Antibodies Research Articles

6588 Insulin Autoimmune Syndrome Presenting with Severe Insulin Resistance and Recurrent Diabetic Ketoacidosis

Abstract Disclosure: E. Looi: None. R. Rosenberg: None. S. Sugar: None. H. Stone: None. A.W. Michels: None. D. Saxon: None. Background: Insulin autoimmune syndrome (IAS) is a rare cause of severe insulin resistance and is typically associated with intermittent hypoglycemia due to insulin antibodies (IA) with high binding capacity and low insulin affinity. However, consideration should be given for atypical presentation of IAS or co-presentation of IAS with type B insulin resistance syndrome (TBIRS) in the absence of hypoglycemia, as described in this case. Case: A 78-year-old African American female (BMI 21 kg/m2) with poorly controlled type 2 diabetes (A1c 11%) experienced recurrent episodes of diabetic ketoacidosis (DKA) despite adherence to escalating insulin doses (310 units daily, 5 u/kg/day). She was referred to endocrinology and continuous glucose monitoring revealed consistently elevated glucose levels (300-400 mg/dL) without hypoglycemia. She had normal renal function, hepatic function, and lipid panel. During a subsequent hospitalization, she required insulin drip rates of 50-70 u/hr. Insulin resistance workup revealed significantly elevated IA level (8.346, normal ≤ 0.010) measured using a fluid-phase radiobinding assay with high insulin binding capacity (1040 U insulin-binding/L blood) and low insulin binding affinity (1.9x107 M), which supported the diagnosis of IAS. Rheumatologic workup for other autoimmune diseases was negative. Due to recurrent DKA episodes, severe insulin resistance without obvious alternative cause, and lack of hypoglycemic events despite low IA binding affinity, co-morbid TBIRS was considered. As there are no commercially available insulin receptor antibody (IRAb) assays, diagnosis could not be confirmed before her condition worsened, and she was admitted for another episode of DKA within a week of being discharged. Due to clinical deterioration and high suspicion for IAS ± TBIRS, treatment with a combination of immunosuppressive agents was initiated in accordance with an NIH protocol (Klubo-Gwiezdzinska J, et al. 2018). Intravenous immune globulin (0.5g/kg/day) was administered over 2 days which resulted in a brief reprieve and followed by significant relapse. Two doses of rituximab (750 mg/m2) and 2 steroid pulses (dexamethasone 40 mg PO daily for 4 days) were then administered 2 weeks apart. Cyclophosphamide 100 mg daily was also started. Her hyperglycemia and insulin requirements subsequently improved, and she was eventually discharged with good glycemic control on a total daily dose of 40 units of insulin (0.5 u/kg/day), an 87.1% reduction in daily insulin requirement. Conclusion: This case demonstrates that IAS - although classically associated with hypoglycemic episodes - may also present with severe insulin resistance and recurrent DKA without hypoglycemia. Whether characterized by high IA binding capacity and affinity or coexisting with IRAb, a combination of immunosuppressants proved effective in treating both TBIRS and IAS in this instance. Presentation: 6/3/2024

Omicron-specific ultra-potent SARS-CoV-2 neutralizing antibodies targeting the N1/N2 loop of Spike N-terminal domain

A multitude of functional mutations continue to emerge on the N-terminal domain (NTD) of the spike protein in SARS-CoV-2 Omicron subvariants. Understanding the immunogenicity of Omicron NTD and the properties of antibodies elicited by it is crucial for comprehending the impact of NTD mutations on viral fitness and guiding vaccine design. In this study, we find that most of NTD-targeting antibodies isolated from individuals with BA.5/BF.7 breakthrough infection (BTI) are ancestral (wildtype or WT)-reactive and non-neutralizing. Surprisingly, we identified five ultra-potent neutralizing antibodies (NAbs) that can only bind to Omicron but not WT NTD. Structural analysis revealed that they bind to a unique epitope on the N1/N2 loop of NTD and interact with the receptor-binding domain (RBD) via the light chain. These Omicron-specific NAbs achieve neutralization through ACE2 competition and blockage of ACE2-mediated S1 shedding. However, BA.2.86 and BA.2.87.1, which carry insertions or deletions on the N1/N2 loop, can evade these antibodies. Together, we provided a detailed map of the NTD-targeting antibody repertoire in the post-Omicron era, demonstrating their vulnerability to NTD mutations enabled by its evolutionary flexibility, despite their potent neutralization. These results revealed the function of the indels in the NTD of BA.2.86/JN.1 sublineage in evading neutralizing antibodies and highlighted the importance of considering the immunogenicity of NTD in vaccine design.

B-246 Pab to mab conversion via mass spectrometry: direct antibody discovery from serum

Abstract Background Navigating the intricacies of discovering unique and functional antibody binders for in vitro diagnostic development comes with several technological challenges, such as limited diversity of antibody repertoires, functional screening challenges, and the absence of suitable in vitro models. A proteomics-driven strategy to antibody discovery is a promising solution to overcome these obstacles by facilitating polyclonal to monoclonal antibody conversion. De novo polyclonal antibody sequencing utilizes mass spectrometry and machine-learning driven bioinformatics to obtain complete antibody sequences from immunoserum or purified protein samples. This approach investigates the entire circulating antibody repertoire at the protein level, thereby broadening the spectrum of potential candidates. It further facilitates functional selection by enabling antibody purification and enrichment techniques. This capability facilitates the identification of antibodies possessing unique functions, crucial for subsequent downstream development. We demonstrate the application and de novo polyclonal antibody sequencing using mass spectrometry and machine learning bioinformatics to enable the next generation of antibody discovery. Methods In this study, we employed a novel mass spectrometry-based approach, REpAb, to de novo sequence polyclonal antibodies directly from the immunized serum of animals or human patients. REpAb integrates mass spectrometry with machine learning-based bioinformatics to de novo sequence and assemble monoclonal antibodies from a polyclonal mixture. Protein lysates underwent analysis using an Orbitrap EclipseTM Series instrument (ThermoFisher Scientific, CA, US) coupled with the LC Evosep One (Evosep, Denmark). Results De novo polyclonal sequencing with REpAb yielded unique antibody binders not found in splenocytes or peripheral blood mononuclear cells (PBMCs). De novo antibody sequencing-discovered antibody sequences from naturally exposed humans showed unrestricted germline usage, compared to restricted germline used in antigen-immunized animals. Additionally, a proteomics-based approach for antibody discovery enables functional antibody selection through standard protein A/G purification, followed by negative and positive selection enrichment strategies to isolate antibody clones specific to the region of interest on the antigen. Conclusions Sequencing antibodies at the protein level directly reflects the circulating antibody repertoires, which expands the antibody diversity from the BCR repertoire. Proteomic-based antibody discovery empowers the functional selection of antibodies via meticulously designed enrichment strategies. This approach allows for the identification of a diverse pool of candidates possessing specific functions, such as anti-idiotypic features.

B-075 Evaluation of patients with monoclonal gammopathy of undetermined significance using Optilite Freelite assays

Abstract Background Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant disorder with a progression risk to multiple myeloma of 1% per year. Patients with MGUS may produce monoclonal intact immunoglobulins (Ig) and/or free light chains (FLC). The International Myeloma Working Group (IMWG) recommendations for the use of FLC measurements in MGUS are to establish the diagnosis of light chain only MGUS (LC-MGUS) and further to risk-stratify newly diagnosed MGUS. Once identified, MGUS is longitudinally monitored at intervals dependent on the risk of transformation. The IMWG recommends serum protein electrophoresis (SPEP) for monitoring of known MGUS with intact Ig, however others have published benefits of additionally monitoring with FLC. In this study, we evaluated the diagnostic sensitivity and specificity of the Optilite® Freelite® Kappa Free Kit and the Optilite Freelite Lambda Free Kit (The Binding Site, part of Thermo Fisher Scientific) in patients with MGUS, and the monitoring performance of Freelite results measured in serial samples from patients with clinically stable and progressive MGUS. Methods Serum samples from 234 patients with clinically diagnosed MGUS and 140 disease controls, taken at single time points, and serial samples (≥3) from 49 patients with MGUS were tested with the Freelite assays. Intact M-protein presence and isotype was confirmed in MGUS samples with immunofixation electrophoresis (IFE). In patients clinically diagnosed with LC-MGUS, the presence of detectable M protein on IFE was not required. The disease control cohort (non-MGUS) had disorders commonly associated with elevated polyclonal immunoglobulins (e.g autoimmune, liver, and renal diseases), confirmed by total IgG and/or IgA and/or IgM results and the absence of M-protein by IFE. Patients represented a diverse population from both European and US sources. Results The overall diagnostic sensitivity of the Optilite Freelite assays was 59.4%, calculated as the frequency of abnormal free light chain ratio (rFLC) in intact immunoglobulin MGUS, and abnormal rFLC with concurrent elevated involved FLC level in LC-MGUS. The diagnostic sensitivity was: 58.0% in non-IgM MGUS, 41.7% in IgM MGUS, 66.7% in biclonal MGUS and 100% in LC-MGUS. In intact (IgG/IgA/IgM) kappa MGUS, the diagnostic sensitivity was 67.5%, versus 41.5% in intact (IgG/IgA/IgM) lambda MGUS. The diagnostic specificity was 86.4%. In the 49 patients with serial samples, concentrations of involved FLC were stable in 93.3% of patients with clinically stable MGUS, whereas in patients with clinically progressive MGUS an increasing trend was seen in 50% of cases. Conclusions These results demonstrate that the Optilite Freelite Kappa and Lambda assays produce clinically relevant results that are useful in the evaluation of MGUS.

Access and adherence to cancer care through the Veteran Affairs Close to Me cancer care service.

111 Background: Diagnosing and treating more than 56,000 Veterans annually, the Veterans Affairs (VA) National Oncology Program (NOP) is one of the nation’s largest integrated providers of oncology services. Within VA, oncology care and parenteral therapies are primarily offered at VA Medical Centers (VAMCs) in urban areas, often inaccessible to rural patients. Issues with travel distance, parking, time, and caregiver support can prevent patients from adhering to treatment appointments as evidenced by historical annual No-Show rates for VAMC infusion clinics that range from 1.1-6.7%. To mitigate potential barriers to access, NOP launched the Close to Me (CTM) cancer care service. The service deploys Registered Nurses (RNs) to local VA Community Based Outpatient Clinics (CBOCs) to administer cancer treatments. VA has opened 23 CTM clinics across 5 geographical regions since October 2021. Methods: The CTM service was implemented at VAMCs with NOP providing direct project management, safety assessment and resources such as a comprehensive implementation toolkit, standard operating procedures, care coordination tools, a tiered risk drug matrix and a standardized Healthcare Failure Mode and Effect Analysis risk evaluation. RNs followed a standardized workflow to assess patient eligibility and verify treatment clearances. Access to care was reported by numbers of unique patients and treatment visits. Patient appointment adherence was monitored by no-show rates documented in the electronic health record. Infusion reactions and medical emergencies were reported by RNs. Travel miles saved was calculated by subtracting the difference in miles round trip from the patient’s home to main VAMC and from home to CBOC, then multiplied by number of treatment visits to CBOC. Results: From October 2021 through April 2024, 23 new CTM clinics opened across 5 regions. 515 unique patients were enrolled for a total of 1,916 patient visits. Treatment appointment no-show rate was 1% across all CTM clinics. The most commonly administered medication classes include gonadotropin-releasing hormone agonist (30%), immune globulin (14.5%), immune checkpoint inhibitors (9.3%) and bisphosphonates (8.3%). Zero infusion reactions or medical emergencies were recorded across 1,430 treatments administered. A total of 204,000 travel miles were saved by patients enrolled in this program. Conclusions: The CTM cancer care service safely optimizes current VA CBOC infrastructure to increase access points to oncology treatment for patients across various geographical regions. CTM reduced travel burden for patients and increased ability to adhere to treatment appointments as evidenced by the service’s low no-show rate. These consistent results across CTM services at VAMCs nationwide have cemented CTM as a safe and effective treatment delivery model to expand access. CTM will be scaled to an additional 30 regions by end of 2025.

Role of Antibodies and Their Specificities in Atherosclerotic Cardiovascular Disease.

Atherosclerosis is a lipid-driven chronic inflammatory disease that is modulated by innate and adaptive immunity including humoral immunity. Importantly, antibody alterations achieved by genetic means or active and passive immunization strategies in preclinical studies can improve or aggravate atherosclerosis. Additionally, a wide range of epidemiological data demonstrate not only an association between the total levels of different antibody isotypes but also levels of antibodies targeting specific antigens with atherosclerotic cardiovascular disease. Here, we discuss the potential role of atherogenic dyslipidemia on the antibody repertoire and review potential antibody-mediated effector mechanisms involved in atherosclerosis development highlighting the major atherosclerosis-associated antigens that trigger antibody responses.

Single-hit genome editing optimized for maturation in B cells redirects their specificity toward tumor antigens.

T-cell-based adoptive immunotherapy is a new pillar of cancer care. Tumor-redirected B cells could also contribute to therapy if their manipulation to rewire immunoglobulin (Ig) genes is mastered. We designed a single-chain Ig-encoding cassette ("scFull-Ig") that redirects antigen specificity when inserted at a single position of the IgH locus. This design, which places combined IgH and IgL variable genes downstream of a pVH promoter, nevertheless preserves all Ig functional domains and the intrinsic mechanisms that regulate expression from the IgM B cell receptor (BCR) expression to Ig secretion, somatic hypermutation and class switching. This single-locus editing provides an efficient and safe strategy to both disrupt endogenous Ig expression and encode a new Ig paratope. As a proof of concept, the functionality of scFull BCR and/or secreted Ig was validated against two different classical human tumor antigens, HER2 and hCD20. Once validated in cell lines, the strategy was extended to primary B cells, confirming the successful engineering of BCR and Ig expression and the ability of scFull-Ig to undergo further class switching. These results further pave the way for future B cell-based adoptive immunotherapy and strategies to express a therapeutic mAb with a variety of switched H-chains that provide complementary functions.

Rapid affinity optimization of an anti-TREM2 clinical lead antibody by cross-lineage immune repertoire mining

We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.

Urban wastewater contains a functional human antibody repertoire of mucosal origin

Wastewater-based surveillance of human disease offers timely insights to public health, helping to mitigate infectious disease outbreaks and decrease downstream morbidity and mortality. These systems rely on nucleic acid amplification tests for monitoring disease trends, while antibody-based seroprevalence surveys gauge community immunity. However, serological surveys are resource-intensive and subject to potentially long lead times and sampling bias. We identified and characterized a human antibody repertoire, predominantly secretory IgA, isolated from a central wastewater treatment plant and building-scale wastewater collection points. These antibodies partition to the solids fraction and retain immunoaffinity for SARS-CoV-2 and Influenza A virus antigens. This stable pool could enable real-time tracking for correlates of vaccination, infection, and immunity, aiding in establishing population-level thresholds for immune protection and assessing the efficacy of future vaccine campaigns.

Viral antibody response predicts morbidity and mortality in alcohol-associated hepatitis.

Alcohol-associated hepatitis (AH) is associated with very high mortality despite abstinence from alcohol; up to 40% of patients die within 6 months of diagnosis. Patients with AH are especially prone to infections, which can lead to multiorgan dysfunction and poorer prognosis. We performed comprehensive serological profiling of the viral and bacterial infection history of 36 healthy controls, 48 patients with alcohol use disorder, and 224 patients with AH from 2 multicenter observational studies. We used systematic viral and bacterial epitope scanning by VirScan, a phage-display immunoprecipitation and sequencing technology that detects the peptides recognized by antibodies in patient sera, to comprehensively analyze antiviral and antibacterial antibodies and identify serologic biomarkers to predict patient outcomes. We found significant differences in the serological profiles of the 3 populations. The number of serum antibody epitopes in patients with alcohol use disorder during abstinence was increased compared with during active alcohol use. A decreased number and diversity of viral and bacterial antibody targets were detected in the sera of patients with AH, particularly those with a higher Child-Pugh score. In patients with AH, a decrease in the serum antiviral, but not antibacterial, antibody repertoire was associated with decompensation and mortality. Ninety-day mortality in AH could be predicted using a serum viral epitope signature. Abstinence from alcohol is associated with a significant increase in serum viral and bacterial antibody response. Decreased serum antiviral antibody repertoire is predictive of decompensation of liver disease and mortality in patients with AH.

Evaluation of a Quadrivalent Shigella flexneri Serotype 2a, 3a, 6, and Shigella sonnei O-Specific Polysaccharide and IpaB MAPS Vaccine

Background: Shigellosis is the leading cause of diarrheal deaths worldwide and is particularly dangerous in children under 5 years of age in low- and middle-income countries. Additionally, the rise in antibiotic resistance has highlighted the need for an effective Shigella vaccine. Previously, we have used the Multiple Antigen-Presenting System (MAPS) technology to generate monovalent and quadrivalent Salmonella MAPS vaccines that induce functional antibodies against Salmonella components. Methods: In this work, we detail the development of several monovalent vaccines using O-specific polysaccharides (OSPs) from four dominant serotypes, S. flexneri 2a, 3a, and 6, and S. sonnei. We tested several rhizavidin (rhavi) fusion proteins and selected a Shigella-specific protein IpaB. Quadrivalent MAPS were made with Rhavi-IpaB protein and tested in rabbits for immunogenicity. Results: Individual MAPS vaccines generated robust, functional antibody responses against both IpaB and the individual OSP component. Antibodies to IpaB were effective across Shigella serotypes. We also demonstrate that the OSP antibodies generated are specific to each homologous Shigella O type by performing ELISA and bactericidal assays. We combined the components of each MAPS vaccine to formulate a quadrivalent MAPS vaccine which elicited similar antibody and bactericidal responses compared to their monovalent counterparts. Finally, we show that the quadrivalent MAPS immune sera are functional against several clinical isolates of the serotypes used in the vaccine. Conclusions: This quadrivalent MAPS Shigella vaccine is immunogenicity and warrants further study.

A transgenic mouse with a humanised B cell repertoire mounts an antibody response to influenza infection and vaccination.

The development of a universal influenza vaccine likely requires an understanding of previous exposure to influenza virus (through vaccination or infection) and how that shapes the antibody repertoire to vaccination, sometimes called Original Antigenic Sin or antigenic imprinting. Whilst animal models can have a much more defined exposure history, they lack a human B cell repertoire. Transgenic mice with the complete human immunoglobulin locus enable studies of controlled infection history leading to human-like antibody evolution. Here we evaluated responses to influenza in the Intelliselect Transgenic mouse (the Kymouse). We show the Kymouse is susceptible to disease following infection with either H1N1, H3N2 or B/Yam influenza viruses and that it induces a robust binding and neutralising antibody response to all three strains of influenza virus. This study demonstrates that human B cell repertoire mice can be used for influenza virus studies, providing a tool for further interrogation of the antibody response.

Large B-cell lymphomas with CCND1 rearrangement have different immunoglobulin gene breakpoints and genomic profile than mantle cell lymphoma

Mantle cell lymphoma (MCL) is genetically characterized by the IG::CCND1 translocation mediated by an aberrant V(D)J rearrangement. CCND1 translocations and overexpression have been identified in occasional aggressive B-cell lymphomas with unusual features for MCL. The mechanism generating CCND1 rearrangements in these tumors and their genomic profile are not known. We have reconstructed the IG::CCND1 translocations and the genomic profile of 13 SOX11-negative aggressive B-cell lymphomas using whole genome/exome and target sequencing. The mechanism behind the translocation was an aberrant V(D)J rearrangement in three tumors and by an anomalous IGH class-switch recombination (CSR) or somatic hypermutation (SHM) mechanism in ten. The tumors with a V(D)J-mediated translocation were two blastoid MCL and one high-grade B-cell lymphoma. None of them had a mutational profile suggestive of DLBCL. The ten tumors with CSR/SHM-mediated IGH::CCND1 were mainly large B-cell lymphomas, with mutated genes commonly seen in DLBCL and BCL6 rearrangements in 6. Two cases, which transformed from marginal zone lymphomas, carried mutations in KLF2, TNFAIP3 and KMT2D. These findings expand the spectrum of tumors carrying CCND1 rearrangement that may occur as a secondary event in DLBCL mediated by aberrant CSR/SHM and associated with a mutational profile different from that of MCL.

Spatial multi-omics reveal intratumoral humoral immunity niches associated with tertiary lymphoid structures in pancreatic cancer immunotherapy pathologic responders.

Integrated multi-modal spatial profiling of human PDAC tumors from neoadjuvant immunotherapy clinical trials reveal diverse spatial niches enriched in TLS.TLS maturity is influenced by tumor location and the cellular neighborhoods in which TLS immune cells are recruited.Unsupervised machine learning of genome-wide signatures on spatial transcriptomics data characterizes the TLS-enriched TME and associates TLS transcriptomes with survival outcomes in PDAC.Interactions of spatially variable gene expression patterns showed TLS maturation is coupled with immunoglobulin distribution and ECM remodeling in pathologic responders.Intratumoral plasma cell and immunoglobin gene expression spatial dynamics demonstrate trafficking of TLS-driven humoral immunity in the PDAC TME. We report a spatial multi-omics atlas of PDAC tumors from a series of immunotherapy neoadjuvant clinical trials. Intratumorally, pathologic responders exhibit mature TLS that propagate plasma cells into malignant niches. Our findings offer insights on the role of TLS-associated humoral immunity and stromal remodeling during immunotherapy treatment.

Abstract C147: Impact of genetic ancestry in triple-negative breast cancer subtypes

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive BC subtype characterized by significant molecular and clinical heterogeneity, influenced by genetic, phenotypic, and environmental factors, as well as interactions of the tumor with its surrounding milieu. A significant racial disparity has been observed between African American (AA) and European American (EA) women regarding the incidence and clinical progression of TNBC. TNBC frequently occurs in young parous women who self-identify as AA and in women who harbor a germline BRCA1 mutation. Notably, AA women are diagnosed with TNBC at a younger age and exhibit a higher risk of developing stage IV disease. Studies examining gene expression signatures that distinguish AA and EA TNBC have reported distinct tumor-associated immunologic profiles in AA patients. We have developed a novel cell type-driven subtyping system based on a racially diverse TNBC sample set that identified three TNBC subtypes, each with distinct biology and prognosis: CC1- immune signaling/T-cell response; CC2- pro- migratory/inflammatory; CC3- fatty acid and hormone and nuclear receptor signaling. In this study, we propose to examine ancestry-inferred differences within each TNBC subtype between patients with African and European ancestry. Methods: RNA-Sequencing data for the Louisiana Tumor Registry, a TNBC cohort of 250 diverse women [EA: 123 and AA: 127], was obtained from our collaborators at Louisiana State University Health Sciences Center. Genetic admixture was performed on 227/250 patients using the method described by Pakstis, A. J. et al. Differentially expressed genes (DEGs) were identified in pairwise comparisons at p-adj ≤ 0.05. Survival was assessed using log-rank tests. Results: We investigated DEGs within each subtype between patients with >60% African (Aa, n=102) or >60% European ancestry (Ea, n=99) (identified via admixture analysis). In all three subtypes, upregulated genes, and their associated enrichment in Aa were related to immune pathways, while DEGs in Ea patients were related to epithelial cell regulation. In CC1, Aa was associated with the upregulation of immunoglobulin genes involved in immunoglobulin production and antigen recognition, while Ea was associated with the upregulation of embryonic development genes. In CC2, Aa showed enriched complement activation and immunoglobulin genes, while Ea exhibited upregulation of basal cytokeratin and growth factor EGFR. In CC3, Aa was associated with the upregulation of inflammatory genes, whereas Ea showed upregulation of cellular senescence genes. Survival analysis demonstrated favorable outcomes for Aa in CC1 and less favorable outcomes for Aa in CC2. Conclusion: Our study highlights significant ancestry-inferred differences in gene expression within TNBC subtypes, with Aa patients showing immune pathways enrichment and Ea patients exhibiting differential epithelial cell regulation. These findings underscore the importance of considering genetic ancestry in TNBC prognosis and treatment strategies. Citation Format: Zahra Mesrizadeh, Kavitha Mukund, Fokrul Hossain, Jovanny Zabaleta, Denise Danos, Luis De Valle, Xiao-Cheng Wu, Chindo Hicks, Augusto Ochoa, Lucio Miele, Victoria L. Seewaldt, Shankar Subramaniam. Impact of genetic ancestry in triple-negative breast cancer subtypes [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C147.

Abstract A045: Pan-viral serological characterization in African Americans and European Americans with Hepatocellular Carcinoma

Abstract Hepatocellular carcinoma (HCC) is often diagnosed at late-stages, particularly among African Americans (AA). Poor screening and diagnosis are key factors that contribute to worse prognoses and disproportionately high mortality rates. We propose investigating viral exposure history as a potential useful screening method. Additionally, by accounting for neighborhood deprivation index (NDI), a metric for differences in socio-economic statuses, we can potentially mitigate this disparity. With NDI and viral exposure history, our study aims to assess HCC prognoses in racially disparate populations. We used a technique called VirScan, which conjugates Phage-Immunoprecipitation (PhIP) and DNA-sequencing, to profile the antibody repertoire against a viral epitope library. Analyzing serum and plasma samples from population controls (PC), individuals at high-risk for developing HCC (HR), and HCC cases, we performed VirScan on 524 AA (272 PC, 205 HR, and 47 HCC) and 342 European Americans (EA) (141 PC, 130 HR, and 71 HCC) participants from the NCI-UMD cohort between 2009-2020 from the greater Baltimore area (NCT00913757; clinicaltrials.gov). We used XGBoost, a machine- learning model, to classify viral features that can discriminate HCC cases from PC group. XGBoost predicted 31 important viral features which distinguish HCC from PC. Of the 31 viral features, 6 viruses were different between AA and EA (FDR < 0.05). To determine the extent to which socio-economic factors contribute to the cancer disparities, we gathered variables from the 1999 census tract as contributors to deprivation. We used Principal Component Analysis (PCA) to determine loadings of these contributor variables; composite of the final variables is derived as NDI. Comparing across groups, we find that average NDI is highest in HR group (x = 0.33), followed by HCC group (x = 0.21), then PC group (x = 0.19). We find that NDI for AA is significantly higher in HCC group (p = 1.3e-09), HR group (p < 2.2e-16), and PC group (p = 5.0e-10) when compared to that of EA. Thus, we demonstrate that both the viral exposure history and NDI of an individual might be useful in prognoses of HCC. This approach could be used to examine the disparities we see in both incidence and mortality of HCC between AA and EA. Citation Format: Theressa Ewa, Whitney Leet, Limin Wang, Marshonna Forgues, Xin Wei Wang. Pan-viral serological characterization in African Americans and European Americans with Hepatocellular Carcinoma [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr A045.

Smoltification of Atlantic Salmon (Salmo salar L.) Is Associated with Enhanced Traffic and Renewal of B Cell Repertoire.

The smoltification of farmed Atlantic salmon is commonly associated with mild immunosuppression. However, B cells may deviate from this trend, showing increased proliferation and migration during this period. This study assessed the effects of smoltification and adaptation to seawater in a controlled experiment. Analyses were conducted on the head kidney, spleen, gill, and both visceral and subcutaneous fat (VAT, SAT) across four time points: parr, early and complete smoltification, and twelve weeks post-seawater transfer. Gene expression analysis was performed to track the distribution and developmental changes in their B cells. Expression profiles of three types of immunoglobulins (ig), including membrane-bound and secreted forms of igm, as well as B cell-specific markers pax1 and cd79, showed strong correlations and contrasted with profiles of other immune cell markers. The highest levels of expression were observed in the lymphatic tissue, followed by the VAT. Enhanced expression in the gill and adipose tissues of smolts suggested an increase in B cell populations. Parallel sequencing of the variable region of the IgM heavy chain was used to track B cell traffic, assessed by the co-occurrence of the most abundant sequences (clonotypes) across different tissues. Smoltification markedly enhanced traffic between all tissues, which returned to initial levels after twelve weeks in the sea. The preferred migration between the head kidney, spleen, and VAT supports the role of abdominal fat as a reservoir of lymphocytes. These findings are discussed in the context of recent studies that suggested the functional significance of B cell traffic in Atlantic salmon. Specifically, the migration of B cells expressing secreted immunoglobulins to virus-infected hearts has been identified as a key factor in the disease recovery and survival of fish challenged with salmon alphavirus (SAV); this process is accelerated by vaccination. Additionally, the study of melanized foci in the skeletal muscles revealed an association between antigen-dependent differentiation and the migration of B cells, indicating a transfer from local to systemic immune responses. Updating the antibody repertoire in the lymphatic and peripheral tissues of smolts may assist in their adaptation to the marine environment and in encountering new pathogens. Emerging evidence highlights B cell migration as an important and previously unrecognized immune mechanism in salmonids.

Longitudinal proteome-wide antibody profiling in Marburg virus survivors identifies wing domain immunogen for vaccine design

Limited knowledge exists on the quality of polyclonal antibody responses generated following Marburg virus (MARV) infection and its evolution in survivors. In this study, we evaluate MARV proteome-wide antibody repertoire longitudinally in convalescent phase approximately every six months for five years following MARV infection in ten human survivors. Differential kinetics were observed for IgM vs IgG vs IgA epitope diversity, antibody binding, antibody affinity maturation and Fc-receptor interaction to MARV proteins. Durability of MARV-neutralizing antibodies is low in survivors. MARV infection induces a diverse epitope repertoire with predominance against GP, VP40, VP30 and VP24 that persisted up to 5 years post-exposure. However, the IgM and IgA repertoire declines over time. Within MARV-GP, IgG recognize antigenic sites predominantly in the amino-terminus, wing domain and GP2-heptad repeat. Interestingly, MARV infection generates robust durable FcɣRI, FcɣRIIA and FcɣRIIIA IgG-Fc receptor interactions. Immunization with immunodominant MARV epitopes reveals conserved wing region between GP1 and GP2, induces neutralizing antibodies against MARV. These findings demonstrate that MARV infection generates a diverse, long-lasting, non-neutralizing, IgG antibody repertoire that perturbs disease by FcɣR activity. This information, along with discovery of neutralizing immunogen in wing domain, could aid in development of effective therapeutics and vaccines against Marburg virus.

121 Copy number variation: From mapping to variance components in Holsteins

Abstract Copy number variations (CNVs) modify transcription levels mainly through changing genes and regulatory elements dosages. Hence, CNVs can contribute to the additive genetic variance of quantitative traits, which might not be fully captured by SNPs. Here, CNVs were mapped from 3,601 Holsteins and their contribution to complex traits was explored by i) quantitative trait loci (QTL) enrichment analysis, and ii) variance component analysis for metritis (1 = disease, 0 = no disease). The 4,113 CNVs derived from high-density (777k) genotypic information were compiled into 1,184 CNV regions (CNVRs) overlapping 7,832 QTLs reported in dairy cattle and available in the AnimalQTLdb. Fisher’s exact test was applied to perform the QTL enrichment analysis followed by False Discovery Rate (FDR) correction. Then, QTLs overlapping CNVRs were enriched for traits when the corrected P-value (PFDR) was lower than 0.05. For variance components estimation, genomic information from all CNVs loci was used to build the CNV-derived genomic relationship matrix (CNV_GRM). CNV loci showing double deletions, single deletions, and normal state in the population were coded as 0, 1, and 2, respectively, whereas CNV loci presenting normal state, single duplications, and double duplications were coded as 0, 1, and 2, respectively. Given that some CNVs presented both deletions and duplications at the same position in the population, these mixed-CNVs were treated as two distinct CNVs loci, one to represent deletions and the other to represent duplications. The variance components for metritis were estimated by fitting either only a SNP-derived GRM (SNP_GRM) or jointly SNP_GRM and CNV_GRM considering 3,272 cows in models that included the female category and farm-year-season as fixed effects. QTLs overlapped with CNVRs were enriched for milk (e.g., milk fat yield, milk yield, and milk protein percentage), reproduction (e.g., calving ease, non-return rate, and heat intensity), exterior (e.g., teat length, udder depth, and udder height), production (e.g., length of productive life, lifetime profit index, and net merit), and health traits (e.g., immune globulin G level, somatic cell, and bovine respiratory susceptibility). Regarding variance components, copy number variations were able to capture a fraction of the additive genetic variance not captured by SNPs alone, increasing the heritability estimate for metritis. In summary, our findings suggest that CNVs overlap QTLs associated with economically important traits more than only by chance and CNVs can capture additional additive genetic variance not fully captured by SNPs alone.

Exploiting human immune repertoire transgenic mice for protective monoclonal antibodies against antimicrobial resistant Acinetobacter baumannii

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era.