bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of extracellular vesicles in AIDS-associated non-Hodgkin lymphoma
Authors: Hegde Priya , Martínez Laura E , Magpantay Larry , Detels Roger , Hussain Shehnaz , Epeldegui Marta
Published: August 2023

Even with the use of anti-retroviral therapy, HIV+ subjects develop non-Hodgkin lymphoma (NHL) at a higher frequency than uninfected subjects. Extracellular vesicles (EVs) secreted by cells may contain immunomodulatory molecules; for example, tumor cells can secrete EVs containing bioactive PD-L1 that can inhibit anti-tumor responses. We have shown EVs bearing PD-L1, TNF-RII or IL-6Rα can be biomarkers of NHL risk. Here we aim to characterize EVs isolated from EBV+ and EBV− lymphoma cell lines to define their involvement in lymphomagenesis and identify more EV-associated biomarkers for NHL risk (like immune checkpoint molecules CD80, CD86, CD28). EVs were isolated from AIDS-NHL and NHL cell lines using a total exosome isolation kit and analyzed by Western blot. A subset of these were also analyzed by proteomics using Metascape, Reactome, and R. EVs were also isolated from archived serum of HIV+ individuals before NHL onset (n = 51) and matched HIV+ controls (n = 53) from participants of the MACS/WIHS Combined Cohort study, with a total exosome isolation kit and characterized by Western blot. EV-specific markers were detected in EVs isolated from cell lines and serum. EVs isolated from EBV+ cell lines largely expressed LMP1, an EBV CD40 homolog that induces EBV-driven B cell transformation. In EVs isolated from serum, levels of CD80, CD86, or CD28 were comparable between pre-NHL samples and matched controls, though control samples tended to have greater CD80 and CD86 expression. Our findings shed light on the EV marker expression profiles of AIDS-NHL and NHL cell lines and show LMP1 expression in EBV+ cell lines. Additionally, this research provides initial insight into the complex composition of EVs and their role in lymphomagenesis.

Supported by grants from NIH (R01CA228157, P30CA016042-S, R01CA228157S)
Studying NF-κB signaling during neuroinflammation in Alzheimer’s Diseases in vitro using hiPSC-derived brain models
Authors: Songkiatisak Preeyaporn , Aqdas Mohammad , Md Toufiqur Rahman Shah , Oh Kyu-Seon , Sung Myong-Hee
Published: August 2023

Neuroinflammation is a key pathological driver of various neurological diseases, including Alzheimer’s disease (AD). NF-κB signaling pathway is widely recognized as a hallmark of inflammation and cellular senescence in which aged microglia may undergo cellular senescence partly influenced by NF-κB. Our previous data indicated that ex vivo microglia comprised of two distinct subpopulations distinguished by morphology and motility. Most microglia cells formed a tight cell cluster, termed “clustered microglia”. The rest was a subpopulation of microglia with smaller cell size and high motility, termed “free-roaming microglia”. In microglia from aged animals, the composition of clustered versus free-roaming subsets was shifted toward a higher prevalence of free-roaming microglia where c-Rel is expressed and the canonical NF-κB signaling is more sustained. To study NF-κB signaling in the context of neuroinflammation, we are developing 3D models, using hiPSC neurons derived from healthy donors of various age groups and co-culturing them with primary microglia from knock-in mice in which their endogenous c-Rel was labeled with a fluorescent protein. Moreover, individuals with APOE-e4 genotype have an increased risk of developing AD. To develop 3D brain models for AD, we will use co-culture of microglia and neurons derived from hiPSCs of APOE-e4 carriers. Live cell imaging and biomarker assessment of microglia and neurons activated by Amyloid β (1–42) will uncover important molecular mechanisms associated with age-dependent interactions between neurons from healthy versus AD-risk individuals, and resident macrophages of the young and aged brains.
An uncommon presentation of primary sclerosing cholangitis leading to the diagnosis of B cell lymphoma and common variable immunodeficiency
Authors: Cox Katie , Miller Holly , Wadera Sheetal , Goode Erin
Published: August 2023

Common Variable Immunodeficiency (CVID) is a disorder of B cells, with hypogammaglobulinemia and an impaired immune response leading to increased risk of infection, autoimmune complications, and risk of malignancy.

A 17-year-old, previously healthy male presented with three months of pruritus, jaundice and transaminitis. Imaging and biopsy showed liver heterogeneity without evidence of cholestasis or ductal dilation. Over time, imaging was consistent with primary sclerosing cholangitis (PSC). The patient eventually required liver transplant after several complications, despite stent therapy.

Explant liver pathology revealed diffuse large B cell and follicular lymphoma, grade 3A with mature monoclonal B cells follicular in origin, negative for MYC rearrangement and MUM1. PCR detected IgH and IgK B cell clonality. He received chemotherapy without complication, for primary hepatic non-Hodgkin’s lymphoma.

Continued flatulence and diarrhea prompted endoscopy, revealing eosinophilic esophagitis. Due to his variable pathology, he was evaluated for a primary immunodeficiency. His labs demonstrated globally diminished immunoglobulins, a low Hib titer and one pathogenic mutation in TNFRSF13B (c.542C>A; p.Ala181Glu), a transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI). Thus, the patient was diagnosed with CVID.

This case highlights a unique presentation of CVID from demonstration of PSC and primary hepatic diffuse large B cell lymphoma. Consideration of immunodeficiencies early on in challenging cases of autoimmune and unique oncologic diagnoses may lead to earlier treatment of underlying conditions and prevention of future complications.
Structural insights into human monoclonal antibodies with pan-coronavirus reactivity
Authors: Barnes Christopher O , Abernathy Morgan
Published: August 2023

Human monoclonal antibodies from convalescent individuals that target the SARS-CoV-2 spike protein have been deployed as therapeutics against SARS-CoV-2. However, the emergence of SARS-CoV-2 variants have diminished the efficacy of almost all antiviral monoclonal antibodies. Moreover, most SARS-CoV-2 specific antibodies are inactive against divergent sarbecoviruses and the larger Orthocoronavirinae subfamily. Thus, continued development of immunotherapies resilient to SARS-CoV-2 viral evolution, and capable of recognizing zoonotic coronaviruses with spillover potential is necessary. Using coldspot-guided antibody discovery, a screening approach that focuses on portions of the virus spike glycoprotein that are both functionally relevant and averse to change, we identified human neutralizing antibodies to highly conserved viral epitopes. We identified and structurally-characterized neutralizing antibodies to the fusion peptide, to the stem helix near the heptad repeat 2 region and to subdomain 1 of the spike glycoprotein, that are broadly cross-reactive and protective in vivo. Collectively, our data adds to the growing body of evidence suggesting the potential use of broadly neutralizing antibodies for prophylaxis or therapy against emerging SARS-CoV-2 VOC and future zoonotic spillover events.

C.O.B. is supported by the Howard Hughes Medical Institute Hanna Gray Fellowship and is a Chan Zuckerberg Biohub investigator.
Single-cell characterization of circulating and synovial immune cells reveals a unique dendritic cell landscape and enrichment of granzyme K+ T cells in Psoriatic Arthritis
Authors: Mullins Briana J , Herrera Alberto , Chang Anthony , Yang Katharine Lu , Girija Parvathy V , Hernandez Anthony Jimenez , Alvarado Luz , Medina Rhina , Scher José , Koralov Sergei B
Published: August 2023

Psoriatic Arthritis (PsA) is an immune mediated disease of the joint. It typically develops within 10 years after the onset of psoriasis, with ~30% of individuals progressing to joint disease. The progression of disease from skin to joint is not fully understood, although it is thought to be orchestrated by T helper 17 immune responses. To understand the immune landscape of PsA, we isolated immune cells from four matched PsA patient blood and synovium samples and performed multimodal single-cell sequencing (ExCITE-seq), which captures surface epitope information and T and B cell receptor rearrangement in addition to transcriptional profile. Our preliminary analysis revealed an enrichment of CD1c+/CD141+ dendritic cells (DCs) in synovium compared to the blood of PsA patients. Differential gene expression of these DC populations showed increased expression of CXCL16 in synovium derived DCs, while analysis of synovial fluid revealed high levels of CXCL10. Differential gene expression of CD8 and CD4 T cells across blood and synovium revealed increased granzyme K (GZMK) in the synovium. Further analyses comparing the GZMK+ T cell populations to their negative counterparts revealed upregulation of CXCR3, suggesting that CXCR3-CXCL10 axis may be responsible for recruitment and/or retention of T cells in the synovium. Pathway analysis for GZMK+ T cells revealed increased cytotoxicity and granulitic activity. Together this data suggests that GZMK+ T cells may contribute to inflammation in the joint of PsA patients, after recruitment to the site of inflammation by CD1c+/CD141+ DCs. Further characterization of additional PsA patients and psoriasis patients at risk for developing PsA is ongoing and will provide insight into PsA pathogenesis.

Supported by a grant from the NIH (I&I Schwab T32) and funding from the National Psoriasis Foundation and the Colton Center for Autoimmunity.
SARS-CoV-2 RBD-specific atypical memory B cells exhibit effector memory-like features in multi-dose mRNA vaccination
Authors: Lee Jeongsoo , Kim Sung-Han , Shin Eui-Cheol , Park Su-Hyung
Published: August 2023

Memory B cell is a heterogenous population and such heterogeneity is a focus of active investigation. How distinct subsets of memory B cells respond to repeated novel antigen challenges such as SARS-CoV-2 in human remains a subject of vigorous research. We employed fluorochrome-conjugated streptavidin tetramers bound with RBD of wild-type and omicron variant (B.1.1.529), respectively, to sort RBD-specific B cells and performed CITE-seq to investigate detailed dynamics of transcriptomes, surface phenotyes and clonality in healthy mRNA vaccination cohort. PBMC samples of 8 BNT162b2 vaccine recipients from 7 consecutive timepoints, before and after each dose of 3-times vaccination, were analyzed. We found pulsating, yet with increasing trend, frequency of atypical memory B cells among tetramer-binding B cells after each dose of vaccine. SCENIC analysis of tetramer-binding memory B cells to find subset-specific regulatory gene networks revealed atypical memory B cells have distinct regulons of genes related to activation such as receptor clustering, signaling and calcium-dependent activation compared with classical memory B cells and found only one shared clone between atypical and classical tetramer-binding memory B cells, suggesting infrequent interconversion between the two subsets. Furthermore, in-vitro polyclonal stimulation of atypical memory B cells showed higher frequency of antibody-secreting cells (ASC) and pre-plasmablasts in flow cytometry and wild-type RBD-specific ASCs in IgG ELISPOT assay, compared with classical memory B cells. These findings highlight effector memory-like features of atypical memory B cells in viral antigen challenge and a potential target of future vaccine design.
Critical COVID-19 disease explained by type I interferon autoantibodies found in patients within the Military Health System
Authors:   EPICC Study Group, Leondaridis Maria A , Yee Debra , Tso Marana , Shaw Elana , Rosen Lindsey B , Samuels Emily , Bastard Paul , Casanova Jean-Laurent , Holland Steven M , Su Helen C , Richard Stephanie , Mende Katrin , Lalani Tahaniyat , Lindholm David , Berjohn Catherine M , Maves Ryan , Colombo Rhonda , Colombo Christopher , Larson Derek , Ewers Evan , Ganesan Anuradha , Huprikar Nikhil , Mody Rupal M , Jones Milissa U , Simons Mark P , Tribble David , Laing Eric , Agan Brian , Pollett Simon , Burgess Timothy , Snow Andrew L
Published: August 2023

Neutralizing auto-antibodies (auto-Abs) that target type I interferons (IFN), a group of cytokines that induce innate immune responses upon viral infection, are found within approximately 10–20% of patients with critical COVID-19. We sought to determine if neutralizing type I IFN auto-Abs contribute to severe COVID-19 in patients within the Military Health System (MHS). The Epidemiology, Immunology, and Clinical Characteristics of Emerging Infectious Diseases with Pandemic Potential (EPICC) cohort collected demographic data, clinical data and sera from SARS-CoV-2 infected patients enrolled across 10 U.S. military treatment facilities. We screened sera collected <21 days post-symptom onset from 214 COVID-19 inpatients and 312 COVID-19 outpatients for IFN auto-Ab positivity and neutralizing activity using Luminex and intracellular flow cytometry, respectively. Similar to previous reports, we detected neutralizing auto-Abs against IFN-α and/or IFN-ω in a significantly higher frequency of inpatients (9 total, 4.2%) versus outpatients (1, 0.32%) (p=0.009). Remarkably, IFN auto-Abs persisted 6–12 months post-infection in most inpatients, including several with a prior history of autoimmune disease. Among inpatients, multivariate logistic regression analyses demonstrated that type I IFN auto-Abs were associated with a greater risk of severe and critical COVID-19 (adjusted odds ratio (aOR) = 16.40 and 6.44, respectively) after adjusting for age, sex and comorbidity burden. Our results confirm a robust association between critical COVID-19 and the presence of type I IFN auto-Abs, which may predispose to other severe respiratory viral infections that cause substantial morbidity and mortality in the MHS.

This work was supported by awards from the Defense Health Program (HU00012020067) and the National Institute of Allergy and Infectious Disease (HU00011920111). The protocol was executed by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense (DoD) program executed by the Uniformed Services University of the Health Sciences (USUHS) through a cooperative agreement by the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF).
Single cell mapping identifies expansion of Cytotoxic CD4 T cells in the atherosclerotic plaque of subjects with Type 2 Diabetes
Authors: Das Dayasagar , Gildea Michael , Eberhardt Natalia , Kaur Ravneet , Fernandez Dawn , Amadori Letizia , Sajja Swathy , Shamailova Roza , Goldberg Ira J , Maldonado Thomas , Rockman Caron B , Berger Jeffrey , Fisher Edward A , Giannarelli Chiara
Published: August 2023

Patients with Type 2 diabetes (T2D) are at greater risk of atherosclerotic cardiovascular disease (ASCVD) and fail to fully respond to standard lipid lowering drugs. While the role of macrophages in T2D plaque pathology has been investigated, no studies have systematically investigated the heterogeneity of CD4 T cells in human plaques and how they respond to cholesterol reduction. To identify the specific T cell alterations in plaques from patients with T2D, we performed scRNAseq of human carotid plaques from 22 subjects (54.5% T2D) were being treated with cholesterol lowering drugs (average LDL cholesterol of 1.2 mmol/L) undergoing carotid endarterectomy. Data were processed and integrated using Seurat and Slingshot algorithms. We identified a total of 24 immune cell clusters by unbiased clustering of CD45+ cells, including CD4 T cells that presented similar frequencies between T2D (~11%) and non-T2D patients (~10%). CD4 T cells comprised of 13 sub clusters: naïve, central memory (CM), effector memory (EM), T regulatory (Tregs), cytotoxic (CTL), and gamma delta (γδ) T cells. One cluster of CD4 CTLs (cluster 5) expressing TRDC and TRGC2 surface markers along with cytotoxicity markers GZMB, GNLY, and PRF1 was significantly expanded in T2D plaques (p=0.032). This CD4CTL expressed high levels of transcription factors (TFs) involved in T cell differentiation such as RUNX3 and ZEB2 and contained sets of genes involved in cytotoxicity, chemotaxis and IL-1 signaling. Trajectory analysis using Slingshot inferred that CD4 CTLs clusters in the plaques differentiate from CD4 EM T cells. Plaques from patients with T2D are highly enriched in CTL/γδ T cells and suggest that their cytotoxic potential contributes to the increased cardiovascular risk.

This Work is supported by AHA SFRN 20SFRN35210252 and the AHA-SFRN Training Program.
Using novel diagnostic antibodies to identify lymphocyte markers of rheumatic fever and heart disease
Authors: Euler Chad W , Fischetti Vincent A
Published: August 2023

Acute rheumatic fever (ARF) is an autoimmune disease that occurs in a subset of patients as a delayed complication of an improperly treated throat infection with a rheumatogenic strain of Streptococcus pyogenes. ARF / Rheumatic heart disease is a leading cause of preventable cardiovascular morbidity and mortality in children worldwide, affecting >33 million people. A method of detecting children susceptible to ARF (3–5% of the total population) would have major global health and economic benefits.

One potential ARF diagnostic is the IgM antibody, D8/17, which binds B cells from ARF patients of diverse ethnic groups and geographical locations at a higher % versus matched controls. While this antibody has had mixed results in the past, no group has identified the antigen that D8/17 binds. To aid in this endeavor, we used recombinant antibody engineering to produce new IgM and IgG1 versions of D8/17 that give more consistent results in our in vitro diagnostic tests.

These new derivatives were then utilized in a multi-omics approach to characterize the differences between immortalized B cell lines of ARF patients and controls. Western blot and MS/MS proteomic analyses of lymphocyte lysates identified cytoskeletal proteins from ARF B cells that cross-react more readily with our recombinant D8/17. RNA sequencing and microarray analysis of B cells from ARF patients versus controls confirmed our proteomic results. Further, we discovered differences in the gene expression of other cell surface proteins, kinases and signaling pathways in ARF B cells that bind these antibodies. We hope our analyses will help identify markers or genetic factors related to ARF development and pathogenesis, or aid in designing diagnostic assays for ARF susceptibility.

Supported by a grant from the NIH /NIAD (SC2 1SC2AI134947-01)
Role of cholinergic anti-inflammatory pathway in the regulation of vascular endothelial permeability and inflammation in type 2 diabetes
Authors: Fazal Nadeem , Tauseef Mohammad
Published: August 2023

Despite tremendous advancement in glycemic control, anti-diabetic medications have failed to revert vascular impairment once triggered by the metabolic disorder. The cholinergic anti-inflammatory pathway (CAP) is a neurophysiological mechanism that regulates the immune system. Studies show that the CAP inhibits inflammation by suppressing cytokine synthesis via release of acetylcholine in organs of the reticuloendothelial system, including the heart, lungs and gastrointestinal tract. Upon release, acetylcholine interacts with α7 nicotinic acetylcholine receptors (α7nAChR), down-regulate pro-inflammatory cytokine synthesis and prevent tissue damage and inflammation. Although a CAP modulated the response to severe inflammation during sepsis, the role of this pathway in the regulation of type-2 diabetes (T2D)-induced increase in endothelial permeability is completely unknown. Our data showed that human coronary artery endothelial cells (HCAECs) from T2D displayed increased endothelial permeability and enhanced pro-inflammatory signaling in response to pro-coagulant thrombin and endotoxin lipopolysaccharide (LPS), respectively. We also found increased expression and activity of acetylcholinesterase enzyme, and no change in expression of α7nAChR in T2D endothelial cells (ECs) compared to ECs from normal subjects. Our research findings showed that the T2D increased the expression and activity of AChE enzyme in HCAECs, and thereby decreasing the availability of ACh at α7nAChR. In summary, our work offered novel insights and therapeutic targets that we think will lead to the development of more effective treatments with improved outcomes.

Grant from CTRE Chicago State University, Chicago, IL
Characterization of Lung Eosinophil Subsets During Respiratory Viral Infection in Mice with Pre-Existing Immunity
Authors: Chang Lauren A , Choi Angela , Rathnasinghe Raveen , Warang Prajakta , Noureddine Moataz , Jangra Sonia , García-Sastre Adolfo , Schotsaert Michael
Published: August 2023

Eosinophils are critical cells in Type 2 immune responses and mucosal tissue homeostasis. Historically, lung eosinophilia has been linked to aberrant Th2 responses in response to inhaled allergens or as a consequence of vaccine-associated enhanced respiratory disease in viral infection. In some animal models of respiratory viral infection, lung eosinophilia has been observed in the absence of overt pathology. Here, we interrogated the phenotype and function of eosinophil subsets recruited to the lung after a sublethal, vaccine-matched intranasal challenge, modeling breakthrough infection. We observed an enrichment for eosinophils in the lungs of TIV-vaccinated mice post-challenge that were phenotypically similar to that of OVA-sensitized allergic mice, but with a marked absence of strong inflammatory cytokine signatures, detectable viral titers, and enhanced morbidity. Moreover, we did not observe an influx of eosinophils in the lungs of challenged unvaccinated mice, which were unable to control viral replication and had strong pro-inflammatory cytokine profiles. This suggests that vaccine-matched viral infection of the respiratory mucosa after priming with antigen in the periphery can result in lung eosinophilia that is neither pathological nor disease-enhancing, but correlates more so with clearance during a breakthrough infection. We longitudinally discerned the differential cellular kinetics of lung eosinophils and the lung microenvironment after breakthrough influenza infection or OVA sensitization across multiple parameters, including pathology. We are further analyzing the eosinophil compartment via RNA-seq, imaging, and spectral flow cytometry for in-depth characterization of this phenomenon.

Supported by NIH Public Health Service Institutional Research T32 Training Award (AI07647) and Centers of Excellence for Influenza Research and Response (CEIRR) contract (75N93021C00014-0-9999-1).
Reduction of cell fusion by deletion in the hypervariable region of the spike protein of mouse hepatitis virus
Authors: Shin Hyun-Jin
Published: August 2023

Deletions in the spike gene of mouse hepatitis virus (MHV) produce several variants with diverse biological characteristics, highlighting the significance of the spike gene in viral pathogenesis. In this study, we characterized the JHM-X strain, which has a deletion in the hypervariable region (HVR) of the spike gene, compared with the cl-2 strain, which has a full spike gene. Cytopathic effects (CPEs) induced by the two strains revealed that the size of the CPE produced by cl-2 is much greater than that produced by JHM-X in delayed brain tumor (DBT) cells. Thus, this finding explains the greater fusion activity of cl-2 than JHM-X in cultured cells, and we speculate that the deletion region of the spike protein is involved in the fusion activity differences. In contrast with the fusion activity, comparison of the virus growth kinetics revealed that the titer of JHM-X was approximately 100 times higher than that of cl-2. We found that the deletion region of the spike protein was involved in fusion activity differences, whereas cl-2 produced significantly higher luciferase activity than JHM-X upon similar expression levels of the spike protein. However, the reason behind the growth difference is still unknown. Overall, we discovered that deletion in the HVR of the spike gene could be involved in the fusion activity differences between the two strains.
Definition and mapping of Cmv5 interval associated effects on the innate immune response to acute Murine Cytomegalovirus infection
Authors: Annis Jessica L , Crittenden Rowena B , Brown Michael G
Published: August 2023

MHC-I molecule H-2Dk confers resistance to acute murine cytomegalovirus (MCMV) infection in both C57L and MA/My mice. M.H2k/b mice are MA/My background aside from a C57L-derived region within the MHC (Cmv5), which diminishes this resistance. Thus M.H2k/b mice lose more weight during infection and present with increased viral load. Additionally, M.H2k/b mice have fewer, more activated NK cells and significant spleen histopathology measured by decreased white pulp area, loss of marginal zone definition, and increasing red pulp acellularity. To better understand how the Cmv5 interval regulates the response to MCMV, we generated several Cmv5 recombinant mouse strains and screened them in vivo, allowing us to narrow the phenotype-associated interval as much as 14 times from the original size of 23.08 Mb. In addition, we sought to further characterize the Cmv5-associated phenotypes in their temporal appearance and potential direct relation to viral load. To this end, we found that many Cmv5 phenotypes could not be fully mirrored in the MA/My mice with increased viral dose, and that marginal zone disruption was one of the first Cmv5 phenotypes to appear, being reliably quantified as early as 2 days post infection (dpi) in the M.H2k/bmice. This suggests that splenic tissue disruption, occurring prior to increase in weight loss or NK cell activation, may be a primary target of the Cmv5 genetic interval and its impact on the response to MCMV. In conclusion, we verified and defined the Cmv5-associated susceptibility to MCMV infection, generated and tested several Cmv5 recombinant strains resulting in a substantially reduced interval size, and determined that the full Cmv5 phenotype is unlikely to be a direct result of increased viral load.

NIH NIAID R01AI50072, Department of Medicine/Nephrology, Center for Immunity Inflammation and Regenerative Medicine, Beirne B Carter Center for Immunology Research.
Gp120 envelope glycoproteins of HIV-1 Group M Subtype A and Subtype B differentially affect gene expression in human vascular endothelial cells
Authors: Suh Andrew J , Suzuki Dante I , Brelidze Tinatin I , Suzuki Yuichiro J
Published: August 2023

While HIV-1 Group M Subtype B is the most prevalent subtype in western countries, the majority of HIV-1 infections in eastern Africa and former Soviet Union countries are caused by Subtype A. However, most of the research on HIV has focused on Subtype B and information on the molecular mechanisms of Subtype A is virtually non-existent. The lack of such knowledge results in health disparities for the development of therapeutic strategies to prevent/treat HIV-associated complications. The present study examined envelope glycoprotein (gp120) of HIV-1 Group M Subtypes A and B. The amino acid sequence alignment analysis determined that gp120 proteins of A and B subtypes are 76% identical and share 86% similarity. The treatment of human vascular endothelial cells with the two gp120 proteins exhibited differential gene expression changes as determined by Proteome Profiler Arrays. Subtype A more potently downregulated prostasin, matrix metalloproteinase-2 (MMP-2), and receptor tyrosine-protein kinase erbB-3/Her3 than did Subtype B, while Subtype B was more effective in downregulating monocyte chemoattractant proteins (MCP-2 and MCP-3). Further, epidermal growth factor (EGF), a mediator of vascular complications, was found to be upregulated by gp120 of Subtype A, but not Subtype B. This is the first report of gp120 proteins affecting human host cells in an HIV subtype-specific manner, opening up the possibility that complications may occur differently in HIV patients throughout the world.

Supported by grants from NIH (R21 AG73919, R03 AG71596)
IL-33 promotes inflammatory responses and exacerbates lung pathology in a mouse model of SARS-CoV-2 infection
Authors: Liang Yuejin , Wang Hui , Hosakote Yashoda Madaiah , Gonzales Casey , Xie Xuping , Shi Pei-Yong , Sun Keer , Soong Lynn , Sun Jiaren
Published: August 2023

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has resulted in more than 6.6 million deaths worldwide as of the end of 2022. Clinical data revealed that dysregulated host immune responses may contribute to disease severity and poor recovery outcomes; however, the mechanisms that underlie immune dysregulation and disease severity are relatively unknown. Interleukin-33 (IL-33), a damage-associated molecular pattern (DAMP) molecule, is mainly expressed in nucleus of epithelial and endothelial cells. Elevated serum IL-33 levels have been recently reported in COVID-19 patients and might be associated with adverse outcomes. In this study, we infected B6 mice with a mouse-adapted SARS-CoV-2 MA10 and found that IL-33 was significantly increased in the lungs at day 2 post-infection, accompanied by highly upregulated inflammatory chemokines CXCL1, CXCL2 and CCL2. Next, we used IL-33−/− mice and demonstrated that IL-33 deficiency resulted in less bodyweight loss at days 2 and 3 and decreased viral burden at day 4. Flow cytometry analyses showed that IL-33−/− mice had reduced numbers of innate immune cells at day 2, including neutrophils, macrophages and NK cells. At day 4, IL-33−/− mice displayed reduced activated T cells in the lungs as compared to WT mice. Lung tissue RNAseq transcriptomics revealed that IL-33 signals contributed to several inflammatory pathways, including type II interferon signaling, pathogen phagocytosis, macrophage activation, cytokine/chemokine and inflammatory responses and oxidative damage. Overall, these data uncover a detrimental role of the DAMP IL-33 in the lungs following SARS-CoV-2 infection and provide new insights to support the development of effective therapeutic strategies for COVID-19.

Supported by grants from NIH (R21 AI153586)
Roles of glycosylation and redox states on SARS-CoV-2 spike protein actions
Authors: Bejoy Jennyfer , Brelidze Tinatin I , Suzuki Yuichiro J
Published: August 2023

The pandemic of Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The viral fusion protein, spike protein, binds to its receptor Angiotensin-Converting Enzyme 2 (ACE2) to enter host cells. Further, the S1 subunit of the spike protein may be cleaved off the virus and affect various organs. In addition to tightly binding to ACE2, S1 spike protein also enhances the peptidase activity of ACE2 and activate cell signaling. The present study compared effects of recombinant S1 spike proteins expressed in HEK cells and in E. coli. The spike protein expressed in HEK cells is heavily glycosylated and binds strongly to ACE2, while the protein expressed in E. coli is not glycosylated and exhibits no ACE2 binding. The SulfoBiotics Protein Redox State Monitoring assay determined that cysteine residues of the spike protein expressed in HEK cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of the protein expressed in HEK cells by Glycopeptidase F attenuates the ACE2 binding, although not completely, and the oxidation of the E. coli protein by hydrogen peroxide enhances the binding. In contrast to the highly distinct ACE2 binding capacities of spike proteins expressed in HEK cells and in E coli, spike proteins expressed in both systems enhance the ACE2 peptidase activity and promote cell signaling. These results suggest that the glycosylated state contributes to the binding of the spike protein to ACE2 and introduce a new concept of the regulation of the spike protein-ACE2 binding by redox mechanisms. Glycosylation and redox states, however, do not define the abilities of the spike protein to enhance the ACE2 enzyme activity or promote cell signaling.

Supported by grants from NIH (R03 AG71596, R21 AG73919)
The role of immune boosting and waning in shaping dengue epidemic dynamics
Authors: Aogo Rosemary , Zambrana Jose Victor , Kuan Guillermina , Balmaseda Angel , Sanchez Nery , Ojeda Sergio , Gordon Aubree , Harris Eva , Katzelnick Leah
Published: August 2023

Infection with one of the four dengue viruses (DENV1-4) induces both virus-specific and broadly reactive antibodies that may either provide protection or increase the severity of disease during secondary infection. Re-exposure in those with multiple prior DENV infections may boost immunity and reduce dengue risk and transmission; however, longitudinal studies investigating re-exposure and protective immunity for dengue epidemic dynamics are still lacking. For instance, does the interaction between long-term waning of immunity between infections and re-exposure to DENV or Zika virus (ZIKV) contribute to the epidemic periodicity? Since measuring re-exposure in highly immune individuals is difficult, previous studies have explored the role of boosting in protection against dengue disease but not using longitudinal measures of herd immunity. Here, we utilize both a longitudinal dataset of antibody titers to DENV and ZIKV measured annually in a Nicaraguan cohort from 2004–2021 and a mathematical model incorporating individual antibody boosting, antibody-dependent enhancement and waning to study the role of boosting on dengue transmission dynamics. Our results support our hypothesis that boosts in highly immune individuals contribute to herd immunity, delaying their contribution into the susceptibles and lowering the rate of dengue cases in future epidemics. However, as their immunity wanes due to lower transmission intensity, the susceptible fraction builds up until a major epidemic that includes re-infection of those with high titers. Our study highlights the interaction of immune boosting and waning in highly exposed adults in shaping dengue epidemics and inform vaccine efforts to maintain immunity over the life-course.

Supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health & by grants from NIH (P01AI106695)
Coronavirus spike protein vaccination history affects SARS-CoV-2 receptor-binding domain-specific memory response in a mouse model
Authors: Brice David , McGargill Maureen A
Published: August 2023

Background

Previous exposure to human common cold coronaviruses (hCCCoVs) has been shown to affect immune responses to SARS-CoV-2. Since most humans have been infected with different hCCoVs throughout their lives, determining exactly how previous immunity to hCCoVs affects anti-SARS-CoV-2 responses, especially to the Spike (S) protein and its receptor-binding domain (RBD), has proven difficult. We, therefore, used a mouse model to investigate how exposure to different coronavirus S alters the immune response to SARS-CoV-2 S vaccination.

Methods

C57BL/6 mice were vaccinated intramuscularly with 30 μg of full-length S from several coronaviruses: hCCCoVs, SARS-CoV, or SARS-CoV-2. All mice were then vaccinated with SARS-CoV-2 full-length S 33 days later. Sera from all mice were collected at multiple timepoints after vaccinations and analyzed by ELISA and microneutralization assays.

Results

Initial SARS-CoV S vaccination, surprisingly, led to significantly higher levels of anti-RBD IgG antibodies compared to SARS-CoV-2 vaccination. Boosting with SARS-CoV-2 S increased SARS-CoV-2 S IgG and IgM levels, regardless of previous vaccination, but increased RBD IgG responses more in SARS-CoV and SARS-CoV-2 S mice and only previous SARS-CoV S vaccination increased in anti-RBD IgM levels. Microneutralization assay results of each timepoint significantly correlated with combined anti-RBD IgG and IgM levels.

Conclusions

SARS-CoV S vaccination leads to more robust anti-SARS-CoV-2 RBD antibody levels in both naïve and SARS-CoV-2 S-vaccinated mice than other coronaviruses studied. Further investigation will help explore which aspects of this SARS-CoV S vaccination could lead to an increased RBD-focused immune response.

David C. Brice was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists.
Unveiling the antiviral activity of interferon epsilon in respiratory infections
Authors: Martinez-Espinoza Ivan , Guerrero-Plata Antonieta
Published: August 2023

Type I interferons (IFN-I), including IFN-α and IFN-β, have demonstrated efficacy in treatments against some viral infections but with a considerable amount of undesirable side effects. IFN-ɛ, a recently discovered member of the IFN-I family, has been found to be expressed constitutively in several tissues, including the lung. However, the antiviral effect of IFN-ɛ against respiratory viral infections is largely unknown. Human respiratory syncytial virus (RSV) is the most important agent responsible for acute lower respiratory tract diseases in infants worldwide. Each year in the United States, RSV leads to approximately 2.1 million outpatient (non-hospitalization) visits among children younger than five years old, with a considerable number of hospitalizations among children younger than five years old and adults 65 years and older. Currently, no vaccine is available to prevent this respiratory tract infections, and new strategies are necessary to treat patients. Therefore, in this study, we aimed to investigate the antiviral effect of IFN-ɛ against respiratory viruses, such as RSV. Using a human epithelial cell line, we assessed the expression, susceptibility, and antiviral effect of IFN-ɛ on RSV infection. Our results demonstrate that RSV is capable of inducing IFN-ɛ. Moreover, RSV is susceptible to the antiviral activity of the rhIFN-ɛ, reducing the number of infected cells as well as the viral replication. That effect is due to the induction of the expression of ISGs with a low inflammatory profile. Overall, our findings demonstrate that RSV is susceptible to the effect of IFN-ɛ, suggesting a potential therapeutic effect of this novel member of the IFN-I family to treat respiratory viral infections.

This work was funded in part by a Louisiana State University School of Veterinary Medicine Competitive Research Program Award
Cytotoxic T cells targeting spike glycoprotein are associated with hybrid immunity to SARS-CoV-2
Authors: Phan Jolie M , Layton Erik D , Yu Krystle K Q , Aguilar Melissa S , Golez Inah , Franko Nicholas M , Logue Jennifer K , Rodda Lauren B , Howard Christian A , Pepper Marion , Gale Michael , Chu Helen Y , Seshadri Chetan
Published: August 2023

mRNA vaccination of individuals with prior SARS-CoV-2 infection provides superior protection against breakthrough infections with variants of concern compared to vaccination in the absence of prior infection. However, the immune mechanisms by which this ‘hybrid immunity’ is generated and maintained are unknown. While genetic variation in spike glycoprotein effectively subverts neutralizing antibodies, spike-specific T cells are generally maintained against SARS-CoV-2 variants. Thus, we comprehensively profiled T cell responses against the S1 and S2 domains of spike glycoprotein in a cohort of SARS-CoV-2-naïve (n = 13) or convalescent (n = 17) individuals who received two-dose mRNA vaccine series and were matched by age, sex, and vaccine type. Using flow cytometry, we observed that the overall functional breadth of CD4 T cells as well as polyfunctional Th1 responses were similar between the two groups. However, polyfunctional cytotoxic CD4 T cell responses against both S1 and S2 domains trended higher among convalescent subjects. Multi-modal single-cell RNA sequencing revealed diverse functional programs in spike-specific CD4 and CD8 T cells in both groups. However, convalescent individuals displayed enhanced cytotoxic and antiviral CD8 T cell responses to both S1 and S2 in the absence of cytokine production. Taken together, our data suggest that cytotoxic CD4 and CD8 T cells targeting spike glycoprotein may partially account for hybrid immunity and protection against breakthrough infections with SARS-CoV-2.
In the absence of DEC-205, Enhanced inflammation in the CNS is Associated with Reduced numbers of IL-10-producing CD4+ T-cells During WNV encephalitis.
Authors: Ristic Fiona Marie , Ramos Damian Stone , Durrant Douglas M
Published: August 2023

West Nile Virus (WNV), a mosquito-borne single-stranded RNA flavivirus, can cause significant human morbidity and mortality. Previous studies have shown that dendritic cells (DCs) within the central nervous system (CNS) are required to reactivate antiviral effector T-cell responses that restrict WNV replication and limit immunopathology within the CNS. Among these DCs, one subset expressing DEC-205 accumulates within the CNS during WNV encephalitis. DEC-205, an endocytic receptor highly expressed on conventional DCs (cDCs), mediates the efficient processing and presentation of antigens in vivo, leading to either tolerance or immunity depending on DC maturation status, suggesting that DEC-205+ DCs play an immunoregulatory role in the CNS during WNV infection. However, the contributions of CNS-localized DEC-205+ DCs in programming WNV-specific T-cell immunity remains poorly understood. In preliminary experiments, WNV-infected DEC-205−/− mice exhibited increased susceptibility to WNV infection, more severe clinical signs of disease, and loss of virologic control with early entry into the CNS despite the increased trafficking, activation, and effector response of CD4+ and CD8+ T cells. We found that this uncontrolled inflammatory response was associated with a lack of IL-10-expressing CD4+ T regulatory cell (T reg) expansion that occurs typically during acute WNV infection. Thus, the enhanced inflammatory response in the absence of DEC-205 was coupled with a failure to protect against WNV infection and neuroinvasive disease. Taken together, DEC-205-expressing DCs regulate the degree and balance of the antiviral immune response to WNV infection within the CNS by activating T regs.

NIH SC3GM130472
Molecular characterization of influenza-specific memory B cell subsets residing in human tissues
Authors: Owiredu Eddie-Williams , Killian John T , King Rodney G , Akther Jobaida , Mousseau Betty , Zumaquero Esther , Zhou Fen , Chen Suki , Nellore Anoma , Rosenberg Alexander , Lund Frances E
Published: August 2023

B cells can be divided into distinct subsets based on origin, activation history and functional attributes. Most human studies, which have characterized bulk polyclonal populations of circulating B cells, suggest heterogeneity between the different B cell subsets. Indeed, we showed that memory B (Bmem) and plasma cells from the blood of the same donor exhibit distinct patterns of isotype distribution, mutational burden and have relatively limited clonal sharing. However, this heterogeneity may not extend to comparisons between the same B cell subset found within different tissues or cells that share the same reactivity. To test this, we examined influenza hemagglutinin (HA)-specific Bmem in multiple donor-matched tissues. We first generated a high-throughput bead array displaying HAs from the seasonal (H1, H3) and pandemic (H2, H5, H7, H9) flu strains and screened the serum from >80 brain-dead human tissue donors. All the donors exhibited high serum IgG reactivity against the circulating strains. We also identified serum reactivity in some donors for HAs that are not circulating in humans, suggesting that these donors likely have HA-specific IgG that are potentially broadly reactive and may have cross-reactive B cells. To directly identify B cells with cross-reactive potential, we enumerated H1 (CA09) and H2 (NETH1999) specific Bmem by flow cytometry. We show that Bmem specific for H1 or H2 are found in the spleen and blood whereas Bmem specific for both H1 and H2 are present in the mediastinal lymph node (LN), spleen and mesenteric LN. The data suggest that the repertoire of HA specific Bmem likely differ between tissues. Going forward, we will characterize the repertoire and molecular signatures of these HA-specific Bmem populations.

Supported by grants from NIH (U19 AI142737,)
IFN-γ mediated epithelial injury during influenza pathogenesis
Authors: Navaeiseddighi Zahrasadat , Schmit Taylor , Guo Kai , Khan Nadeem
Published: August 2023

Influenza pneumonia is developed due to an exuberant host response accompanied by an impaired repair, causing profound airway lung and vascular injuries that impact gas exchange and require hospitalization. However, a significant knowledge gap remains in delineating the host response mechanisms regulating the intricate inflammatory balance in the lung that either favor viral clearance or develops into immune-mediated lung pathology. Substantial emerging data reveal that dysregulated interferon (IFN) signaling, including excessive IFN-II (IFN-g) production, promotes lung injury and impairs lung repair during influenza pathogenesis. In our ongoing work, we have discovered that IFN-g exerts a direct pathologic effect by promoting epithelial injury resulting in the exacerbation of pulmonary inflammation and loss of barrier integrity during influenza. The notable findings from our study are as follows. a.IFN-g causes the cell death of infected (IAV) and un-infected (bystander) alveolar epithelial cells at a comparable rate, contributing to the disruption of pulmonary barrier. b.Molecularly, we identified that IFN-g suppressed IL6-STAT3 axis, which exerts a protective effect against influenza epithelial injury. c.Notably, the protective effect of IFN-g deficiency was abrogated by neutralizing IL-6. d. Finally, using in vitro human and mouse alveolar epithelial cells we show that IL-6 treatment can ameliorate IFN-g mediated injury of IAV-infected or un-infected epithelial cells (bystander injury). Our data is the first to demonstrate suppression of the IL-6-STAT3 axis as a potential molecular basis of pathologic IFN-g response in lung inflammation and repair to the best of our understanding.

National Institute of Health
Vitamin A deficient mice exhibit impaired viral clearance, increased inflammation and altered immune response during respiratory viral infection
Authors: Pritchett Maisie , Ebert Emily , Teague Ryan M , Shornick Laurie P
Published: August 2023

Vitamin A deficiency (VAD) is prevalent in developing countries and is associated with increased mortality after respiratory viral infections. Vitamin A can only be acquired through the diet, and millions of pregnant women and young children are deficient; however, the effects of VAD on the immune response are not completely understood. Pregnant C57BL/6 mice were placed on a VAD diet at gestational day five, pups remained on this diet after birth. At 8 weeks of age, control and VAD mice were infected intranasally with a sub-lethal dose of Sendai Virus (SeV), a natural mouse pathogen. Body weight loss in control mice peaked at day 7 post-infection, while VAD mice continued to lose weight until day 11 post-infection. This was associated with delayed clearance of virus, elevated cytokines TNFα, IL-4, and IL-17, and increased numbers of inflammatory cells in the VAD lungs. Flow cytometry showed significantly increased percentages of neutrophils on days 7 and 21, increased NK cells on day 7, increased macrophages on day 7 and increased MHC Class IIhi macrophages on days 7 and 21. In the adaptive response, CD4+ and CD8+ T cells were decreased on day 7 and increased on day 21. CD4+ T cell subsets showed increased CD4+Foxp3+ T cells on day 7 and increased CD4+Tbet+ T cells on day 21. B lymphocytes were lower at all time points. Together, this suggests that VAD affects multiple cell populations in both the innate and adaptive immune responses to viral infection in the lung,

Supported by Grants from Sigma Xi Grant in Aid of Research, Saint Louis University College of Arts and Sciences Knoedler Award, and Saint Louis University College of Arts and Sciences Graduate Undergraduate Award.