OBJECTIVES/SPECIFIC AIMS: Objective: apply checkpoint inhibitors that are specific to the exhaustive markers expressed on tumor CD8+ T-cells ex vivo in order to improve cytokine release and cytotoxic function in comparison to two control groups: (1.) T-cells that receive no antibodies; (2.) T-cells that receive standard inhibition with PD-1 and CTLA-4 antibodies only. Long-term objective: provide personalized medicine in the treatment of HCC by using checkpoint inhibitors that are specific to the receptors expressed by an individual tumor. METHODS/STUDY POPULATION: The study population includes patients undergoing liver transplantation or surgical resection for HCC. Two grams of tumor, two grams of healthy liver tissue at least one centimeter from the tumor margin, and 50 milliliters of blood will be obtained. Solid tissue will be mechanically and enzymatically disrupted and CD8+ T-cells will be isolated from all sites. Using flow cytometry, the expression of surface receptors PD-1, CTLA-4, LAG-3, TIM-3, BTLA, CD244, and CD160 will be categorized in each tissue to identify which receptors are upregulated in the tumor microenvironment. Up to three antibodies specific to the upregulated receptor(s) on the tumor T-cells will be applied per specimen. The experimental arm will receive these antibodies and co-stimulation with CD3/CD28 and will be compared to two controls. One control will receive only CD3/CD28, and the other will receive CD3/CD28 in addition to the standard combination of PD-1 and CTLA-4 inhibitors. From each condition, flow cytometry will be used to assess the mean production of interleukin-2, tumor necrosis factor-α, interferon-γ, granzyme B, and perforin expression as an assessment of T-cell function. RESULTS/ANTICIPATED RESULTS: Preliminary data from the peripheral blood of healthy controls confirms that the developed flow cytometry panels effectively identify the surface receptors and cytokine production of CD8+ T-cells. Two patients have successfully been enrolled in this study. It is predicted that T-cells extracted from the tumor will express more inhibitory receptors than normal liver or peripheral blood and will have increased function after they are targeted with checkpoint inhibitors that are specific to the inhibitory surface receptors they express. DISCUSSION/SIGNIFICANCE OF IMPACT: HCC is the second leading cause of cancer-related death worldwide and therapeutic options are limited for patients who are not surgical candidates. T-cells are a critical component of the anti-tumor response to HCC. However, T-cells can develop an exhausted phenotype characterized by up-regulated inhibitory receptors (PD-1, CTLA-4, LAG-3, TIM-3, CD-244, CD-160, BTLA) and decreased function, allowing for immune escape. Clinical trials using combined checkpoint inhibition with PD-L1 and CTLA-4 antibodies have been considered a breakthrough for patients with advanced HCC, as up to 25% show an objective tumor response. The explanation for the varied susceptibility to checkpoint inhibition remains unknown and is hypothesized to be secondary to inconsistencies in the expression of surface inhibitory receptors. Although inhibitory receptor expression has been shown to be upregulated under conditions of hepatitis and/or HCC, there has been no single study to effectively investigate the expression of all known inhibitors in order to better explore the interplay between them. It will be of great academic interest and clinical purpose to evaluate individual receptor expression and engage the correlating antibodies given the possibility of synergism between receptors and the need for a more profound anti-tumor T-cell response in HCC.

OBJECTIVES/SPECIFIC AIMS: The central goal of this proposal is to characterize the mechanisms that mediate success or failure of immature intestinal barrier in necrotizing enterocilitis. METHODS/STUDY POPULATION: To do this, I will utilize stem cell derived human intestinal organoids (HIOs), an innovative model of the immature intestine, and a cohort of bacterial isolates collected from premature infants who developed NEC to interrogate the cause-effect relationship of these strains on maintenance of the intestinal barrier. I hypothesize that the epithelial response to bacterial colonization is strain-dependent and results in differences in inflammatory signaling that shape epithelial barrier function in the immature intestine. RESULTS/ANTICIPATED RESULTS: Preliminary data shows that colonization of HIOs with different bacteria leads to species-specific changes in barrier function, and some species selectively damage the epithelial barrier while others enhance epithelial barrier function. I have identified key inflammatory signals that serve as central drivers of intestinal barrier function. DISCUSSION/SIGNIFICANCE OF IMPACT: Characterization of this process is expected to substantially advance scientific understanding of early events in NEC pathogenesis and lead to new opportunities for targeted therapeutic intervention to accelerate barrier maturation or prevent hyperinflammatory reactivity in the neonatal intestine. The research proposed in this application represents an entirely novel approach to studying host-microbial interactions in the immature. Conceptually, this novel translational approach will help to define the pivotal role of colonizing bacteria in initiating epithelial inflammation in NEC patients.

ABSTRACT IMPACT: If immune checkpoint blockade increases bacterial clearance with or without antibiotics in vitro, clinical application would be almost immediate and dramatic creating a seismic shift in the current therapeutic paradigm of periprosthetic joint infection. OBJECTIVES/GOALS: Periprosthetic joint infection (PJI) is a major cause of failure after joint replacement. Currently, the treatment of PJI relies on removing biofilm contaminated implants. Some of the bacteria within biofilm undergo a phenotypic shift becoming small colony variants (SCVs). SCVs induce local immunosuppression through PD-1/L1 signaling. METHODS/STUDY POPULATION: We will infect cultured human macrophages and bone marrow aspirate with stable Staphylococcus aureus SVCs and treat with anti-PD-1 or anti-PD-L1 monoclonal antibodies with and without antibiotics (e.g., gentamycin, cefazolin, vancomycin, rifampicin) and assess the residual bacterial viability. We will utilize multiplexed ion beam imaging to quantify PD-1/L1 expression in human tissue from patients with a chronic PJI and compare those to patients undergoing an aseptic revision. Patients with a chronic PJI are likely to have increased expression of PD-1/L1 as their tissue samples are prospectively screened. RESULTS/ANTICIPATED RESULTS: SCVs reduce the phagocytic activity of macrophages and can survive intracellularly. SCVs also induce anti-inflammatory M2-macrophage polarization and recruit a heterogeneous group of immature monocytes and granulocytes called myeloid-derived suppressor cells (MDSC) to the periprosthetic microenvironment. M2-macrophages and MDSCs then produce an immunosuppressive cytokine milieu characterized by increased IL-10 and decreased TNF-α. Clinically isolated SCVs up-regulate the expression of PD-L1 and PD-L2 on the surface of macrophages, representing a mechanism by which SCVs induce host immunosuppression and survive immune clearance. Our preliminary data show PD-L1 expression during septic PJI, but not in aseptic revisions. DISCUSSION/SIGNIFICANCE OF FINDINGS: If immune checkpoint blockade is shown to increase bacterial clearance with or without antibiotics, host immunomodulation would represent a novel class of therapeutic adjuvants to assist surgical debridement and antibiotic administration that could be superimposed on existing treatment algorithms to improve PJI related outcomes.

OBJECTIVES/SPECIFIC AIMS: To create a new semantically correct high-throughput phenotyping (HTP) platform. To demonstrate the utility of the HTP platform for observational research and can allow clinical investigators to perform studies in 5 minutes. To demonstrate the improved accuracy of observational research using this platform when compared with traditional observational research methods. To demonstrate that patients who have Roseacea are at increased risk of having obstructive sleep apnea (OSA). METHODS/STUDY POPULATION: This population is a set of 212,343 patients in the outpatient setting cared for in the Buffalo area over a 6-year period. All records for these patients were included in the study. Structured data was imported into an OMOP (OHDSI) database and all of the notes and reports were parsed by our HTP system which produces SNOMED CT codes. Each code is designated as a positive, negative or uncertain assertion and compositional expressions are automatically generated. We store the codified data 750,000,000 codes in Berkley DB, a NOSQL database, and we keep the compositional graphs in both Neo4J and in GraphDB (a triple store). Labs are coded in LOINC and drugs using RxNorm. We have developed a Web interface in .Net named BMI Search, which allows real-time query by subject matter experts. We analyzed the accuracy of structured Versus unstructured data by identifiying NVAF cases with ICD9 codes and then looked for any additional cases based on the SNOMED CT encodings of the clinical record. This was validated by 2 clinical human review of a set of 300 randomly selected cases. Separately we ran a study to determine the relative risk of OSA with and without Rosacea using the data set described above. We compared the rates using a Pearson χ2 test. RESULTS/ANTICIPATED RESULTS: We are able to parse 7,000,000 records in an hour and a half on 1 node with 4 CPUs. This yielded 750,000,000 SNOMED CT codes. The HTP data set yielded 1849 cases using ICD9 codes and another 873 using the HTP-NLU data, leading to a final data set of 2722 cases from our population of 212,343 patients. In total, 580 patients had Rosacea;5443 patients had OSA without Rosacea and 51 patients had OSA with Rosacea. Patients with Rosaca had an 8.8% risk of OSA whereas patients without Rosacia only had a 2.6% risk of OSA. This was highly statistically significant with a p<0.0001 (Pearson χ2 test). The number needed to test was only 12. DISCUSSION/SIGNIFICANCE OF IMPACT: HTP can change how we do observational research and can lead to more accurate and more prolific investigation. This rapid turn around is part of what is necessary for both precision medicine and to create a learning health system. Patients with Rosacea are at increased risk of and should be screened for OSA.

OBJECTIVES/GOALS: Despite a disproportionate impact of COVID-19 on minority and under-resourced communities, nearly all COVID-19 resources have only been online in English. A statewide coalition of community and academic partners used community-engaged strategies to provide tailored outreach to diverse populations. METHODS/STUDY POPULATION: The STOP COVID-19 CA statewide team had a workgroup focused on communications. Members of this group represented different sectors, racial/ethnic groups, disciplines, and regions across the state. They had regular meetings to discuss and strategize how to overcome the impact of historic and structural racism on access to COVID-19 resources, including testing, vaccines, and protective equipment. The team also shared regular updates about changes in community concerns and needs as well as new, tailored resources. RESULTS/ANTICIPATED RESULTS: Together, the team has been able to reach diverse populations across the state, including providing information about COVID-19 in multiple languages and formats, from radio to virtual town halls to local health fairs. The multiple sites also increased access to vaccines and testing through trusted community leaders and locations, including church-based locations to bringing vaccines and testing directly to workplaces. These community pop-up vaccination sites have helped to vaccinate large numbers of diverse populations, some of whom were initially unsure about getting the vaccine, which has helped to reduce the gaps in community vaccination rates by race/ethnicity. DISCUSSION/SIGNIFICANCE: This network of community-engaged strategies utilized for rapid COVID-19 response could also be used to for responses to future public health emergencies, addressing chronic diseases (e.g., diabetes, hypertension), or even other complex issues that affect society and health (e.g., climate change).

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

OBJECTIVES/GOALS: Epilepsy with myoclonic-atonic seizures (EMAS) is a childhood onset epilepsy disorder characterized by seizures with sudden loss of posture, or drop seizures. Our objective was to use short-read genome sequencing in 40 EMAS trios to better understand variants contributing to the development of EMAS. METHODS/STUDY POPULATION: Eligibility for the cohort included a potential diagnosis of EMAS by child neurology faculty at Children’s Hospital Colorado. Exclusion criteria included lack of drop seizures upon chart review or structural abnormality on MRI. Some individuals had prior genetic testing and priority for genome sequencing was given to individuals without clear genetic diagnosis based on previous testing. We analyzed single nucleotide variants (SNVs), small insertions and deletions (INDELs), and larger structural variants (SVs) from trio genomes and determined those that were likely contributory based on standardized American College of Medical Genetics (ACMG) criteria. RESULTS/ANTICIPATED RESULTS: Our initial analysis focused on variants in coding regions of known epilepsy-associated genes. We identified pathogenic or likely pathogenic variants in 6 different individuals involving 6 unique genes. Of these, 5 are de novo SNVs or INDELs and 1 is a de novo SV. One of these involve a de novo heterozygous variant in an X-linked gene (ARHGEF9) in a female individual. We hypothesize the skewed X-inactivation may result in primarily expression of the pathogenic variant. We anticipate identifying additional candidate variants in coding regions of genes previously not associated with EMAS or pediatric epilepsies as well as in noncoding regions of the genome. DISCUSSION/SIGNIFICANCE OF IMPACT: Despite the genetic heterogeneity of EMAS, our initial analysis identified de novo pathogenic or likely pathogenic variants in 15% (6/40) of our cohort. As the cost continues to decline, short read genome sequencing represents a promising diagnostic tool for EMAS and other pediatric onset epilepsy syndromes. CONFLICT OF INTEREST DESCRIPTION: The authors have no conflicts of interest to disclose. SD has consulted for Upsher-Smith, Biomarin and Neurogene on an unrelated subject matter. GLC holds a research collaborative grant with Stoke therapeutics on unrelated subject matter.

Objectives/Goals: This study aims to explore the relationship between plasma biomarkers (GFAP, NF-L, and IL-1β) and cognitive impairment in moderate to severe TBI patients. We will assess biomarker levels and their link to neurocognitive outcomes at acute and chronic stages of injury. Methods/Study Population: We will recruit 100 patients aged 21 years and older with moderate to severe TBI (Glasgow Coma Score 3–12) from a trauma hospital. Blood samples will be collected at 24–72 hours post-injury and again at 3 and 6 months. Plasma levels of GFAP, NF-L, and IL-1β will be measured using multiplex ELISA. Neurocognitive tests will be administered at 3 and 6 months to assess cognitive function. Correlations will be made between biomarker levels, neurocognitive performance, and disability scores (Disability Rating Scale and Glasgow Outcome Scale). Exosome isolation from plasma will allow for detailed analysis of astrocyte-derived biomarkers and their association with long-term cognitive impairment and recovery. Results/Anticipated Results: We anticipate that plasma levels of GFAP, NF-L, and IL-1β will be elevated in the acute phase of moderate to severe TBI and will correlate with injury severity. At 3 and 6 months, higher levels of IL-1β, in particular, are expected to be strongly associated with cognitive deficits. We also anticipate that biomarkers in astrocyte-derived exosomes will provide more specific insights into long-term neuroinflammation and its impact on cognitive function. These findings could pave the way for targeted, personalized interventions to improve recovery in TBI patients. Discussion/Significance of Impact: This research focuses on inflammation’s role in cognitive impairment and disability in TBI patients. We propose using multiple biomarkers – GFAP, IL-1β, NF-L – paired with advanced techniques like exosomes and multiplex analyses to identify novel therapeutic targets, aiming for personalized treatment strategies, as well as prognosis.

Objectives/Goals: pT217-tau is a novel fluid biomarker that predicts onset of Alzheimer’s disease (AD) symptoms, but little is known about how pT217-tau arises in brain, as soluble pT217-tau is dephosphorylated postmortem in the humans. Aging macaques naturally develop tau pathology with the same qualitative pattern and sequence as humans, including cortical pathology. Methods/Study Population: The etiology of pT217-tau in aging brains can be probed in rhesus macaques, where perfusion fixation allows capture of phosphorylated proteins in their native state. We utilized multi-label immunofluorescence and immunoperoxidase and immunogold immunoelectron microscopy to examine the subcellular localization of early-stage pT217-tau in entorhinal cortex (ERC) and dorsolateral prefrontal cortex (dlPFC) of aged rhesus macaques with naturally occurring tau pathology and assayed pT217-tau levels in blood plasma using an ultrasensitive nanoneedle approach. Results/Anticipated Results: pT217-tau labeling is primarily observed in postsynaptic compartments, accumulating in: 1) dendritic spines on the calcium-storing smooth endoplasmic reticulum spine apparatus near asymmetric glutamatergic-like synapses and 2) in dendritic shafts, where it aggregated on microtubules, often “trapping” endosomes associated with Aβ42. The dendrites expressing pT217-tau were associated with autophagic vacuoles and dysmorphic mitochondria, indicative of early neurite degeneration. We observed trans-synaptic pT217-tau trafficking between neurons within omega-shaped bodies and endosomes, specifically near excitatory, but not inhibitory synapses. We also examined pT217-tau in blood plasma in macaques across age-span and observed a statistically significant age-related increase in pT217-tau. Discussion/Significance of Impact: We provide direct evidence of pT217-tau trafficking between neurons near synapses to “seed” tau pathology in higher brain circuits, interfacing with the extracellular space to become accessible to CSF and blood. The expression of pT217-tau in dendrites with early signs of degeneration may help to explain why this tau species can herald future diseases.

OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

OBJECTIVES/SPECIFIC AIMS: Inflammatory Bowel Disease (IBD) is a chronic, life-long condition characterized by inflammation of the intestine that greatly affects an individual’s quality of life. While biologic therapy directed against TNFα (anti-TNFα, Infliximab) and α4β7 integrin (anti-α4β7; Vedolizumab) is used to treat IBD, a substantial number of patients remain non-responsive. Using a comprehensive bioinformatics approach, the aim of this study was to characterize immune cell profiles and altered molecular pathways in IBD patient non-responders to anti-TNFα and anti-α4β7 therapy to determine potential mechanisms and/or indicators of treatment non-response. METHODS/STUDY POPULATION: Publicly available whole transcriptomes from 65 healthy control and IBD endoscopic biopsies were assessed (NCBI GEO GSE73661). Specifically, transcript profiles from responders or non-responders to anti-TNFα and anti-α4β7 therapy were utilized. Differentially expressed transcript profiles were obtained by comparing responders or non-responders prior to receiving therapy versus healthy controls using NCBI’s GEO2R after adjustment with Benjamini and Hochberg testing (p<0.05). Immune profiling of DEgs were analyzed by the core LM22 immune signature for subsets of B-, T-, dendritic-, mast-cells, macrophages, and neutrophils (CIBERSORT, cibersort.stanford.edu) (p<0.05). Networks, functional analysis, and interpretation of transcriptomic data were performed using Ingenuity Pathway Analysis (IPA) (Qiagen) (p<0.05). RESULTS/ANTICIPATED RESULTS: Initially, we determined colonic immune profiles in responders and non-responders to anti-TNFα and anti-α4β7 therapy. Compared to responders, in both anti-TNFα and anti-α4β7 non-responders we found elevated neutrophil levels (p<0.05). Specific to anti-TNFα treatment, non-responders demonstrated substantially reduced Treg cells (p<0.05); whereas, exclusive to anti-α4β7 treatment, non-responders showed elevated dendritic cells, activated CD4 T cells, and reduced M2 macrophages (p<0.05). Next we profiled differentially expressed transcripts to determine molecular pathways associated with therapy non-response. In both anti-TNFα and anti-α4β7 non-responders, we observed alterations in pathways specific to cellular growth and metabolism. Among cell growth pathways we found activated growth hormone, Wnt, ErB, and IGF-1 signaling; whereas, among metabolic regulation we found altered triglyceride, tryptophan, and leptin signaling. Moreover, unique to anti-TNFα non-responders, we found activated sphinogosine-1-phosphate and paxillin pathways. While non-response to anti-α4β7 indicated activation of SAPK/JNK and IL-9 signaling. DISCUSSION/SIGNIFICANCE OF IMPACT: Together these data define specific immune profiles and molecular pathways observed in non-responders to anti-TNFα and anti-α4β7 therapy. Our analysis identified substantial alterations in pathways specific to cellular growth and metabolism, identifying a link between non-response to biologic therapy and specific cell functions. These data suggest particular alterations in immune profiles and molecular pathways could play a role in non-response to biologic therapy, highlighting a future direction for personalized treatment regimens that could lead to more targeted use of existing therapies and more favorable patient health outcomes.

Objectives/Goals: Transgender women who have sex with men (TGWSM) have higher HIV risk. The rectal mucosal (RM) immune environment of TGWSM who choose feminizing hormone therapy (FHT) has been shown to be distinct from the RM of cisgender men who have sex with men (MSM). We studied the impact of FHT on the adaptive immune cellular composition of the RM. Methods/Study Population: We sampled cross-sectional and longitudinal cohorts of TGWSM and cisgender MSM from The Silom Clinic in Bangkok, Thailand from December 2020 to December 2023. We included participants aged >18 years, all cisgender MSM and TGWSM with FHT levels in the therapeutic range for cisgender women. We performed RM biopsies and analyzed the adaptive immune cell characteristics via flow cytometry. We will perform binary linear regression to assess the association between systemic FHT levels and the percentage of CD4+ T cells expressing key biomarkers. Primary outcomes include the percentage of CD4+ T cells that express CCR5, with a secondary outcome of the percentage of CD4+ T cells that express Ki67. Results/Anticipated Results: The cross-sectional cohort included 100 TGWSM on FHT and 50 cisgender MSM. The longitudinal cohort included 25 TGWSM who were initiating FHT. Similar primary and secondary outcomes are to be elucidated in both cohorts. We anticipate the RM environment of TGWSM using FHT in both cohorts compared to the RM environment of cisgender MSM in the cross-sectional cohort will be associated with greater percentages of activation/co-receptor expression of CD4+ T cells that express biomarkers of interest. In the longitudinal cohort, we similarly anticipate increased percentages and activation/co-receptor expression of CD4+ T cells expressing biomarkers of interest in TGWSM after in comparison to before initiating FHT. Discussion/Significance of Impact: This is the largest study of its kind to compare HIV target cells in RM of TGSWM, which challenges prevailing perspectives suggesting to group cisgender MSM with TGWSM. Anticipated results will inform HIV prevention strategies and future vaccine studies in this high-risk population.

ABSTRACT IMPACT: Our data demonstrate that VC2 oncolytic virotherapy has significant clinical potential. OBJECTIVES/GOALS: Use our novel oncolytic herpes simplex virus type I (HSV-1), VC2, to understand how oncolytic virotherapy affects the immunosuppressive tumor microenvironment as a mechanism of efficacy. METHODS/STUDY POPULATION: We tested the efficacy of VC2 as an oncolytic virotherapy (OVT) in a syngeneic B16F10-derived mouse model of melanoma. We modified the B16F10 to express nectin-1 (B16F10n-1), the major receptor for HSV-1. Engrafted B16F10n-1 tumors were intratumorally treated with either phosphate-buffered saline (PBS) or 1x10^6 pfu VC2. At indicated time points, treated tumors were excised and processed for immunohistochemistry or flow cytometry analysis. For our experimental metastasis studies, mice were intravenously challenged with B16F10n-1 cells. For our depletion studies, CD4+ and CD8+ T cells were depleted in mice by treatment with mouse anti-CD4 and anti-CD8 monoclonal antibodies respectively, while the control mice were given Rat IgG2b isotype. RESULTS/ANTICIPATED RESULTS: We found that VC2 slowed tumor growth rates and significantly enhanced survival times over control treated mice. VC2-treated mice that survived initial tumor engraftment were able to reject a second tumor challenge and were also resistant to lung colonization (experimental metastasis) of tumor cells. Furthermore, VC2 treatment promoted increased intratumoral T cell infiltration and induced a strong antitumor effect that decreased growth rates of distant, untreated tumors. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our data demonstrate that VC2 OVT has significant clinical potential. Furthermore, due to the increased survival rates and CD8+ T cells dependence, our model will enable study of the immunological correlates of protection for VC2 OVT and OVT in general, as well as to inform the rational design of future OVs with improved therapeutic potentials.

OBJECTIVES/GOALS: The objective is to assess the effect of pesticide exposure (individually and pesticide mixtures) on the immune response to COVID-19 in children. The goal is to improve scientific knowledge on factors affecting COVID-19 and identify a potentially modifiable factor to reduce disparities in COVID-19 morbidity. METHODS/STUDY POPULATION: Blood samples will be obtained from 50 children with asthma two time points; baseline and 12 months later. SARS-CoV-2 infection or vaccination will be determined with blood exposome RNA analyses.. Immunological response will be measured using neutralizing, phagocytizing, and NK-activating anti-body responses biomarkers. Pesticide exposure will be measured via urinary pesticide metabolites (UPMs). For individual metabolites multivariable analyses for each pesticide will be conducted using generalized estimating equation (GEE) models with compound symmetry correlation to account for the repeated measures design. To assess the pesticide mixture, weighted quantile sum regression (WQS) will be used. RESULTS/ANTICIPATED RESULTS: The main hypothesis is that increased pesticide exposure results in a reduction in the immunological response to SARS-CoV-2 infection and the COVID-19 vaccine. Therefore, we anticipate that increasing concentrations of individual UPMs as well as the increasing index will result in reductions in markers of the immune response to SARS-CoV-2 infection and the COVID-19 vaccine. DISCUSSION/SIGNIFICANCE: Exposure to pesticides is a modifiable environmental factor. If pesticides are found to alter the immune response to COVID-19 infection and vaccination, these data will provide an evidence base for efforts to reduce pesticide exposure in children.

OBJECTIVES/SPECIFIC AIMS: We hypothesized that CXCL12, as a biased dimer variant or secreted at dimer-dominant concentrations, would influence PDAC growth and progression. METHODS/STUDY POPULATION: PDAC cells were genetically manipulated to express dimer-promoting levels of CXCL12. These cells were studied in vitro or orthotopically implanted into the mouse pancreas for in vivo studies. As a second approach, recombinant wild-type or engineered CXCL12 monomer or dimer proteins were applied to cells in culture or administered intra-peritoneal to study the effects on tumor growth. RESULTS/ANTICIPATED RESULTS: Mice engrafted with CXCL12-expressing cells had a better survival rate, delayed tumor growth and smaller tumors. Tumors from these mice had significantly less proliferation, measured by Ki-67 staining. In vitro analysis of CXCL12-expressing cells showed decreased viability and growth rates. Percent of cells in the cell cycle G2 phase was also decreased, suggesting cell cycle progression blockade. Viability of human PDAC cells dose-dependently declined upon wild-type CXCL12 treatment, with the non-motile dimer-dominant dose (1000 nM) exhibiting maximal effect. Treatment in an allogeneic mouse model of PDAC with locked-dimer CXCL12, but not wild-type, reduced tumor burden. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results support the notion that biased CXCL12 signaling may be therapeutically exploited to limit pancreatic cancer progression.

OBJECTIVES/SPECIFIC AIMS: The objective of the study was 2-fold; to identify potentially deleterious alleles in a child with Treacher Collins syndrome, and; to demonstrate the value of the iobio analysis platform for intuitively and rapidly analyzing genomic data. METHODS/STUDY POPULATION: We used the iobio suite of web-based applications to analyze quality metrics for the sequencing data and called variants for the proband and his parents. We then visually interrogated variants in genes potentially associated with the syndrome in real-time, using the intuitive gene.iobio application. We sought high impact variants that demonstrated a predicted impact on the protein function, and were simultaneously at low allele frequency in the general human population. Variants were also compared against the ClinVar database of known mutations to identify variants that have already been associated with this, or related syndromes in the literature or clinical studies. Finally, the gene.iobio tool allows users to interrogate the primary sequencing data to ensure that no variants had been missed by the primary variant calling pipeline. This analysis pipeline was performed using intuitive web-based apps in real time, and consequently represents a system that is available to users that traditionally are excluded from these analyses. RESULTS/ANTICIPATED RESULTS: The iobio suite was used to rapidly assess data quality and interrogate genetic variants for a child with Treacher Collins syndrome. A compound heterozygote consisting of 2 missense alleles in the TCOF1 gene was identified as a compelling pathogenic allele, necessitating further functional investigation. The study helped validate the use of the intuitive iobio tools in such analyses, strengthening the case for greater involvement of medical professionals in data analysis. DISCUSSION/SIGNIFICANCE OF IMPACT: The performed analyses demonstrated that the whole genome sequencing data for the family being studied was of a very high quality, although 1 gene demonstrated a local region of almost zero coverage. This ensured that study conclusions can be presented with confidence. A variant associated with Treacher Collins syndrome 1 in ClinVar was uncovered in the TCOF1 gene, however, given it’s benign rating, this variant was not considered further. The most interesting candidate was a compound heterozygote, consisting of 2 missense mutations, also in the TCOF1 gene. These mutations occurred with allele frequencies of 22% and 8% in the general population, and additional molecular and functional studies are currently being pursued.

As the COVID-19 pandemic took hold in the USA in early 2020, it became clear that knowledge of the prevalence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among asymptomatic individuals could inform public health policy decisions and provide insight into the impact of the infection on vulnerable populations. Two Clinical and Translational Science Award (CTSA) Hubs and the National Institutes of Health (NIH) set forth to conduct a national seroprevalence survey to assess the infection’s rate of spread. This partnership was able to quickly design and launch the project by leveraging established research capacities, prior experiences in large-scale, multisite studies and a highly skilled workforce of CTSA hubs and unique experimental capabilities at the NIH to conduct a diverse prospective, longitudinal observational cohort of 11,382 participants who provided biospecimens and participant-reported health and behavior data. The study was completed in 16 months and benefitted from transdisciplinary teamwork, information technology innovations, multimodal communication strategies, and scientific partnership for rigor in design and analytic methods. The lessons learned by the rapid implementation and dissemination of this national study is valuable in guiding future multisite projects as well as preparation for other public health emergencies and pandemics.