bluuebunny/crossref_metadata_2025_split huggingface dataset:
Monoclonal antibodies recognizing a putative immunodominant epitope in the hemagglutinin stalk do not enhance influenza A virus mediated fusion activity (VIR5P.1156)
Authors: Mullarkey Caitlin , Tan Gene , Nachbagauer Raffael , Miller Matthew , Palese Peter 
Published: January 2023

Abstract
The discovery of broadly neutralizing antibodies (Abs) which bind to the conserved hemagglutinin (HA) stalk has led to renewed interest in the development of a universal influenza virus vaccine. In swine, there have been isolated reports of vaccine-induced enhancement of disease in heterologous vaccination and challenge studies. It has been suggested that Abs recognizing the HA stalk promote virus fusion activity, contributing to pathology in pigs. Given that robust data in mouse and ferret models suggests a protective role for stalk Abs, here we examined the relationship between HA-stalk binding Abs and fusion. We generated mouse monoclonal Abs C4 and C6, which recognize a previously identified minimal epitope in the HA stalk. These Abs lack hemagglutination inhibition activity and did not inhibit or enhance virus-mediated fusion in vitro. In a mouse challenge model, we assessed the consequences of pre-exposure administration of a C4/C6 cocktail. No significant differences were observed in animals receiving C4/C6 in morbidity and mortality relative to naïve and isotype controls. We also probed the sera from a cohort of individuals who received an H5N1 vaccine. Although we detected baseline levels of Abs to the minimal stalk epitope associated with increased fusion activity in swine, titers did not increase post-vaccination. Together, our data suggests that stalk Abs do not enhance virus-mediated fusion and thus should not impede progress toward a universal influenza vaccine.
Secondary heterosubtypic influenza infections result in radically different T cell immunodominance hierarchies and clinical outcomes (VIR5P.1137)
Authors: Duan Susu , Meliopoulos Victoria , McClaren Jennifer , Guo Xizhi , Sanders Catherine , Smallwood Heather , Webby Richard , Schultz-Cherry Stacey , Doherty Peter , Thomas Paul 
Published: January 2023

Abstract
When human populations face a novel subtype influenza A virus (IAV) with pandemic potential, the role of CD8+ cytotoxic T lymphocyte (CTL) responses targeting epitopes conserved or cross-reactive between the novel and previously encountered IAVs can be crucial to limit disease severity. To discover immune correlates of protective efficacy, we used a model of H7N9 secondary infections in mice primed by prior H1N1or H9N2 infection. Each priming case significantly, but variably, reduced disease morbidity and mortality, virus load, and time to virus clearance after H7N9 challenge. The sizes of the respective memory CTL pools were the best predictors of protective efficacy. The secondary CTL responses were characterized by earlier, significantly greater airway infiltration of H7N9 virus-specific CTLs but with distinct epitope immunodominance hierarchies among the dominant KbPB1703, DbPA224, and DbNP366 epitopes for each priming case. The epitope conservation between the priming and challenge viruses clearly influenced but alone did not consistently predict the immunodominance. The receptor repertoire of CTLs targeting a crossreactive and non-crossreactive DbNP366 epitope variant were characterized to reveal the nature of epitope cross-reactivity. These findings will contribute to understanding human CTL responses and rational design of CTL-mediated vaccines, where protective efficacy and immunodominance hierarchies may be highly sensitive to the immunological history of hosts.
A model to non-invasively image respiratory viral infections in immunocompromised mice (VIR5P.1139)
Authors: Mostafa Heba , Srinivasan Ashok , Russell Charles 
Published: January 2023

Abstract
To enable real time monitoring of respiratory viral infections in living mice, we have developed a non-invasive bioluminescence imaging system for Sendai virus. Sendai virus is the murine form of human parainfluenza virus 1, a leading respiratory pathogen in children that commonly progresses to the lower respiratory tract (LRT) in immunocompromised patients with high mortality rate. Using this system, we are able to non-invasively monitor the spread of viral infection into the lower respiratory tract (LRT) based on the immune status of the host. In our ongoing studies, we demonstrate that inoculation of 7000 PFU of Sendai virus in 5ul volume mimics infection after natural contact transmission and does not penetrate well into the LRT before clearance. Immunosuppression by Dexamethasone, Cylophosphamide, or both drugs leads to a delay in clearance of infection by 1, 4, and 6 days respectively. Moreover, immunosuppression results in higher LRT viral loads. We are presently dissecting the relationship between local and systemic immune responses that result in enhanced LRT infection and clearance based on drug treatment protocols. An understanding of mechanisms by which immunosuppression leads to LRT disease may assist patients with malignancy, receiving hematopoietic stem cell transplantation, among other immunosuppressive conditions, in prevention and treatment of pneumonia that commonly develops after respiratory viral infections.
Broadly-neutralizing anti-influenza virus antibodies: Enhancement of neutralizing potency in polyclonal mixtures and IgA backbones (VIR5P.1141)
Authors: Miller Matthew , He Wenqian , Mullarkey Caitlin , Duty J. , Moran Thomas , Palese Peter 
Published: January 2023

Abstract
The recent discovery of broadly-neutralizing antibodies that target the hemagglutinin stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. To this end, we have examined the functions of hemagglutinin stalk-binding antibodies in an endogenous setting - as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro, but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. Additionally, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk-specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context, and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design.
Type 1 interferon primes naïve CD8 T cells to respond to lower concentrations and affinities of cognate ligands (VIR6P.1178)
Authors: Urban Stina , Welsh Raymond 
Published: January 2023

Abstract
During an acute virus infection, antigen-specific CD8 T cells undergo clonal expansion and differentiation into effector cells in order to control the infection. Studies have shown that CD8 T cells need to divide at least once before they acquire specific effector functions, including the ability to directly lyse target cells and produce certain cytokines. However, recently we have shown that pre-exposure of the host to type I interferon (IFN) sensitizes CD8 T cells to rapidly acquire effector function without the need to divide. The IFN-primed naïve CD8 T cells produced IFNγ, enhanced TNF and degranulated upon stimulation with high affinity cognate antigen. The threshold required for IFN-induced sensitized CD8 T cells to become activated and produce effector functions was reduced, as primed CD8 T cells produced cytokines in response to lower affinity ligands and lower concentrations of cognate ligands. In addition, an in vivo cytotoxicity assay showed that primed naïve CD8 T cells specifically lysed target cells. The reduced requirement of naïve CD8 T cells to produce effector functions if pre-exposed to IFN-induced environments may increase the overall number of naïve T cells able to respond to any given antigen. IFN-primed increased numbers and response of naïve T cells may help to control antigen load during an acute infection.
Mitochondrial antiviral signaling molecule-mediated mitochondrial calcium regulates CD8 T cell function during acute virus infection (VIR6P.1163)
Authors: Thomas Sunil , Huang Albert , Loo Yueh-Ming , Wilkins Courtney , McMillan Aimee , Gale Michael 
Published: January 2023

Abstract
MAVS is a crucial adaptor molecule through which RIG-I-like receptors (RLR)s signal to mediate downstream innate antiviral and inflammatory responses. However a role for MAVS in directly programming adaptive immunity has not been defined. Here we identified a cell-intrinsic role for MAVS in programming CD8 T cells following virus infection. We utilized a mouse model of acute Lymphocytic choriomeningitis virus (LCMV) infection to define the actions of adoptively transferred WT or MAVS deficient CD8 T cells. Lack of MAVS lead to defects in CD8 T cell expansion as well as function as measured by cytokine and cytotoxic T cell effector molecule production but these parameters were not deficient in double knockout mice lacking the RLRs MDA5 and RIG-I. We identified defects in total cytoplasmic calcium, mitochondrial calcium as well as mitochondrial membrane potential in MAVS-/- CD8 T cells in vivo. We found that the inflammatory conditions of LCMV -induced cell proliferation place a metabolic load on CD8 T cells and their mitochondria such that the absence of MAVS lead to defects in mitochondrial calcium independently of the RLRs. Importantly, the MAVS defect associated with defective nuclear factor of activated T cells c1 (NFATc1) activation and consequently decreased IL-2 production. Our study assigns a cell-intrinsic role for MAVS in regulating adaptive immunity following viral infection by means of regulating mitochondrial calcium and thereby affecting T cell fitness.
Soluble Tim-3 is shed from CD8+ T cells by the sheddase ADAM10, is increased in plasma during untreated HIV infection, and correlates with HIV disease progression (VIR6P.1165)
Authors: Clayton Kiera , Douglas-Vail Matthew , Rahman A. , Medcalf Karyn , Xie Irene , Chew Glen , Tandon Ravi , Lanteri Marion , Norris Philip , Deeks Steven , Ndhlovu Lishomwa , Ostrowski Mario 
Published: January 2023

Abstract
Chronic HIV infection results in a loss of HIV-specific CD8+ T cell effector function, termed “exhaustion”, mediated, in part, by the co-inhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized and its role in HIV disease is unknown. Here we show that Tim-3 is shed from the surface of responding CD8+ T cells by the matrix metalloproteinase, ADAM10, producing a soluble form of the co-inhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma, and was significantly elevated during early and chronic untreated HIV infection, but was not found differentially modulated in HAART treated HIV-infected subjects or in elite controllers, when compared to HIV-uninfected subjects. Plasma sTim-3 levels positively correlated with HIV viral load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8+ T cells in terms of IFN-γ production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis with implications for novel therapeutic interventions.
Peptidomic analysis of type-1 diabetes associated HLA-DQ molecules and impact of HLA-DM editing (APP5P.108)
Authors: Zhou Zemin , Reyes-Vargas Eduardo , Escobar Hernando , Chang Kuan , Rockwood Alan , Delgado Julio , He Xiao , Jensen Peter 
Published: January 2023

Abstract
HLA-DM is critical in editing peptide repertoires presented by APC, thus influencing the selection/activation of CD4+ T cells. We previously reported that type-1 diabetes (T1D) associated HLA-DQ2, 8 & DQ2/8 molecules (DQs) are relatively resistant to DM editing of the CLIP peptides. In this study, we further studied DM sensitivity of T1D-associated DQs by peptidomic analysis. Compared to a T1D-protective DQ6, the percentages of shared peptides among T1D-associated DQs were significantly higher. There were greater numbers of nested peptide sets and long peptides (&gt;20 aa) eluted from T1D-associated DQs. Predicted peptide binding motifs of DQ2, 8 & 2/8 shared charged anchor residues, while hydrophobic anchors were present in DQ6 peptides. Competition binding assays comparing the eluted peptides from DM sensitive, dependent, or resistant groups showed that the binding affinities of the DM sensitive peptides were significantly lower than that of CLIP1 peptide. The increased sDQ8-CLIP1 complex stability, with the intrinsic half-life of 58 h measured by fluorescence polarization assay, was 8 times longer than that of sDQ6-CLIP1 (7 h), further supporting the DM resistance of T1D associated DQs. Collectively, T1D associated DQs share distinct characteristics, including binding of peptides from known T1D auto-antigens. Our new data further support the hypothesis that DM resistance in T1D associated DQs affects the peptide repertoires, thus contributing to the pathogenesis of T1D.
Varying structural environments of an ectopic epitope influence CD4+ T cell immunogenicity (APP5P.117)
Authors: Rust Blake 
Published: January 2023

Abstract
Preliminary studies have indicated that epitopes adjacent to large flexible loops of antigens are often specialized for the production of interferon gamma (IFNγ). Variants of the HIV envelope glycoprotein gp120 protein were created that feature the ectopic CD4+ T-cell epitope 2W1S swapped in at two points in a variable loop (V4) of gp120. The variants (T38 and T39) were designed to place the epitope in different environments of conformational “depth” within gp120. Both antigen sequence and antigen structure contribute to CD4+ T cell immunodominance through their effects on MHCII binding and antigen processing, respectively. In order to isolate one of these two factors, antigen variants with the same ectopic CD4+ T cell epitope at different structural locations within the protein were created to eliminate the variation in MHCII binding affinity. We hypothesize that 2W1S-specific CD4+ T cells that arise from T38 immunization will display increased IFNγ expression as compared to mice immunized with T39 due to the conformationally “shallow” context of the epitope. However, any interpretation of epitope immunogenicity must take into account the global stability of the protein, and not just the local context of epitope in the native gp120. Preliminary studies indicate that T39 adopts a conformation that is more similar to that of native gp120 as compared to T38. Current studies are investigating immunogenicity of both ectopic and endogenous epitopes by multiple assays.
Processing of native or citrullinated myelin oligodendrocyte glycoprotein peptides in B cells is affected by EBV infection: implications for multiple sclerosis (APP5P.118)
Authors: Morandi Elena , Jagessar Anwar , Dijkman Karin , t` Hart Bert , Gran Bruno 
Published: January 2023

Abstract
Multiple Sclerosis is a neurodegenerative disease with an immune-mediated pathogenesis in which Epstein Barr Virus (EBV) is a risk factor. EBV infects human B cells, in which it becomes latent. Our hypothesis is that infection renders B cells potent APC for autoreactive T cells, through influences on intracellular proteolysis and post-translational modifications of self antigens. The aim of this study is to assess the effect of EBV infection of B cells on processing of myelin oligodendrocyte glycoprotein (MOG). We investigated the processing of recombinant extracellular MOG and of the immunodominant peptides MOG1-20, MOG35-55 and through SDS PAGE gel analysis in the presence or absence of Cathepsin inhibitors. We also studied the effect of MOG35-55 citrullination in position 41 and 46. In addition, we determined the proteolytic activity of Cathepsin G and H by activity assay. We found that EBV infection increases the activity of Cathepsin G and H, leading to increased degradation of MOG35-55 by B cells. However, infection also rescues rhMOG from total degradation. By contrast, inhibition of Cathepsin G or citrullination of Arg 46 abrogated the degradation of MOG35-55. These results indicate that citrullination of an immunodominant peptide of MOG protects it from destructive processing in EBV infected B cells. This could facilitate presentation of a disease-relevant myelin autoantigen that may be involved in disease induction and progression.
Peptide sequence determinants in HLA-DM susceptibility (APP5P.105)
Authors: Reyes-Vargas Eduardo , Barker Adam , Zhou Zemin , He Xiao , Jensen Peter 
Published: January 2023

Abstract
HLA-DM catalyzes peptide exchange reactions in MHCII molecules. DM functions by accelerating the peptide loading process and editing the epitopes that are presented to CD4 T cells. The catalytic potency of DM differs by MHCII-peptide complex such that some are variably edited while others are immutable. To date, the catalytic mechanism and peptide sequence determinants of susceptibility to DM editing remain unresolved. In this study, we use an FP assay to measure the effect of DM on the binding kinetics of HLA-DR1 to peptides with anchor residues of neutral, disfavored, or favored amino acids. We provide evidence for a model in which the peptide sequence determines the “effective affinity” of an MHCII-peptide complex for DM and specifies DM catalytic potency. Our results support a mechanism whereby interactions between peptide and MHC regions far from the DM contact interface can alter the effective binding affinity of the complex. We show that the peptide P1 anchor impacts kcat, which implies that the peptide N-terminus may occupy the binding groove in the “functional” catalytic complex. We show that association rates are not sensitive to peptide sequence, indicating that DM has little effect on the association of peptide to empty class II molecules. Together, these findings provide insight into the DM catalytic mechanism and suggest that stable interactions with DM at the contact surface are linked to unstable interactions in the peptide-binding groove and vice versa.
Interleukin 6 (IL6) is an important upstream contributor towards the progression of SLE-like disease in BXSB.Yaa mice (BA11P.134)
Authors: Jain Shweta , Adkins Elisabeth , Christianson Gregory , Sproule Thomas , Roopenian Derry , Morse Herbert 
Published: January 2023

Abstract
Systemic lupus erythematosus (SLE) is a multigenic autoimmune disorder that results in the production of autoantibodies (autoAbs) and deposition of immune complexes leading to tissue inflammation and organ damage. Typical disease characteristics include glomerulonephritis, splenomegaly and monocytosis. To understand disease mechanism, we studied spontaneous autoimmune BXSB.Yaa strain in which male mice develops SLE-like features with intricacy paralleling human SLE. It was shown that an extended pseudo-autosomal region on Y chromosome carrying a duplication of Tlr7 gene is responsible for the disease. TLR7 binds with ssRNA that consequently leads to production of inflammatory cytokines. We previously reported that IL21, produced by CD4 T follicular helper cells (TFH), plays a critical role in the disease of BXSB.Yaa mice. Since TFH cells require IL6 for their differentiation and maintenance from naïve T cells and dysregulated IL6 production has pathological consequences in SLE patients, we sought to determine if IL6 plays important upstream role in disease progression. We found that increased serum levels of IL6 in BXSB.Yaa mice correlated with the production of autoAbs, Ig secretion and hyperactive B cells. Moreover, abrogating IL6 signaling not only improved survival of BXSB.Yaa mice but they also had reduced numbers of TFH and lesser B cell activation. Thus, we conclude that IL6 signaling is an important determinant in the progression of SLE-like disease in BXSB.Yaa mice.
Selective deletion of estrogen receptor alpha in FoxP3+ CD4+ T cells influences peripheral T cell homeostasis (BA11P.139)
Authors: McKarns Susan 
Published: January 2023

Abstract
Being female confers a greater risk of developing an autoimmune disease than any other known genetic or environmental risk factor. Given that a loss of regulatory T cell (Treg) function contributes to autoimmunity and estrogen receptor alpha (ERα) promotes Tregs and suppresses experimental autoimmune encephalomyelitis (EAE), ERα-induced Tregs offers a new strategy to treat autoimmune disorders. The objective of these studies was to determine whether selective ablation of ERα in FoxP3+ Tregs dysregulates peripheral T cell homeostasis. To the end, mice harboring a floxed Esr1 gene (ERαf/f) were bred to mice expressing a YFP-Cre fusion protein driven by an IRES following foxp3 to obtain FoxP3+ Treg restricted deletion of Ers1. Thymic and peripheral CD4+ and CD8+ T cell frequency appeared unaffected with Esr1 ablation except for a sex-biased reduction in the total number of thymic and peripheral FoxP3+ Tregs and an increase in total number of splenocytes in ERαf/f FoxP3Cre mice compared to ERα+/+ FoxP3Cre mice. Spotaneous chronic inflammation and autoimmunity was undetected in ERαf/f FoxP3Cre mice. In a CD4+ T cell transfer model of experiment colitis, selective ablation of the ERα in FoxP3+ Tregs resulted in exacerbated inflammation compared to mice harboring ERα-sufficient Treg cells. Overall, these preliminary results suggest that FoxP3+ Treg-specific ERα is essential for the function of peripheral Tregs and immune homeostasis in a sex-biased manner.
Tolerance induction in recent thymic emigrants (BA11P.121)
Authors: Friesen Travis , Ji Qingyong , Fink Pamela 
Published: January 2023

Abstract
Recent thymic emigrants (RTEs) comprise the subset of peripheral T cells that have most recently undergone thymic maturation and egress to the lymphoid periphery. RTEs are an important lymphocyte population to study because they represent the majority of T cells in neonates and contribute to T cell diversity throughout life. Using RAG2p-GFP transgenic (Tg) mice to identify RTEs in the periphery, our lab has discovered that T cell development continues for several weeks after cells have left the thymus and entered the lymphoid periphery. We hypothesize that post-thymic maturation represents a period of heightened susceptibility to tolerance induction. We tested this notion using T cell receptor (TCR) Tg ovalbumin (OVA)-specific CD4 (OT-II) and CD8 (OT-I) RTE and MN T cells transferred into mice expressing a membrane-bound form of the OVA protein in the pancreatic islets and kidney proximal tubular cells (RIP-mOVA Tg). Following co-transfer of congenically marked OT-II and OT-I RTE and MN T cells to RIP-mOVA Tg mice, we found that, compared to mature T cells, both CD4 and CD8 RTEs proliferate, accumulate, and secrete less IL-2 and IFNg, but express elevated levels anergy-associated genes. Finally, both OT-II and OT-I RTEs were less diabetogenic than their mature T cell counterparts when transferred into RIP-mOVA adoptive hosts. These results further support our hypothesis that post-thymic maturation represents a period of heightened susceptibility to tolerance induction.