bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous detection and characterization of antigen-specific B cells and CD4+ and CD8+ T cell responses upon natural infection and vaccination
Authors: Inekci Dilek , Hansen Bjarke Endel , Brix Liselotte
Published: August 2023

An important lesson learned from the COVID-19 pandemic is the need for a more comprehensive assessment of the immune status upon a natural infection and vaccination. In addition to measuring antibody titers, monitoring the status of antigen-specific B and T cell must be taken into consideration to gain a more complete understanding of the elicited immune response and its durability.

The objective of this study was to develop an immune monitoring assay based on the Dextramer® technology for simultaneous monitoring of antigen-specific B and T cells (CD4 and CD8). The assay can be applied in combination with flow cytometry and in single cell RNA-seq analysis.

The assay builds on Dextramer® reagents displaying full-length spike protein (specific B cell assessment) or MHC-peptide complexes displaying virus peptides (detecting specific CD8 or CD4 T cells).

The assay was evaluated by longitudinal assessment of immunity in individuals vaccinated against SARS-CoV-2 by combining the assay with flow cytometry (n=5) and the Chromium 10× RNA-seq platform (n=2). The latter allows an in-depth view of the immune status by simultaneous investigation of the transcriptome, surface proteins and receptor sequences of antigen-specific cells.

Our results show that by using the virus-specific Dextramer® assay we could monitor changes in SARS-CoV-2-specific B- and T-cell responses before and after primary vaccination and booster doses using flow cytometry and single cell RNA-seq analysis. These results show that the Dextramer® assay can aid in understanding the elicitation and persistence of antigen-specific responses. Moreover, the assay can easily be tailored to any antigen-specificity by replacing the ligands coupled to the Dextramer® reagents.
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

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

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

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

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>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

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

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

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

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

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

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

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 >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

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.
The effect of neutrophils on limited hepatitis C virus vaccine immunogenicity in people who inject drugs
Authors: Roznik Katerina , Rousseau Kimberly E , Thompson Elizabeth A , Cox Andrea L
Published: August 2023

In the only efficacy trial of a hepatitis C virus (HCV) vaccine, the vector-based vaccine lowered peak HCV RNA levels in people who inject drugs (PWIDs) but did not prevent chronic HCV infection. The same vaccine regimen tested in healthy controls (HCs) resulted in a larger percentage of individuals responding to vaccination (100 vs. 78%) and a 3-fold higher magnitude of T cell responses compared to PWIDs. We evaluated baseline immune characteristics of PWIDs to understand the reduced responses, ruling out differences in baseline anti-vector immunity, subclinical HCV exposure, and increased Tregs. However, we found markedly elevated neutrophil counts in PWIDs at baseline compared to HCs. Elevated neutrophils were previously linked to nonresponse to hepatitis B vaccination in HCs, yet this phenomenon has been largely understudied. We analyzed baseline neutrophil phenotypes using flow cytometry and identified elevated PD-L1, CD11b, and CD63 on neutrophils from PWIDs compared to HCs. Stimulation of HC neutrophils with opioid receptor (OR) agonist DAMGO significantly upregulated PD-L1 and CD11b, an effect partially reversed with an OR antagonist. We also observed metabolic reprogramming of neutrophils in PWIDs compared to HCs, with higher expression of glycolytic machinery (GLUT1/HK2) and mitochondrial membrane protein VDAC1, indicative of increased mitochondrial mass and use of oxidative phosphorylation. Elevated baseline CD11b and CD63 suggest dysregulation and activation of neutrophils, with increased PD-L1 potentially contributing to poor T cell responses in PWIDs. Mechanistic studies of dysregulated neutrophil interactions with T cells may provide insights into vaccine-induced responses and future vaccine efforts.

Supported by NIH grant U19 AI159822.
Determining antigen-specific T cell phenotypes in cases of Hepatitis C virus clearance versus chronic infection
Authors: Stavrakis Georgia , Rousseau Kimberly , Thompson Elizabeth , Cox Andrea L
Published: August 2023

Globally, approximately 1.5 million new Hepatitis C virus (HCV) infections occur annually. A vaccine to prevent chronic HCV infection would limit HCV transmission and pathogenesis of HCV. To understand immunity to HCV infection in high risk populations, we prospectively followed HCV antibody negative high-risk people who inject drugs (PWIDs) in a protocol designed for monthly follow-up. We demonstrated previously that HCV reinfection in PWIDs is associated with a reduction in the magnitude and duration of viremia versus the initial infection and broadened cellular immune responses, consistent with protective immunity against chronic disease. We have characterized CD8+ HCV-specific T cells from the acute phase of HCV using high dimensional spectral flow cytometry to define the simultaneous expression of 25 intracellular metabolic and cell surface molecules. We found that HCV-specific CD8+ T cells from those who progress to chronic infection have a unique acute phase phenotype that clusters separately from those who later successfully control HCV. Acute phase CD8+ HCV-specific T cells from those who subsequently clear an initial HCV infection show increased expression of CD127 and CXCR3 and decreased expression of GLUT1, PD-1 and CD39 compared to acute phase T cells in those who later progress to chronic infection. Understanding HCV specific T cell phenotypes in successful control of HCV infection and reinfection will guide goals for vaccine induced immunity to prevent chronic HCV infection.

Supported by grants from the NIH (U19 AI159822)
TNFR2-targeting peptide for cancer immunotherapy
Authors: Chen Xin , Liao Ping , Chen Zhonghao
Published: August 2023

There is compelling evidence that tumor necrosis factor receptor type II (TNFR2) is preferentially expressed by highly immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in tumor-infiltrating lymphocytes (TILs). These findings led us and others to propose and experimentally proved that targeting TNFR2 with antibodies could induce anti-tumor immune responses. In this study, we identified a TNFR2-targeting peptide P20 through phage display. P20 had in vitro activity to block TNF-TNFR2 interaction and inhibit TNFR2-mediated activation/expansion of Treg cells. The in vivo effect of P20 on tumor growth was examined with mouse tumor models including CT26 colon cancer and OVA-EG7 lymphoma. The results showed that the treatment with P20 (i.p.) resulted in a marked inhibition of tumor growth, accompanied by a reduced number of Tregs and an increased number of IFN-γ+ CD8+ cytotoxic T lymphocytes in the tumor microenvironment. Furthermore, P20 could markedly enhance the efficacy of anti-PD-L1 in the inhibition of tumor growth in a syngeneic MC38 mouse colon cancer model. Thus, P20 may be a useful anti-tumor immunotherapeutic agent and merits further investigation. (Funded by FDCT 0099/2021/A2, UM MYRG2022-00260-ICMS, UM CPG2023-00025-ICMS)

There is compelling evidence that tumor necrosis factor receptor type II (TNFR2) is preferentially expressed by highly immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs), especially those present in tumor-infiltrating lymphocytes (TILs). These findings led us and others to propose and experimentally proved that targeting TNFR2 with antibodies could induce anti-tumor immune responses. In this study, we identified a TNFR2-targeting peptide P20 through phage display. P20 had in vitro activity to block TNF-TNFR2 interaction and inhibit TNFR2-mediated activation/expansion of Treg cells. The in vivo effect of P20 on tumor growth was examined with mouse tumor models including CT26 colon cancer and OVA-EG7 lymphoma. The results showed that the treatment with P20 (i.p.) resulted in a marked inhibition of tumor growth, accompanied by a reduced number of Tregs and an increased number of IFN-γ+ CD8+ cytotoxic T lymphocytes in the tumor microenvironment. Furthermore, P20 could markedly enhance the efficacy of anti-PD-L1 in the inhibition of tumor growth in a syngeneic MC38 mouse colon cancer model. Thus, P20 may be a useful anti-tumor immunotherapeutic agent and merits further investigation
Blocking Respiratory Syncytial Virus infection utilizing decoy ICAM-1 receptor proteins
Authors: Geanes Eric S , McLennan Rebecca , Khanal Santosh , Bradley Todd
Published: August 2023

Respiratory syncytial virus (RSV) infections result in a significant number of hospitalizations for young children and the elderly each year, and current infection rates continue to rise, leading to a substantial public health burden. While numerous vaccine candidates are in clinical trials, there is currently no approved RSV vaccine to prevent infection or antiviral treatment after infection. There is an urgent need to find new therapeutics and strategies that reduce severe RSV disease that could improve health and save lives. The RSV fusion (F) protein is critical for binding host cells and mediating infection and is the target of most candidate vaccines. The RSV F protein has been shown to interact with several host-cell surface receptor proteins such as Intercellular Adhesion Molecule 1 (ICAM-1), NCL, EGFR, IGF1R, and infect respiratory epithelial cells. In this study, we confirmed ICAM-1 could bind RSV F protein and inhibit RSV infection via an RSV neutralization assay. Using RSV F protein as bait, we immunoprecipitated RSV F interacting proteins from A549 respiratory cells and performed mass spectrometry analysis to identify novel host proteins that bind RSV F protein. Finally, we engineered a mRNA-lipid nanoparticle (LNP) expressing soluble ICAM-1. The goal is to utilize the ICAM-1 mRNA-LNP as a passive intranasal vaccine that would result in over-expressed soluble ICAM-1 in respiratory mucosal surfaces to function as a decoy receptor that would reduce RSV infection. We are evaluating this therapeutic approach in preclinical models as a prophylactic or treatment after infection. ICAM-1 mRNA-LNP may provide a novel defensive strategy to reduce RSV infection or disease severity and augment future RSV vaccines that become approved.

Children’s Mercy Research Partners
Oral vaccination with live attenuated pathogenic bacteria protects immunocompromised hosts from lethal colitis
Authors: Wang Shuyu , Wan Fengyi
Published: August 2023

Enteropathogenic Escherichia coli(EPEC) and enterohemorrhagic E. coli(EHEC) are significant causative agents for diarrheal diseases globally. Vaccination has been deemed as the most effective strategy for preventing EPEC and EHEC caused foodborne illnesses; however, FDA has not yet licensed any vaccine against EPEC or EHEC. Of note, live attenuated bacterial vaccine strategy has been scarcely investigated for against EPEC or EHEC infection, in particular in immunocompromised populations where the infections are associated with severe symptoms and outcomes. Infection with Citrobacter rodentium(CR), the murine equivalent of EPEC and EHEC, in mice has been widely used to develop novel vaccine strategies and evaluate vaccination efficacy. CR infection in immunocompromised mice such as interleukin-22 knockout (Il22−/−) mice leads to severe symptoms and mortality, which mirrors the severe outcomes of EPEC and EHEC infections in immunocompromised populations. We explored whether oral inoculation with live CR mutants with rationally attenuated virulence could protect lethal infection in susceptible Il22−/− mice. We showed that oral administration of live attenuated CR strains elicited efficient systemic and humoral immunity against variant CR virulence proteins, hence protecting otherwise lethal CR infection, in Il22−/− mice. Our findings provide proof of concept evidence for live attenuated vaccination strategy to prevent EPEC/EHEC infections in immunocompromised hosts associated with more severe symptoms and lethality, and reveal novel CR-specific antibody subtypes and virulence determinants that are critical for live attenuated vaccination offered immune protection.
Vaccine antigen archiving by lymphatic endothelial cells boosts archived antigen-specific CD8 T cell memory responses during a pathogenic challenge
Authors: Doan Thu A , Sheridan Ryan , Forward Tadg , Tamburini Beth
Published: August 2023

Numerous studies have shown viral antigens can persist in lymph nodes after the resolution of the infection. We demonstrated that lymphatic endothelial cells (LEC), which comprise the lymphatic vasculature necessary for antigen drainage from the tissue, are the predominant cell type required for the persistence of both vaccine and viral antigens within the lymph node. We termed this process antigen archiving due to the ability of LEC to actively archive antigens to which an immune response has occurred. This process involves antigen acquisition, retention, and exchange of the antigen between LECs and dendritic cells (DC), resulting in the presentation of the archived antigens to archived antigen-specific CD8 memory T cells. In this study, we evaluated the effect of LEC archived antigen on archived antigen-specific memory T cells during a pathogenic challenge. We predicted that a pathogenic challenge would result in the release of the archived antigen and subsequent archived antigen presentation. Following a pathogenic challenge, we found a significant increase in archived antigen-specific endogenous or transferred memory CD8 T cells, compared to bystander activation seen by memory T cells of an unrelated specificity. The phenotype of the archived antigen-specific memory CD8 T cells was more effector-like and led to a significant increase in protection against Listeria monocytogenes (LM)-expressing the archived antigen, in the local area. In conclusion, these findings suggest that archived antigen may be released during a pathogenic challenge. As such, the archived antigen may be acquired by migratory DCs as they traffic through the lymphatics in order to present the archived antigen to memory CD8 T cells acting as an effective boost.

NIH R01 AI121209
The mature c-terminal domain of filamentous hemagglutinin is sufficient to provide protection against Bordetella pertussis in the murine nasal cavity
Authors: Pyles Gage Michael , Weaver Kelly , Witt William , Huckaby Annalisa , Mateu-Borras Margalida , Damron Fredrick , Barbier Mariette
Published: August 2023

Bordetella pertussis is the causative agent of whooping cough. The transition from whole cell (wP) to acellular pertussis vaccines (aPs) triggered an increase in pertussis incidence despite wide-spread vaccine coverage. aPs contain protein antigens purified from B. pertussis: pertactin, fimbriae, filamentous hemagglutinin (FHA), and pertussis toxin (PT). Antibodies are critical for protection against B. pertussis, and have been demonstrated to trigger opsonization, block bacterial adhesion, and neutralize disease symptoms. Following aP-vaccination, however, antibodies target a diminished number of functional B cell epitopes as the antigens are chemically denatured during aP manufacturing. The effects of denaturation on PT are well documented, but this has not been studied for other vaccine antigens, such as FHA. Additionally, FHA is a large antigen, and most of the protein does not play a role in bacterial adhesion. To direct antibody responses toward non-denatured, functionally relevant portions of FHA we aimed to test the Mature C-terminal Domain (MCD) of FHA as vaccine antigen. Protection provided by MCD was compared to several vaccine preparations, including aP and native FHA. We observed that aP and wP vaccines lead to the induction of antibodies that recognize FHA, MCD and B. pertussis. Using a CD-1 murine model of vaccination and challenge we demonstrated that MCD is immunogenic and sufficient to decrease bacterial burden in the murine airway, providing ~85% reduction in B. pertussis present in the nasal cavity. Overall, our data supports the use of MCD as a vaccine antigen for protection against B. pertussis. Ongoing studies focus on the selection of the adjuvants and route of administration to optimize vaccine efficacy.

Mariette Barbier is funded by NIH grant 5R01AI141671-04. Fredrick Damron is funded by NIH grant 5R01AI137155-04. The Vaccine Development Center Research Challenge Grant (no. HEPC.dsr.18.6) from the Division of Science and Research, WV Higher Education Policy Commission also contributed support.
Mucosal chemokine adjuvant supports heterologous protection against Delta and Omicron VOCs in the context of a SARS-CoV-2 wild-type DNA vaccine
Authors: Gary Ebony Nicole , Tursi Nicholas J , Warner Bryce M , Parzych Elizabeth M , Ali Ali R , Frase Drew , Moffat Estella , Embury-Hyatt Carissa , Smith Trevor R F , Broderick Kate E , Humeau Laurent , KobasA Darwyn , Patel Ami , Kulp Daniel W , Weiner David B
Published: August 2023

Emerging variants of concern (VOCs) and waning vaccine-induced immunity challenge SARS-CoV-2 control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces could enhance vaccine efficacy and potentially reduce transmission. We immunized mice with DNA plasmids expressing a wild-type SARS-CoV-2 spike protein (pS) alone, or with the DNA-encoded mucosal chemokine adjuvant cutaneous T cell-attracting chemokine (pCTACK). Mice co-immunized with pS and pCTACK exhibited increased spike specific IFNy T cell responses compared to those receiving the pS alone. While both groups showed similar levels of spike receptor binding domain (RBD)-specific IgG in their serum, in the mucosa, co-immunization with pCTACK enhanced RBD-specific IgA, and cecal extracts from pCTACK co-immunized animals neutralized pseudotyped viruses. When ACE2 transgenic mice were immunized, rested for 3 months, and challenged with a heterologous SARS-CoV-2 Delta variant, co-immunization with pCTACK supported 100% survival while immunization with wild-type pS antigen alone supported 60% survival. Enhanced protection from morbidity was also observed as 100% of pCTACK co-immunized animals did not exhibit infection-induced weight loss. When animals were immunized similarly and challenged with Omicron BA.2, pCTACK co-delivery resulted insignificantly decreased viral loads compared to pS-only immunization. These studies demonstrate that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites with significant impact on heterologous challenge. These data have significant implications for the design of improved vaccines targeting SARS-CoV-2.

This work was supported by NIH NCI award T32 CA09171 (ENG) and NIH NIAID award T32-AI-055400 (EMP) and The Wistar Institute coronavirus discovery fund with additional support from CEPI/Inovio Pharmaceuticals.
Acute and chronic viral infection generate brain-resident memory T cells with distinct phenotypic, functional, spatial, and metabolic profiles
Authors: Mix Madison R , Epping Lecia L , Hancox Lisa S , Merrill Ronald A , Taylor Eric B , Badovinac Vladimir P , Harty John T
Published: August 2023

The rising incidence of post-viral neurological sequelae represents a substantial burden on global public health. Therefore, there is an urgent need to investigate alterations in brain immune composition after acute and chronic viral infection. While virus-specific tissue-resident memory (TRM) CD8+ T cells are observed in human health and neurological disease, these populations remain understudied in the brain. Here, we leveraged the comparative model system of lymphocytic choriomeningitis virus (LCMV) strain Armstrong vs. Clone 13 infection in mice to generate brain TRM with quiescent and exhausted profiles respectively. We then interrogated the 1) phenotype, 2) functionality, 3) localization, and 4) metabolism of anti-viral TRM in the brain. Importantly, brain TRM exhibit higher expression of exhaustion markers (PD-1, LAG-3, TIM-3, TOX) compared to T circulating memory (TCIRCM) cells in the same host. Furthermore, brain TRM in chronically infected hosts are functionally impaired in cytokine production and cytolytic capacity. Anti-viral brain TRM seed distinct neuroanatomical sites, such as the cerebrospinal fluid, parenchyma, and choroid plexus, often adopting aggregate-like structures in solid compartments. Compared to TCIRCM, quiescent and exhausted brain TRM also demonstrate higher expression of surface amino acid transporters with disparate amino acid uptake. These findings are contextualized by whole brain metabolomic profiling indicating global perturbations in amino acid metabolism post-acute and chronic viral infection. Altogether, these data highlight brain-intrinsic memory T cell signatures and offer broad insight into how brain TRM may influence post-viral neurological outcomes.

Supported by grants from the NIH (AI042767, AI114543, AI167847, 5T32GM007337) and the University of Iowa Post-Comprehensive Research Fellowship.
Rhesus macaque cytomegalovirus deploys viral Fcγ receptors to evade antibody-mediated cellular immunity
Authors: Otero Claire E , Petkova Sofia , Taher Husam , Scheef Elizabeth , Moström Matilda , Sprehe Lesli , Kendall Savannah , Papen Courtney , Früh Klaus , Kaur Amitinder , Permar Sallie , Hengel Hartmut , Malouli Daniel , Kolb Philipp
Published: August 2023

Cytomegalovirus (CMV) is a top priority in vaccine development due to the high burden of disease in immunocompromised populations and congenitally infected infants. Evidence suggests that CMV-specific Fc-mediated antibody effector responses, such as antibody dependent cellular phagocytosis and cytotoxicity, are key for prevention of multiple CMV disease outcomes. However, human CMV expresses three Fcγ receptors that have been shown in vitro to synergistically interfere with anti-CMV IgG engagement of host Fcγ receptors (FcγRs). We recently identified three functionally homologous viral Fcγ receptors (vFcγRs) in rhesus macaque (RM) CMV (RhCMV), which allow us to validate the function of vFcγRs in vivo. Using a full-length (FL) RhCMV backbone (Taher, et al. 2020), we produced a mutant RhCMV lacking all identified vFcγRs (ΔvFcγR) and compared infection of CMV-seronegative male RMs with ΔvFcγR RhCMV (n=4) to FL RhCMV (n=3). RMs infected with ΔvFcγR and FL RhCMV experienced similar peak DNAemia levels 1 week post infection but ΔvFcγR RhCMV DNAemia was contained by week 3 while FL RhCMV remained detectable in plasma through week 9. The observed early control of infection is concurrent with the development of RhCMV-specific IgG responses which peak 3–4 weeks post infection in both groups. The kinetics of RhCMV-specific binding and neutralizing antibody responses were similar between the two groups of RMs, suggesting that anti-RhCMV Fc-mediated antibody effector responses may have been more effective against the RhCMV deleted of vFcγRs. Thus, vFcγRs are promising candidates for vaccine immunogens to improve Fc mediated effector responses and efficacy to reduce incidence of CMV-associated birth defects and transplant complications.

Supported by grants from NIH P01-AI129859 and P01-AI129859_S1, NIH T32-CA009111, National CMV Foundation ECRA
T-bet controls the maintenance and differentiation potential of flu-induced effector memory B cells
Authors: Risley Christopher A , Schultz Michael D , Allie S Rameeza , Peel Jessica N , Liu Shanrun , Fucile Chris , Scharer Christopher D , Rosenberg Alex , Boss Jeremy M , Randall Troy D , Lund Frances E
Published: August 2023

Although the transcription factor (TF) T-bet is expressed by antigen-experienced human B cells, including the extrafollicular IgDnegCD27neg memory-like DN2 cells and the IgDnegCD27+ effector memory cells (eBmem), it is unknown whether T-bet expression influences the development, maintenance, or function of Bmem cells. Using oligo-labeled recombinant influenza nucleoprotein (NP) tetramers and paired single cell RNA and V(D)J sequencing, we characterized NP-specific Bmem in the draining LN of flu-infected memory mice. We identified 7 transcriptionally distinct clusters of mature non-replicating β Bmem cells. While none of the Bmem clusters expressed TFs required for plasma cell (PC) lineage commitment, one cluster, containing clonally-expanded somatically-mutated Bmem cells, expressed significantly higher levels of the T-bet gene, Tbx21, and was transcriptionally very similar to human T-bet+ eBmem. The NP+ Tbx21+ Bmem upregulated gene networks associated with metabolic reprogramming, protein synthesis and the mTOR-dependent unfolded protein response, suggesting that these cells might represent Bmem that are metabolically poised for rapid PC differentiation. Using constitutive and inducible models to delete Tbx21 specifically in B cells we showed that the rapid differentiation potential of Bmem is controlled by T-bet and that T-bet is required for the development and persistence of NP+ eBmem subsets in LN and lung. Thus T-bet not only marks Bmem with effector potential but also regulates the persistence and function of flu-induced NP+ Bmem that are transcriptionally poised to provide rapid humoral protection from infection.

Supported by a grant from the NIH (R01 AI110508)