# data[].attributes.title
Neutralizing antibody pressure on the evolution of SARS-CoV-2 variants."

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National Institute of Allergy and Infectious Diseases (NIAID)

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/ ABSTRACT: Rationale: SARS-CoV-2 evolution has led to the emergence of viral variants that evade existing immunity in the human population, which continue to pose a threat to global public health. It has proven challenging to predict which mutations will arise in future dominant viral variants, and, as a result, it is difficult to design vaccines that provide adequate protection against viruses that will circulate in future waves of infections. Our preliminary data show that viral variant evolution is most closely correlated with evasion of serum neutralizing antibodies, and that serum neutralizing antibody responses are shaped by immune imprinting to the ancestral D614G strain. This mentored career project aims to leverage these observations to develop an in vitro model for predicting where mutations will appear in the virus and to use that information to design updated vaccines. Candidate: As a Transfusion Medicine fellow with a PhD in Genomics and Computational Biology and two years of experience in virology and immunology research, I bring a unique complement of perspectives and skills to the analysis of viral and antigenic evolution and to vaccine design strategies. Further training in advanced BSL-2-compatible pseudovirus culture, mouse immunization, and computational structural biology will be central to the completion of the proposed project and to my development as an independent physician-scientist aiming to improve our understanding and mitigation of pathogen evolution. My primary mentor, Dr. David Ho, an international leader in virology, and my complementary multidisciplinary advisory team, will ensure my research and career development progress. Environment: The Ho laboratory at the Columbia University Irving Medical Center (CUIMC) is a world leader in the study of pandemic viruses, including SARS-CoV-2, with expertise in the characterization of viral variants and serum antibody analysis. The Ho lab has access to abundant resources and many collaborators, including leaders in pseudovirus construction, structural biology, and vaccine design. CUIMC also has a long track record of supporting junior physician-scientists on their paths to successful independent careers in academic medicine. Approach: We will test the central hypothesis that widespread early exposure to ancestral SARS-CoV-2 shapes the evolutionary trajectory of the virus and that such a trajectory can be modeled in vitro. In Aim 1 we will assess the impact of mouse serum neutralizing antibodies elicited by different immunization histories on mutational profiles in cultured BSL-2-rated pseudoviruses and correlate mutations with historical public health databases. In Aim 2, we will identify whether particular epitopes on the spike protein are particularly susceptible to the emergence of mutations under serum antibody selective pressure. In Aim 3, we will design and test novel COVID-19 vaccine candidates in mice based on observed mutations that arise in pseudoviruses. This project will enhance our understanding of SARS-CoV-2, provide a framework for in vitro modeling of antigenically variable pathogens, and contribute to vaccine design strategies.

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Modeling the Effect of Apolipoprotein LI Risk Variants on CVD Risk in African American E-cigarette Users Using Human Induced Pluripotent Stem-Cell-Derived Endothelial Cells

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National Heart Lung and Blood Institute (NHLBI)

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African American individuals face a disproportionately higher risk of tobacco-related cardiovascular diseases (CVD) than other races, a disparity not fully explained by traditional and socioeconomic risk factors. Despite lacking approval from the U.S. Food and Drug Administration (FDA) for their safety, e-cigarettes (e-cigs) have become increasingly popular, particularly among youth, and are now among the most commonly used tobacco products alongside traditional cigarettes. Approximately half of African American individuals carry at least one of two genetic variants (G1 and G2) of the apolipoprotein L1 (APOL1) gene, which are exceedingly rare in other populations. APOL1 is widely expressed, particularly in the vasculature. We have shown that carriers of APOL1 G1 and G2 variants have increased susceptibility to tobacco-related CVD, including stroke and coronary heart disease. Dysfunction of vascular endothelial cells (ECs) is a critical precursor to CVD. EC dysfunction also plays a key role in APOL1-associated pathology, including exacerbated renal issues and increased susceptibility to sepsis and severe COVID-19. Recent research indicates that, similar to cigarettes, both e-cigs and menthol—a flavor popular in the African American community—independently impair endothelial function. While studies, including those using induced pluripotent stem cell (iPSC)-derived ECs, demonstrate these effects, the specific impact of APOL1 risk variants on vascular health in African American tobacco product users remains unknown. The goal of the proposed research is to determine the effects of e-cigs, with and without menthol, on endothelial health, compare them to the effects of cigarettes, and identify potential molecular markers and pathways associated with CVD in African American users, with a focus on the APOL1 genotype. As such, this application aims to expand my background and expertise in modeling the CVD risk from tobacco products and to provide specific training in tobacco product-related in vitro assays, iPSC methodology, gene editing, and computational techniques. Building on my prior work in human studies, this research extends to the cellular level to address significant gaps in knowledge regarding the adverse effects of the most popular tobacco products, with and without menthol, among the African American population—a demographic long targeted by the tobacco industry marketing. To achieve this goal, I will use a robust in vitro platform of human iPSC-ECs to address the following aims: Aim K1) to determine the effect of e-cigs and cigarettes on markers of EC dysfunction in G1/G1 iPSC-ECs, Aim R1) to determine the effect of e-cigs and cigarettes on endothelial function in G2/G2 iPSC-ECs, and Aim R2) to determine the effect of e-cigs and cigarettes on inflammatory markers and lipid mediators of inflammation in G1/G1 and G2/G2 iPSC-ECs. This project will deepen our understanding of the adverse effects of widely used tobacco products on vascular health in the CVD-burdened African American population. It also aims to identify molecular markers of cardiovascular injury in this high-risk group, providing insights into the mechanisms of tobacco-related cardiovascular damage and supporting the development of targeted interventions.

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United States Food and Drug Administration

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Modeling SARS-CoV-2 variant emergence from immunocompromised hosts

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National Institute of Allergy and Infectious Diseases (NIAID)

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This proposal describes a five-year research training program that will facilitate my ongoing transition from an applied mathematician to an independent, quantitative, multidisciplinary biomedical researcher. I will work closely with clinicians, mathematical modelers, bioinformaticians, evolutionary biologists, virologists and immunologists to develop a suite of mathematical models which will be validated against viral, immune, phylogenetic and epidemiolocal datasets. The goals of my proposal will be to: 1) understand the mechanisms facilitating generation of SARS-CoV-2 variants of concern (VOC) during prolonged infections in immunocompromised (IC) individuals, and 2) identify key bottle necks that limit the number of VOCs that predominate in the general population. SARS-CoV-2 is the appropriate virus for which to develop this modeling framework due to the availability of data, and ongoing incidence, but the approach will be widely applicable to other pathogens.  The training program includes an outstanding group of mentors and collaborators. My scientific advisory committee consists of experts in modeling infectious diseases (Dr Josh Schiffer and Dr Dan Reeves), clinical care for IC individuals (Dr Josh Schiffer and Dr. Alpana Wahgmare), epidemiology (Dr Cheryl Cohen and Dr Dobromir Dimitrov), viral evolution (Dr JT McCrone and Dr Mahan Ghafari), and biostatistics and machine learning (Dr Ollivier Hyrien). This group is dedicated to ensuring the success of my project, and my career development as an independent researcher. The specific learning goals required for my successful transition to biomedical research will be accomplished through didactic coursework in virology, immunology, epidemiology and phylogenetics as well as conferences and professional training in the skills of a successful mentor and group leader.  The research plan addresses a critically important clinical and public health issue. Prolonged SARS-CoV- 2 infections in IC individuals are the most likely source of most novel VOC, which have extended and strongly exacerbated the impact of the pandemic. Though these infections have had an outsized public health impact, clear guidance regarding clinical management and safety measures is lacking. Understanding the within-host evolution of SARS-CoV-2 is paramount to addressing these issues. Through accomplishing the aims of this proposal, Dr Owens will address critical gaps in our knowledge of SARS-CoV-2 evolution and create an in silico framework to study SARS-CoV-2 interventions at both individual and population level. Ultimately, this proposal will allow Dr Owens to influence the future of pandemic response research as well as build a self-sustaining program at the interface of mathematical modeling, immunology, viral dynamics, and viral evolution.

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United States National Institutes of Health

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Human upper airway immune memory kinetics and durability against respiratory pathogens

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National Institute of Allergy and Infectious Diseases (NIAID)

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Peripheral blood is the primary sample type collected to evaluate a vast range of medical conditions. However, peripheral blood testing may not reflect processes occurring in tissue, like the upper airway. The SARS- CoV-2 (SARS2) pandemic highlighted the importance of being able to sample the upper airway for purposes including evaluation of viral infection and clearance. Although the upper airway represents the primary site of infection for many human pathogens, critical questions remain regarding upper airway immunity and protection. Nasal cavity swab sampling can be used to bridge knowledge gaps regarding upper airway immunity to important respiratory pathogens like SARS2 and to address important immunologic questions where peripheral blood sampling is inherently insufficient. I established novel methods for reproducible, longitudinal sampling of upper airway immune cell populations using swabs and demonstrated that sufficient numbers of viable immune cells could be collected to allow for high resolution downstream analyses, including multiparametric flow cytometry and single cell RNA sequencing. Upper airway resident memory B and T cell populations, including SARS2- specific memory B and T cells were characterized using these methods.  There is great interest in developing next generation vaccines, including vaccines that can elicit robust mucosal immune responses. This requires knowledge of the immunologic contexts in which mucosal immunity is generated and the requirements for maintenance of upper airway immune memory. I hypothesize that local antigen exposure is required to develop durable upper airway immune memory, and intramuscular immunization alone may fail to elicit upper airway immunity despite generating circulating immunity. I will test this hypothesis by studying upper airway memory B and T cell frequencies, diversity, kinetics and durability in distinct immunologic contexts including SARS2 breakthrough infection (BTI) and COVID-19 booster vaccination using this award. Other respiratory pathogens of public health importance will also be examined.  If funded, this K08 will assist with my transition to becoming a successful independent physician scientist investigator in human immunology and infectious diseases. The La Jolla Institute for Immunology (LJI) is a superb training environment and located on the campus of another excellent academic research and medical institution, the University of California, San Diego (UCSD). I will receive ongoing mentorship from an established investigator with a track record for producing successful mentees, Professor Shane Crotty, PhD, a world- renowned expert in the field of immunology. LJI and UCSD are closely affiliated, and Dr. Crotty has an adjunct appointment at UCSD. I will have access to resources at both LJI and UCSD for this career development award. As an Associate Physician in UCSD’s Division of Infectious Diseases I will receive additional mentorship in academic career development, and maintain my clinical acumen by providing part-time medical care and consultation services for medically complex patients.

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The Impact of Changes in Primary Care Clinicians' Work Effort on the Health of Older Adults

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There are demonstrated benefits of comprehensive, continuous, and coordinated primary care for older adults, ranging from higher rates of appropriate preventive care receipt to lower rates of hospitalization and mortality. However, the benefits of strong primary care are threatened by an impending primary care workforce crisis, exacerbated by prevalent trends of primary care physician (PCP) workforce attrition and clinical effort reduction. Partly in response to these trends, there is increasing representation of NPs and PAs, collectively referred to as advanced practice clinicians (APCs), in the primary care workforce. However, burnout, intent to leave, and intent to reduce clinical effort are also prevalent among primary care APCs. These trends across primary care clinicians (PCCs; comprising physicians, NPs, and PAs) may significantly threaten quality of and access to care for older adults. At present, there is limited evidence to inform healthcare leaders and policy makers about how primary care workforce disruptions impact access to and quality of primary care received by older adults. There is additionally insufficient information on the actionable factors associated with PCC turnover and PCC reductions in clinical effort. In this grant, we will leverage data from Medicare fee-for-service and Medicare Advantage, which together provide coverage for 93% of older adults, in order to: 1) quantify the number of Medicare patients impacted by PCC turnover and sustained reductions in billed clinical effort and identify factors associated with these work effort changes; 2) assess the impact of PCC turnover and PCCs’ sustained reductions in billed clinical effort on patterns of primary care receipt and non-primary care utilization, including emergency department visits and hospitalizations; and 3) assess the impact of PCC turnover and sustained reductions in billed clinical effort on quality of care for older adults. All analyses will be conducted for the overall population of older adults as well as for subgroups of more vulnerable older adults, including those with dementia, multiple chronic conditions, and from underserved groups (e.g., dually eligible for Medicaid). Additionally, analyses will be conducted for physicians and APCs separately, and for the overall study period and comparing the pre- and post-COVID periods. The results from this study will elucidate how changes in PCCs’ work patterns influence the care of the growing US population of older adults. They will provide actionable insights for leaders seeking to design clinical systems and policies that enhance primary care for older adults. Overall, this proposal will enhance the ability of clinical, operational, and policy leaders to maintain the effort of the primary care workforce and optimize care for older adults.

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Discovery and Optimization of Broadly Protective Merbecovirus Neutralizing Antibodies

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National Institute of Allergy and Infectious Diseases (NIAID)

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Middle East respiratory syndrome coronavirus (MERS-CoV) infection has the highest mortality rate (36%) of any of the known human-pathogenic Betacoronaviruses. MERS-CoV continues to circulate in the Middle East, and due to global travel has spread to 27 other countries making it a global health priority. A number of viruses related to MERS-CoV have been identified in mammals worldwide, and several have been found to utilize human DPP4- or ACE2-receptors for cell entry. Therefore, the MERS-related betacoronaviruses (Merbecoviruses, MERBs) have considerable zoonotic potential. While many medical countermeasures like vaccines and antibody therapeutics were developed to fight SARS-CoV-2, these countermeasures do not prevent disease caused by MERBs. The long-term goal of this proposal is to generate such MERB broadly neutralizing antibody (bnAb) therapeutics. The significance of this project includes the identification of broadly protective antibodies (Abs) and their epitopes, enabling rational design of antibody-based therapeutics and vaccines against a deadly virus. While previous research has focused on identifying potent neutralizing antibodies against MERS-CoV, no antibodies have been shown to be cross- protective against MERS-CoV and other MERBs. In preliminary studies in vaccinated rhesus macaques (RMs), we elicited robust cross-binding and cross-neutralizing plasma Abs against multiple MERBs. Post- vaccination RM B cells bound both MERS-CoV receptor binding domain (RBD) and bat MERS-CoV-related virus NL140422 RBD. Individual Abs from these RMs bound to as many as 7 different MERS-CoV-related RBDs and MERS-CoV. The objectives of this study are to 1) determine the neutralization breadth of monoclonal nAbs from these vaccinated RMs, 2) determine the critical features of the binding interface between cross-reactive nAb and virus spike RBD, and 3) determine the cross-protective efficacy of the mAbs. The innovations of this project include human, bat, and pangolin live-virus MERB models (MERS-CoV, PDF2180, NL140422 (MERS 422), MjHKU4r, HKU-5), 13 genetically diverse MERB spike and RBD antigens, MERS-CoV and MjHKU4 mouse challenge models, and the use of a machine-learning Ab optimization to improve natural Abs. The impact of this project includes the demonstration that vaccination can elicit broad MERB nAbs against multiple conserved epitopes, the development of trispecific immunotherapies, and the definition of bnAb epitopes that inform rational design of immunogens targeting cross-protective B cells. Our Abs will be potential tools to combat MERS-CoV outbreaks and prepare for future outbreaks from pre- emergent MERBs.

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Middle East Respiratory Syndrome Coronavirus

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Decoding cellular networks governing respiratory mucosal IgA immunity

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National Institute of Allergy and Infectious Diseases (NIAID)

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Abstract/summary The factors and mechanisms driving robust respiratory mucosal immunity, particularly respiratory IgA responses, post-infection or vaccination are largely unknown. This represents a significant gap in our understanding necessary for designing future vaccination strategies aimed at providing enhanced mucosal protection against respiratory viral infections including new SARS-CoV-2 variants. This RO1 grant proposal aims to address this critical knowledge gap. Our central hypothesis is that the generation of mucosal IgA and respiratory protective immunity is contingent upon the localized interactions among pulmonary macrophages, CD4 T cells, and B cells within the respiratory tract. Three specific aims (SA) are proposed. Aim 1: Identify the associated mechanisms by which respiratory CD4+ T cells promote IgA production in situ. Aim 2: Elucidate TGFβ-dependent macrophages and B cell interactions in mucosal IgA production. Aim 3: Define the molecular and functional signatures of mucosal cross-reactive IgA-producing B cells. We believe that the insights obtained will be crucial in developing next-generation mucosal vaccines designed to effectively counter SARS-CoV-2 variants and other respiratory pathogens, significantly enhancing public health prevention strategies against respiratory infections. .

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Protective mRNA Vaccines Against Tuberculosis

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National Institute of Allergy and Infectious Diseases (NIAID)

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Protective mRNA Vaccines Against Tuberculosis. Summary. Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tb), remains a significant global health challenge, affecting approximately one-third of the world’s population and resulting in nearly 1.4 million deaths annually. The existing vaccine, M. bovis BCG (BCG), offers variable protection, with efficacy ranging from 0% to 80%. Our previous research has identified several innovative platform technologies aimed at enhancing vaccine development for major infections impacting both human and animal health. Notably, we have developed unique nano-adjuvant systems (NAS) that have demonstrated effectiveness against respiratory infections, including coronavirus and M. avium. In this project, we will utilize our expertise in tuberculosis vaccine development and nanoparticle vaccine platforms to assess the protective efficacy of a novel combination vaccine against TB. Our approach incorporates cutting-edge mRNA vaccine technology delivered via QuilA-DOTAP (QTAP), a novel lipid nanoparticle delivery adjuvant that ensures stable mRNA transcript delivery at various temperatures suitable for use in TB- endemic regions. Preliminary analyses of QTAP-adjuvanted combination mRNA vaccine encoding three mycobacterial antigens (Ag85B, Hsp70, and EsxH), referred to as QRNA, have shown robust protective immunity in mouse models challenged with both low and high doses of the virulent M. tb Erdman strain. In this project, we plan to First; examine the safety and immunogenicity of QRNA vaccines in variable murine models using both immune-compromised (Rag1-/-) and immune-competent (C3HeB/FeJ) murine models. Second; analyze the protective role of QRNA vaccine as a homologous or heterologous vaccine primed with BCG against challenge with M. tb Erdman (lineage 4, laboratory strain) or HN878 (lineage 2, hypervirulent clinical strain). Finally, we will assess protective immunity of QRNA vaccines in guinea pigs to identify vaccine-induced immune correlates of protection elicited by the mRNA vaccine candidates in guinea pigs, a TB model that mimic human infection. Once achieved, results from those aims will enhance our understanding of RNA-based immunization against TB. Future projects will further dissect the generated immunity in non-human primates, a more relevant model for human TB.

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United States National Institutes of Health

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Advancing iPSC-derived Thymic Epithelial Cells as Cell Therapy for T Cell Immune Reconstitution in Vulnerable Populations(original application ID AI190181-01)

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National Institute of Allergy and Infectious Diseases (NIAID)

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| NARRATIVE The thymus instructs T cell immunity and central tolerance, yet its therapeutic potential remains clinically untapped as the signals that drive thymic epithelial cell (TEC) differentiation remain incompletely understood. The thymic epithelium comprises a highly specialized set of cells that attract lymphoid progenitors, promote their proliferation and maturation into thymocytes, and facilitate the selection of a diverse, self-tolerant T cell receptor (TCR) repertoire. The role of the thymus in building immune identity begins before birth. The organ peaks in size in infancy and then structurally and functionally involutes over time. This process causes the decline in immune competence with age (immune senescence). The impact of this phenomenon was exposed during the COVID-19 pandemic when waning immunity left the elderly more vulnerable to adverse outcomes. Thymus insult also occurs in many patients through medications, radiation, infections and graft-versus-host disease. The most severe form of thymic compromise is congenital athymia, the inborn absence of the thymus due to genetic mutations. Genetic or acquired thymic injury leads to immunodeficiency, autoimmunity, inflammation and increased cancer risk. Regenerating thymic function, e.g., through human induced pluripotent stem cell (iPSC)-derived regenerative thymic tissues holds greatest therapeutic promise for these patients. We have used single-cell transcriptomics of human fetal anterior foregut-derived organs to uncover the signals that drive TEC differentiation. We have translated these insights into a novel differentiation platform for the derivation of TECs from iPSCs in vitro. When iPSC-derived TEC organoids are transplanted into athymic NSG nude (NSG-Foxn1-/-) mice engrafted with human hematopoietic stem cells, they function like the human thymus, giving rise to human ab-T cells with a diverse TCR repertoire, gd-T cells and regulatory T cells. In this application, we now seek to advance the translation of iPSC-derived TECs (iTECs) by testing their safety and efficacy as cell therapy for vulnerable patient populations in need of improved T cell immunity. In Aim 1, we will determine the capacity of iTECs to promote T cell reconstitution, functional antigen-specific T cell responses and the development of a broad TCR repertoire in vivo. In Aim 2, we will assess if T cells educated on iTEC are tolerant to “self” but respond to “non-self”. In addition, we will directly analyze the HLA-associated peptide repertoire presented on iTECs using immunopeptidomics. In Exploratory Aim 3, we will test if HLA-editing of iPSCs for iTECs derivation affects antigen-specific immune responses, TCR repertoire, and immunopeptidome in vivo. Advancing the translation of iPSC-derived TECs into a cell therapy is an entirely new strategy to leverage the therapeutic potential of T cells from inside the body and could begin a new chapter of immunotherapeutics.

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A Microfluidic-Free Droplet Technology for Rapid and Quantitative Airborne Pathogen Monitoring

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National Institute of Allergy and Infectious Diseases (NIAID)

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Pathogen transmission via indirect routes such as fomite, waterborne, and airborne transmission are hallmarks of both endemic and pandemic spread. Viruses such as Influenza, SARS-CoV-2, and measles are notable examples, in addition to deadly microbes such as Bordetella pertussis, Mycobacterium tuberculosis, and Coccidioides species. However, the rapid detection and analysis of airstreams as part of biosurveillance, public health monitoring, or epidemiological research remains challenging. Current state-of-the-art methods rely on bulky apparatuses for both the collection and detection of airborne agents; most of these methods are ill suited to rapid response point-of-testing within medical facilities, workplace locals, or public spaces. Further, these technologies are based on bulk Polymerase Chain Reaction (PCR) and Loop-mediated isothermal amplification (LAMP) methods that have limited quantitative accuracy. Thus, more portable and accurate platforms are needed to address the types of rapid response and ubiquitous monitoring that is required to minimize outbreaks in the 21st century. Towards this objective, this grant will address the need for an improved method of airborne pathogen detection through the development of the digital droplet Aerosol Capture & Quantification (ddACQ) system. The ddACQ system consists of two novel technologies, a filter particle array and a droplet buoyancy counter. The filter particle array allows for the capture of airborne pathogens or biological agents and generation of microfluidic droplets when mixed with an oil and water reagent solution. A phone-powered heat block then drives a one-step isothermal digital droplet reaction. Digital techniques have several key advantages over classical quantitative PCR and LAMP techniques, namely single molecule detection and direct quantification without a standard curve. Finally, the droplet buoyancy counter allows for smartphone based read out of the reaction immediately at the testing site in under an hour. Together, the innovations within and implementation of the ddACQ system represents a novel application of microfluidic principles and an enabling technology for both pathogen transmission research and public health monitoring applications.