bluuebunny/crossref_metadata_2025_split huggingface dataset:
Capturing the Functional Antibody Repertoire from Multiple B-cell Subsets Following Antigen Stimulation
Authors: Kenney John Stephen , Santibanez-Vargas Leonel , Chang Rick , Keshner Emily 
Published: August 2023

Abstract
Antigen stimulation of mature B-cells induces activated B-cells followed by differentiation to either memory B-cells or plasmablasts. Each subset has a unique phenotype and potentially unique immunoglobulin repertoire that can be mined for therapeutic antibody candidates. As new targets for therapeutic antibodies have become more challenging, primary B-cell technologies have arisen to meet this challenge, but, their use has typically been limited to one of the B-cell subsets. We sought to develop a platform that could be used generally to interrogate all of the B-cell subsets (i.e., activated, memory and plasmablasts) that are potential sources of useful antibodies. Using fluorescence activated cell sorting (FACS), we have identified and sorted antigen-specific activated and memory B-cells by staining for CD19, surface IgG, and fluorochrome-tagged antigen. Antigen-specific plasmablasts have been identified and sorted after encapsulation of cells in microdroplets, capture of secreted IgG, CD19/CD138 staining, and fluorochrome-tagged antigen staining. Additionally, hybridomas have been evaluated using either protocol. The sorted cells have been sequenced by next generation sequencing (NGS) of paired heavy and light chains. With this platform ~10,000–20,000 antigen specific B-cells from a population of a few million viable lymphocytes have been sorted in 1–2 hours. From the sorted population, 100 – 700 unique paired clonotypes have been identified using this platform. The platform enables the capture of immunoglobulin repertoires from multiple B-cell subsets that is comprehensive, flexible, and cost-effective.
Executing precision longitudinal flow to differentiate natural variation of immune populations from treatment response
Authors: Scheiding Sheila , Kus Anna , Long Alice , Wiedeman Alice 
Published: August 2023

Abstract
One challenge in assessing modulation of the immune response following immune therapy is understanding natural variation in the same immune populations of specific patient cohorts. Confident interpretation of data requires standardization of procedures and proper use of appropriate controls. Here, we describe the steps taken to address potential sources of technical variability in a 31-parameter flow cytometry panel built for a Type 1 Diabetes study with over 450 samples. This includes titering and bridging antibodies, selection and integration of multiple compensation bead control options, optimizing machine performance through voltage scans and 8-peak bead matching, and post-acquisition FlowJo gating for data cleanup. When applying this process to our large study, we were able to utilize the coefficient of variation (CV) in combination with our internal control sample to show low overall technical variation. Of the 64 frequency and intensity outputs we monitored, 75% had CVs less than 25%. Higher CVs were seen exclusively in low abundance or intensity populations more subject to measurement error. When looking at the natural variation of the populations we monitored, none of the technical variation we noted exceeded the biological variation present in the trial samples. Furthermore, we calculated CVs for each population over time in each individual and averaged those CVs within their cohorts to quickly identify populations of interest with treatment—in this case, modulation of follicular helper cells. Employing this streamlined process produces clean data that can be analyzed confidently and allows for quick discernment of biological changes in subjects with or without immune perturbation across time.
ITN (NIAID) and TrialNet (NIDDK) funding along with BRI internal HIP Core funds
Combined Infection-Inflammation Microsystem to Quantify NETosis
Authors: Jones Caroline N , Datla Udaya Sree 
Published: August 2023

Abstract
Abundant NETosis has been observed in many human pathologies, including in COVID-19 patients. Excessive neutrophil extracellular trap (NET) production is known to lead to unfavorable coagulopathy and immunothrombosis in these patients. Traditional immunoassays employed to quantify NETosis focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NETs with single-cell resolution in the presence bacteria and inflammatory cytokines. The device was engineered to have open chambers and constricted channels to measure NETosis at varying degrees of mechanical confinement. The microsystem enabled us to take time-lapse images of individual primary human neutrophils releasing NETs in clinically relevant microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosa PAO1 infection when challenged with the inflammatory cytokines Tumor Necrosis Factor-α and Interleukin-6 (but not Leukotriene B4), compared to the infection alone. We confirmed reduced NET release in confined channels in the microsystem. We quantified the temporal dynamics and differences in the areas of NETs released, showing a high percentage of large NETs with Pseudomonas-inflammation mediator treatment. Overall, our NET-array device serves as a proof-of-concept model to further our understanding of the spatiotemporal dynamics of NET release to infection in a defined microenvironment. Our NET-array device is now being used for high-throughput and cost-effective screening of novel immunotherapies on human neutrophils.
NIH NIGMS (R35GM133610-01)
Baseline immunotypes reveal weak and strong antibody responders to influenza vaccination
Authors: Cevirgel Alper , Shetty Sudarshan , Vos Martijn , Nanlohy Nening , Beckers Lisa , Bijvank Elske , Rots Nynke , van Beek Josine , Buisman Anne-Marie , van Baarle Debbie 
Published: August 2023

Abstract
Vaccines are currently the best available interventions to protect individuals from infectious diseases. However, immunosenescence, described as immune dysfunction at older age, leads to lower responsiveness to vaccines. We analyzed a broad set of immune cell subsets (n=129) in whole-blood of 308 individuals (aged 25–98) pre- and post-flu vaccination to identify those with decreased vaccine responsiveness. Detailed T, B, NK cell and monocyte subsets were examined and associated with day 28 hemagglutination inhibition antibody titers. None of the 129 immune cell subsets at day 0 associated with antibody titers (FDR adj.p&gt;0.05). Previously, based on 59 immune cell subsets we stratified individuals using an unsupervised approach into 9 immune clusters which we named immunotypes. We identified two specific immunotypes that were associated with either a significantly weak and or a strong antibody response 28 days after vaccination. These weak responders were characterized by a lower CD4+ T naïve/memory (Tn/Tm) cell ratio, lower percentage of non-classical (anti-inflammatory) monocytes and higher number & percentage of HLA-DR+ CD4+ & CD8+ T cells, a clear ageing-related immune signature. In contrast, strong responders showed a higher Tn/Tm ratio and higher percentage of stem cell like memory CD4+ & CD8+ T cells. A weak antibody response was also associated with a lower percentage of follicular HLA-DR+ and follicular CD38+ T cells, and higher CD4+ T effector regulatory cells 7 days after vaccination. Overall, we identified pre-vaccination immunotypes that were associated with distinct vaccine responsiveness profiles that may be useful for future risk profiling
Aging is associated with accumulated T follicular helper cells with decreased metabolic function
Authors: Fisher Jacob S , Lancaster Jessica 
Published: August 2023

Abstract
The aging immune system responds poorly to infection and vaccination, producing reduced numbers of germinal centers, low affinity antibody titers, and reduced B cell memory. Prolonged contacts between activated B cells and CD4+ T cells are crucial to germinal center formation and T follicular helper cell (Tfh) differentiation, but it is unclear how aging impacts the quality of this interaction. Peptide immunization confirmed that aged mice have less expansion of antigen-specific germinal center B cells and reduced antibody titers. Furthermore, aging was associated with decreased CD4+ T conventional cells and accumulated Tfh cells and regulatory T cells, even in naïve mice. Despite increased numbers, our results indicate that aged Tfh have decreased metabolic function, which may have repercussions on germinal center responses. To determine the contribution of cell-extrinsic influences on antigen-specific Tfh induction, we adoptively transferred young, antigen-specific B and CD4+ naïve T cells into aged hosts prior to peptide immunization, which resulted in reduced antigen-specific germinal center B cell and Tfh expansion and reduced antibody titers when compared to young hosts. The results from these experiments emphasize the importance of the lymphoid microenvironment in germinal center initiation and maintenance. We hypothesize that age-associated Tfh accumulation may impair antigen-specific Tfh induction and subsequent germinal center responses. Current studies use live-cell 2-photon microscopy to track the impact of age on lymphocyte motility and Tfh:B interactions, in order to determine the relative contributions of aged APCs, non-specific aged Tfh, and aged regulatory T cells to germinal center deficiency.
Research supported by the Mayo Clinic Robert and Arlene Kogod Center on Aging Innovation Award UL1R002377
Pre-vaccination CD56dim CD16+ NK cell abundance and Th1/Th17 ratio predict responsiveness to conjugated pneumococcal vaccine in older adults
Authors: Ravichandran Sathya Baarathi Shanthi , Diaz Fernando E , Karakaslar Onur E , Marches Radu , Rossi Robert , Nahm Moon H , Chaussabel Damien , Kuchel George , Banchereau Jacques , Ucar Duygu 
Published: August 2023

Abstract
Older adults are at high risk of morbidity and mortality from Streptococcus pneumoniae (pneumococcus) infections. There are two available vaccines for pneumococcus: T-cell-independent capsular polysaccharide Pneumovax and T-cell-dependent conjugated Prevnar. However, how older adults respond to these vaccines at the cellular level and whether there are baseline predictors for responsiveness, is not known. To address this, we recruited older adults (60+ yrs), who are vaccinated with Prevnar (n=19) or Pneumovax (n=20). Vaccine responsiveness was quantified using opsonophagocytic assays, which revealed that both vaccines induce strong responses. Interestingly, sex was associated with Prevnar responses, where women mounted stronger responses than men. Pre-vaccination flow cytometry data showed that Th1 cells positively, Th17 cells negatively correlated with Prevnar responses. Furthermore, bulk RNA-seq data from PBMCs showed that baseline expression levels of cytotoxic genes (NCAM1, GNLY, PRF1) are negatively associated with Prevnar responses. scRNA-seq data from top and bottom responders showed that this cytotoxicity signature stems from CD56dim CD16+ NK cells, where having more of these cells are detrimental to responses. Interestingly, women had significantly higher Th1, lower Th17 and lower CD16+ NK cells compared to men, which explains their stronger Prevnar responses. This is the first study to uncover older adults’ responses to two pneumococcal vaccines; we uncovered an activated immune phenotype of Th and NK cell subsets that impedes responses to Prevnar. Interestingly, this phenotype only affected adjuvanted vaccine responses, providing an opportunity for precision vaccinology for pneumococcus disease.
Supported by grants from National Institute of Health (R35 GM124922, R01 AG052608) and JAX cancer center (JAX-CC).
Characterizing the cellular and humoral immune responses to Tdap during pregnancy compared to non-pregnant controls
Authors: Hall Jesse M , Caution Kyle J , Yuan Yan , Holt Kia , Gupta Yash , Mir Umul , Stafford Cameron , Skoff Tami , Hariri Susan , Pan Xueliang , Worly Brett , Dubey Purnima 
Published: August 2023

Abstract
Maternal Tdap vaccination during pregnancy is recommended to protect infants from pertussis, pre-vaccination. However, it is unclear whether maternal immune responses improve following Tdap booster. We present analysis of immune responses pre- and post-Tdap booster, of pregnant women (Pw) primed by whole cell vaccines, who received Tdap booster during pregnancy, compared to Tdap boosted non-pregnant women (NPw). Tdap antigen specific serum antibody reactivity and avidity increased following booster in both groups. Reactivity and avidity were higher in NPw compared to Pw. Activation-induced marker assay of peripheral blood revealed that pertussis specific Tmem cells increased following Tdap booster in Pw and NPw. After Tdap booster, the percentage of CD4+CD25+OX40+ T cells was approximately 2.5-fold higher in NPw. In contrast, the proportion of CD4+CD25+PDL1+ T cells post-booster were 3-fold higher in Pw compared to NPw, suggesting that Tdap booster may elevate Tregs in Pw. Interim analysis suggests that booster vaccination increases immune responses, although the magnitude and phenotype differ between Pw and NPW. Antibody function, race related immune profiles, and detailed phenotyping of T cell responses are in progress.
Passive antibody transfer from pregnant women to their fetus are maximized after SARS-CoV-2 vaccination irrespective of prior infection
Authors: Lauritsen Cody , Trinh Ivy , Desai Srushti , Clancey Erin , Murrell Amelie , Rambaran Saraswatie , Chandra Sruti , Elliott Debra , Smira Ashley , Stone Addison , Agbodji Ayitevi , Dugas Courtney , Satou Ryosuke , Pridjian Gabriella , Longo Sheri , Ley Sylvia , Piedimonte Giovanni , Norton Elizabeth , Robinson James , Gunn Bronwyn 
Published: August 2023

Abstract
Pregnancy is associated with a higher risk of adverse symptoms and outcomes for most infectious diseases, including SARS-CoV-2. These adverse risks are also observed in neonates. Antibodies provide protection against SARS-CoV-2 infection, and have been reported after maternal vaccination, maternal infection, and through passive transfer from mother to fetus in utero. However, it is unclear whether the magnitude or quality of maternally-derived fetal antibodies differs in the context of infection or vaccination. Here, we evaluated 93 paired maternal and neonatal umbilical cord blood plasma samples collected between October 2020 and February 2022 from a birth cohort of pregnant women from the greater New Orleans area. Histories of SARS-CoV-2 infection or vaccination were obtained. Humoral immunity was profiled for the levels of spike-specific antibodies and induction of antiviral humoral immune functions, including neutralization and Fc-mediated innate immune effector functions. We found that SARS-CoV-2 vaccination or infection during pregnancy increased the levels of antiviral antibodies from naïve subjects. Importantly, vaccinated mothers and cord samples had the highest anti-spike antibody levels and antiviral function independent of the time of vaccination during pregnancy. These results show that the most effective passive transfer of functional antibodies against SARS-CoV-2 in utero is achieved through vaccination, highlighting the importance of vaccination in pregnant women.
Supported by grants from NIH (NCI U54CA260581-02, NHLBI Grant HL-061007) and USDA (APHIS NBAF NSTP)
SARS-CoV-2 Vaccine-Elicited Antibody Responses in Breastmilk
Authors: Williams Caitlin , Gross Mackensie , Herbek Savannah , Permar Sallie , Yang Yawei 
Published: August 2023

Abstract
SARS-CoV-2 mRNA-LNP immunizations significantly reduce severe COVID19. However, our understanding of the immune response within the context of pregnancy and breastfeeding is limited. The overarching goal of this study is to delineate the antibody kinetics, binding breadth, and neutralization capacity of SARS-CoV-2 mRNA-LNP vaccine-elicited antibodies within the breastmilk compartment and compare to serum
All participants had detectable IgG in breastmilk. The median serum: breastmilk fold difference of SARS-CoV-2 Spike-specific IgG is 368.2 and 583.51 higher in serum than milk after a first and second dose of vaccine respectively. Concentration of Spike-specific IgG is strongly correlated between serum and milk throughout the study period (Spearman r=0.9429). Although there were no self reported cases of COVID19, 4/13 participants had anti-Nucleoprotein (anti-N) IgG. In participants with anti-N IgG above the cutoff. Vaccination against SARS-CoV-2 induced a 4-fold higher median plasma concentration of anti-Spike IgG compared to participants with no N protein reactivity. The Spike specific IgG in breastmilk was 7-fold higher in participants with anti-N compared to those without anti-N IgG. Pseudovirion neutralization increased with a second dose, and there was a 13-fold median decrease in neutralization at 6 months post dose 1.
Post-partum immunization with a SARS-CoV-2 mRNA-LNP vaccine elicits a robust IgG response in serum which is transferred to breastmilk. Serum was able to neutralize pseudovirus after two doses, but neutralization waned in participants without any history of infection. We plan to continue to characterize these antibodies by conducting breastmilk neutralization assays, against the omicron variant
NIH: 1 R01-AI161008
Naive phenotype antigen-specific CD4+ T cells persist in the memory repertoire after vaccination
Authors: Bartolo Laurent , Xu Ruozhang , Pan Yi-Kan 
Published: August 2023

Abstract
Immunization leads to activation and differentiation of antigen-specific T cells, resulting in the development of immunological memory. However, how the human immune repertoire changes after vaccination remains incompletely understood. Here, we used the live attenuated yellow fever virus (YFV) vaccine or the SARS-CoV-2 mRNA vaccine as models to track post-vaccine kinetics in healthy donors. We quantified and characterized 36 distinct YFV-specific T cell populations and 25 SARS-CoV-2-specific populations by direct ex vivo peptide-MHC tetramer staining. Unexpectedly, we found naïve-phenotype vaccine-specific T cells reappearing after vaccination. On average, 20% of T cells retained a naive phenotype by CD45RO and CCR7 staining although each population had previously expanded after vaccination. A subset of naïve-like T cells expressed other differentiation markers consistent with T memory stem cells (TSCM), but some naïve-like T cells were negative for all memory markers tested. These naïve-phenotype T cells were able to respond to antigens and showed an increased expression of a recent thymic immigrant marker CD31. In conclusion, our results provided the surprising finding that the post-exposure T cell repertoire contains naïve antigen-specific T cells. We plan to test the possibility that the thymus continues to add new T cells into the adult immune repertoire in future experiments.
NIH (R01 AI134879) NIH (R01 AO66358)
Differential dengue antibody responses in recipients of a Zika DNA vaccine are governed by dengue immune history
Authors: Raimundi Guillermo Luis , Lowman Kelsey Elizabeth , Mpingabo Patrick , Katzelnick Leah 
Published: August 2023

Abstract
Dengue virus (DENV 1–4) and Zika virus (ZIKV) are closely related flaviviruses that induce immunological cross-reactivity. The VRC5283 Zika virus wild type DNA vaccine is the first to complete a Phase 2b trial (NCT03110770). The objective of this study was to test if VRC5283 induced potent ZIKV-specific neutralizing antibodies (NAbs) while avoiding priming of cross-reactive antibodies that could potentially enhance dengue disease. Here, we measured NAbs to DENV1–4 and ZIKV in serum samples obtained from individuals living in dengue-endemic (Puerto Rico) and non-endemic areas (Texas) immunized with VRC5283 at weeks 0, 4, and 8. We performed plaque reduction neutralization tests (PRNT50) with sera collected at weeks 0 and 16 after final vaccination. Sera were tested for ZIKV isolated in Brazil, and DENV1–4 isolated in Sri Lanka. Most individuals from dengue-endemic areas had NAbs &gt;1:20 to DENV1–4 and ZIKV prior to vaccination, with the highest NAb titers to DENV2. VRC5283 boosted immunity across all flaviviruses in the dengue-endemic cohort, with an 8.47-fold increase in ZIKV, 2.76-fold increase in DENV1, 1.67-fold increase in DENV3 and 2.06-fold increase in DENV4 titers, but did not significantly boost DENV2 titers. Sera from non-endemic areas had no NAbs to DENV1–4 or ZIKV prior to vaccination, and a boost in NAbs following vaccination was observed only to ZIKV. Thus, VRC5283 only induces dengue cross-reactive NAbs in individuals with prior dengue immunity. Ongoing testing with binding assays will further evaluate whether vaccination induced beneficial or enhancing antibodies to DENV1–4.
This work was supported by the Intramural Research Program of NIAID.
Identification and characterization of proteome-wide T cell epitopes from Mtb in active tuberculosis infection
Authors: Panda Sudhasini , Morgan Jeffrey , Peters Bjoern , Arlehamn Cecilia Lindestam 
Published: August 2023

Abstract
Tuberculosis caused by Mycobacterium tuberculosis is one of the leading causes of death from a single infectious agent. Identifying dominant epitopes and comparing their reactivity in different states of tuberculosis (TB) infection can help in designing both diagnostics and vaccines. We performed proteome-wide screen of 21,220 Mtb derived peptides in 21 mid-treatment active TB patients using IFN-γ fluorospot assay. The overall breadth of IFN-γ responses in active TB recognized an average of 10 individual epitopes. In total, we identified 175 individual epitopes, and 37 of those (21.1%) were recognized by 2 or more individuals. These responses were predominantly directed against antigens part of the cell wall and cell processes category. We also found differential reactivity in active TB and LTBI cohorts in terms of clusters of antigens recognized and recognition of TB vaccine candidate antigens. Interestingly, we have identified 9 novel antigens, which have not been described as antigens for Mtb in the past. Additionally, we created a pool of peptides only recognized by individuals with active TB. This active-specific pool has shown higher T cell response in a new active TB cohort compared to individuals with LTBI suggesting a better pool for distinguishing these disease states. Therefore, identifying disease state-specific epitopes and antigens will help in the development of diagnostics, vaccine candidates and correlate of protection.