in dire need. In 2021, we published a watershed paper describing how we can leverage cell surface glycans that the SARS- CoV-2 virus uses to bind and infect cells, to capture it onto rapid test strips for sensitive detection of the virus (Kim et al, ACS Central Science). Inspired and motivated by our success with SARS-CoV-2 sensing, we propose a novel lateral flow strip assay (LFSA) device for rapid and reliable point-of-care antigen-based detection capable of differentiating between HSV-1 and HSV-2 infections and sensing of CNS complications through serum. As cell surface proteoglycans such as heparan sulfate play an important role in the binding and cell entry of HSV, we will leverage it as a universal binder and use type specific cell receptors to distinguish between HSV strains (Specific Aim 1). Higher selectivity will be achieved by exploring sensitivity to sulfonation of heparan sulfate and other glycocalyx proteins. Sensor performance will be evaluated in complex fluids such as human genital fluids or saliva, and in genital washings of HSV-infected mice. To enhance our ability to identify and subtype HSV, we will engineer tailored cell membranes to optimize their interactions with viral envelope proteins, and strip and print these cell-derived membranes on paper test strips in Specific Aim 2. In Specific Aim 3, we will develop a blood test for the rapid quantitative screening of glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL), upregulated biomarkers upon CNS damage. We will incorporate electrochemical signals for quantitative assessment. This Bluetooth device will enable early and fast triage of patients for further screening. Together, these devices will enable us rapid and cost-effective screening of high-risk populations, accurate subtyping, and a swift connection of patients with treatment.

# data[].attributes.keywords[]
Centers for Disease Control and Prevention (U.S.)

# data[].attributes.title
Inhibiting Chikungunya Virus Protease using MTase-like Domain Interactions for Novel Antiviral Therapies.

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.abstract
Chikungunya (CHIKV) is an RNA alphavirus that infects 3 million people in 45 countries including the US annually. Acute infection is flu-like, but in 40% of infections, debilitating joint pain emerges that can last for years. Infection during pregnancy also results in severe encephalopathy in newborns or aborted fetuses. Viral proteases are effective antiviral drug targets and are the standard of care for viral diseases (e.g HIV, hepatitis C, SARS-CoV- 2). The activity of the nsP2 protease from CHIKV (CHIKVP) is vital for infection. Inhibition of CHIKVP blocks processing of the viral polyprotein, prevents viral replication, lowers viral titers and stops disease progression. Thus, CHIKVP is an excellent antiviral drug target. To date, no effective antivirals of CHIKVP have been approved for acute or chronic infection. Our ultimate goal is to use insights into CHIKVP structure and dynamics to develop an inhibitor to oppose CHIKV infection, the resulting chronic pain and prevent pediatric neurological syndromes.  CHIKVP is composed of a protease domain and a methyltransferase-like domain (MTL). To date, no functions of the MTL have been identified. In a search for novel CHIKVP binders, we identified ligands that bind to the MTL at an elongated cavity and allosterically inactivate the protease. The site shares structural homology with S-adenosyl methionine (SAM) cofactor binding sites, but does not bind SAM. The allosteric site binds to GTP, which suggests that a function such as RNA binding may be conserved in the MTL.  Here we propose a research strategy for the development and direct comparison of CHIKVP active-site and allosteric inhibitors. We will build compounds derived from a large compound screen and also build from MTL-binding fragments we have already identified. We have developed NMR approaches that allow us to readily distinguish active-site from allosteric inhibitors. Importantly, we have developed approaches that allow us to monitor activity, binding and dynamics in solution without having to rely on freezing samples which is required for other structural techniques, to inform our inhibitor design. Recent data have suggested that RNA plays a critical role in CHIKVP function, enhancing protease activity. We have identified a site that we hypothesize binds RNA and describe a series of studies to understand the mechanism by which RNA impacts protease function. We will bring all these structural insights into our inhibitor development approach. At each step of development, we will closely monitor efficacy against viral infection for CHIKV and other related alphaviruses to determine whether pan-alphaviral inhibition is achievable with a given class of compounds. Critically, we will also implement a directed evolution approach across both domains of CHIKVP to predict the susceptibility of our inhibitors to resistance mutations. This will enable us to develop enduring antivirals and will also address longstanding unanswered questions about the favorability of allosteric inhibition in antiviral drug development.

# data[].attributes.title
Identification of RNAi-independent antiviral genes through biased genetic screen in C. elegans

# data[].attributes.funder_divisions[]
National Institute of Allergy and Infectious Diseases (NIAID)

# data[].attributes.awardee_organization.name
LOUISIANA STATE UNIV A&M COL BATON ROUGE

# data[].attributes.abstract
Viruses, especially RNA viruses, are formidable pathogens to cellular hosts. Owing to the error-prone nature of their replicases, RNA viruses rapidly accumulate large numbers of genetic mutations in their genome, enabling them to evade immune detection. Some RNA viruses, such as influenza viruses and coronaviruses, can also generate genome variants through genome reassortment or genome recombination mechanisms. It is thus important to study antiviral mechanisms intrinsically resistant to genetic mutations in viral genome, which may lead to the development of novel antiviral strategies.  In plants, insects and vertebrates, there are antiviral mechanisms triggered by non-dsRNA products of invading viruses. These antiviral mechanisms provide another layer of protection in case viral dsRNA-triggered immunity is compromised by the invading viruses. In supporting this hypothesis, many plant and animal viruses have been found to produce diverse types of dsRNA binding proteins that are able to bind and sequester viral dsRNA to block immune detection.  So far, RNAi is known as the most important antiviral defense mechanism in Caenorhabditis elegans. However, some recent studies on worm antiviral defense suggest that viral infection in C. elegans triggers transcriptional programs that in return provide protection against invading viruses in an RNAi-independent manner. Therefore, very much like what has been demonstrated in plants and insects, RNAi-independent antiviral defense (RiAD) may provide worms another layer of protection against virus in case antiviral RNAi is compromised.  To fill the knowledge gap in our understanding of worm RiAD and as proof of principle, the PI’s lab has recently carried out a biased genetic screen of limited scale, aiming to identify genes specifically contributing to worm RiAD. This genetic screen identified 8 candidate genes that confer RiAD targeting a flock house virus (FHV) replicon. 5 of these candidate genes also mediate RiAD against Orsay virus, which naturally infects C. elegans (21).  In this application, we propose to continue the biased genetic screen and finish it on a much greater scale to ensure double coverage on all genes involved in RiAD. We will then map and identify the candidate genes through whole genome sequencing combined with feeding RNAi and function rescue assay. Since approximately 70% of C. elegans genes have human homologs, function and mechanism study of the identified genes may not only lead to the identification of novel conserved mechanisms of antiviral innate immunity across kingdoms but also inform the development of novel antiviral strategies.