Determining Cellular and Molecular Mechanisms of Multivalent Soluble Antigen Arrays that Contribute to Therapeutic Efficacy Against a Murine Model of Multiple Sclerosis

A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgAPLP:LABL), consisting of a hyaluronic acid (HA) linear polymer backbone co-grafted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous in vivo studies established that SAgAPLP:LABL was therapeutic in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. This dissertation sought to elucidate SAgA therapeutic cellular mechanisms while identifying therapeutic molecular properties. In Chapter 2, the role of two-signal co-delivery was explored by evaluating EAE in vivo results in conjunction with in silico molecular dynamics simulations and nanomaterial properties for various covalent and physical combinations of HA, PLP, and LABL. We found that co-delivery of both primary autoantigen and secondary inhibitory signal was necessary for therapeutic efficacy against EAE. In Chapters 3 and 4, the SAgAPLP:LABL cellular mechanism was investigated in a model B cell system by evaluating binding, specificity, and signaling modulation in vitro. Here, we developed click-conjugated cSAgAPLP:LABL which, unlike SAgAPLP:LABL, employed a non-hydrolyzable linker chemistry to conjugate PLP and LABL to HA. cSAgAPLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgAPLP:LABL. We found that cSAgAPLP:LABL acted through high avidity, antigen-specific B cell binding, targeting the B cell receptor (BCR) to dampen BCR-mediated signaling. Our conclusions point to induction of antigen-specific B cell anergy as the cSAgAPLP:LABL therapeutic mechanism and present a promising option for ASIT

Multivalent antigen arrays exhibit high avidity binding and modulation of B cell receptor-mediated signaling to drive efficacy against experimental autoimmune encephalomyelitis

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biomacromolecules, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biomac.7b00335.A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgAPLP:LABL), consisting of a hyaluronic acid (HA) linear polymer backbone co-grafted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgAPLP:LABL, employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgAPLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using non-hydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule’s action at the cell surface to improve efficacy. ‘Click SAgA’ (cSAgAPLP:LABL) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgAPLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgAPLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgAPLP:LABL. Furthermore, c SAgAPLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgAPLP:LABL, achieving equivalent efficacy at one quarter of the dose. These results indicate that non-hydrolyzable conjugation increased the avidity of cSAgAPLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling.NIH T32 GM008545Madison and Lila Self Graduate Fellowship at the University of KansasHoward Rytting pre-doctoral fellowship from the Department of Pharmaceutical Chemistry at the University of Kansa

Routes of Administration and Dose Optimization of Soluble Antigen Arrays in Mice with Experimental Autoimmune Encephalomyelitis

Soluble Antigen Arrays (SAgAs) were developed for treating mice with experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. SAgAs are composed of hyaluronan with grafted EAE antigen and LABL peptide (a ligand of ICAM-1). SAgA dose was tested by varying injection volume, SAgA concentration, and administration schedule. Routes of administration were explored to determine the efficacy of SAgAs when injected intramuscularly, subcutaneously, intraperitoneally, intravenously, or instilled into lungs. Injections proximal to the central nervous system (CNS) were compared to distal injection sites. Intravenous dosing was included to determine if SAgA efficiency results from systemic exposure. Pulmonary instillation was included since reports suggest T cells are licensed in the lungs before moving onto the CNS1,2. Decreasing the volume of injection or SAgA dose reduced treatment efficacy. Treating mice with a single injection on day 4, 7, or 10 also reduced efficacy compared to injecting on all three days. Surprisingly, changing the injection site did not lead to a significant difference in efficacy. Intravenous administration showed efficacy similar to other routes, suggesting SAgAs act systemically. When SAgAs were delivered via pulmonary instillation, however, EAE mice failed to develop any symptoms, suggesting a unique lung mechanism to ameliorate EAE in mice

Acute B-Cell Inhibition by Soluble Antigen Arrays Is Valency-Dependent and Predicts Immunomodulation in Splenocytes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Biomacromolecules, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acs.biomac.9b00328.Antigen valency plays a fundamental role in directing the nature of an immune response to be stimulatory or tolerogenic. Soluble Antigen Arrays (SAgAs) are an antigen-specific immunotherapy that combats autoimmunity through the multivalent display of autoantigen. While mechanistic studies have shown SAgAs to induce T and B-cell anergy, the effect of SAgA valency has never been experimentally tested. Here, SAgAs of discrete antigen valencies were synthesized by click chemistry and evaluated for acute B-cell signaling inhibition as well as downstream immunomodulatory effects in splenocytes. Initial studies using the Raji B-cell line demonstrated SAgA valency dictated the extent of calcium flux. Lower valency constructs elicited the largest reductions in B-cell activation. In splenocytes from mice with experimental autoimmune encephalomyelitis, the same valency-dependent effects were evident in the downregulation of the costimulatory marker CD86. The reduction of calcium flux observed in Raji B-cells correlated strongly with downregulation in splenocyte CD86 expression after 72 hours. Here, a thorough analysis of SAgA antigenic valency illustrates that low, but not monovalent, presentation of autoantigen was ideal for eliciting the most potent immunomodulatory effects.Madison and Lila Self Graduate Fellowship at the University of KansasNIH T32 GM00854

Factors influencing terrestriality in primates of the Americas and Madagascar

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (bodymass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use

Factors influencing terrestriality in primates of the Americas and Madagascar

Among mammals, the order Primates is exceptional in having a high taxonomic richness in which the taxa are arboreal, semiterrestrial, or terrestrial. Although habitual terrestriality is pervasive among the apes and African and Asian monkeys (catarrhines), it is largely absent among monkeys of the Americas (platyrrhines), as well as galagos, lemurs, and lorises (strepsirrhines), which are mostly arboreal. Numerous ecological drivers and species-specific factors are suggested to set the conditions for an evolutionary shift from arboreality to terrestriality, and current environmental conditions may provide analogous scenarios to those transitional periods. Therefore, we investigated predominantly arboreal, diurnal primate genera from the Americas and Madagascar that lack fully terrestrial taxa, to determine whether ecological drivers (habitat canopy cover, predation risk, maximum temperature, precipitation, primate species richness, human population density, and distance to roads) or species-specific traits (body mass, group size, and degree of frugivory) associate with increased terrestriality. We collated 150,961 observation hours across 2,227 months from 47 species at 20 sites in Madagascar and 48 sites in the Americas. Multiple factors were associated with ground use in these otherwise arboreal species, including increased temperature, a decrease in canopy cover, a dietary shift away from frugivory, and larger group size. These factors mostly explain intraspecific differences in terrestriality. As humanity modifies habitats and causes climate change, our results suggest that species already inhabiting hot, sparsely canopied sites, and exhibiting more generalized diets, are more likely to shift toward greater ground use

Multivalent Soluble Antigen Arrays Exhibit High Avidity Binding and Modulation of B Cell Receptor-Mediated Signaling to Drive Efficacy against Experimental Autoimmune Encephalomyelitis

A pressing need exists for antigen-specific immunotherapies (ASIT) that induce selective tolerance in autoimmune disease while avoiding deleterious global immunosuppression. Multivalent soluble antigen arrays (SAgA_PLP:LABL), consisting of a hyaluronic acid (HA) linear polymer backbone cografted with multiple copies of autoantigen (PLP) and cell adhesion inhibitor (LABL) peptides, are designed to induce tolerance to a specific multiple sclerosis (MS) autoantigen. Previous studies established that hydrolyzable SAgA_PLP:LABL, employing a degradable linker to codeliver PLP and LABL, was therapeutic in experimental autoimmune encephalomyelitis (EAE) in vivo and exhibited antigen-specific binding with B cells, targeted the B cell receptor (BCR), and dampened BCR-mediated signaling in vitro. Our results pointed to sustained BCR engagement as the SAgA_PLP:LABL therapeutic mechanism, so we developed a new version of the SAgA molecule using nonhydrolyzable conjugation chemistry, hypothesizing it would enhance and maintain the molecule’s action at the cell surface to improve efficacy. “Click SAgA” (cSAgA_PLP:LABL) uses hydrolytically stable covalent conjugation chemistry (Copper-catalyzed Azide–Alkyne Cycloaddition (CuAAC)) rather than a hydrolyzable oxime bond to attach PLP and LABL to HA. We explored cSAgA_PLP:LABL B cell engagement and modulation of BCR-mediated signaling in vitro through flow cytometry binding and calcium flux signaling assays. Indeed, cSAgA_PLP:LABL exhibited higher avidity B cell binding and greater dampening of BCR-mediated signaling than hydrolyzable SAgA_PLP:LABL. Furthermore, cSAgA_PLP:LABL exhibited significantly enhanced in vivo efficacy compared to hydrolyzable SAgA_PLP:LABL, achieving equivalent efficacy at one-quarter of the dose. These results indicate that nonhydrolyzable conjugation increased the avidity of cSAgA_PLP:LABL to drive in vivo efficacy through modulated BCR-mediated signaling

Co-delivery of autoantigen and B7 pathway modulators suppresses experimental autoimmune encephalomyelitis

Autoimmune diseases such as multiple sclerosis (MS) are characterized by the breakdown of immune tolerance to autoantigens. Targeting surface receptors on immune cells offers a unique strategy for reprogramming immune responses in autoimmune diseases. The B7 signaling pathway was targeted using adaptations of soluble antigen array (SAgA) technology achieved by covalently linking B7-binding peptides and disease causing autoantigen (proteolipid peptide (PLP)) to hyaluronic acid (HA). We hypothesized that co-delivery of a B7-binding peptide and autoantigen would suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Three independent B7-targeted SAgAs were created containing peptides to either inhibit or potentially stimulate the B7 signaling pathway. Surprisingly, all SAgAs were found to suppress EAE disease symptoms. Altered cytokine expression was observed in primary splenocytes isolated from SAgA-treated mice, indicating that SAgAs with different B7-binding peptides may suppress EAE through different immunological mechanisms. This antigen-specific immunotherapy using SAgAs can successfully suppress EAE through co-delivery of autoantigen and peptides targeting with the B7 signaling pathway

Codelivery of antigen and an immune cell adhesion inhibitor is necessary for efficacy of soluble antigen arrays in experimental autoimmune encephalomyelitis

Autoimmune diseases such as multiple sclerosis (MS) are typified by the misrecognition of self-antigen and the clonal expansion of autoreactive T cells. Antigen-specific immunotherapies (antigen-SITs) have long been explored as a means to desensitize patients to offending self-antigen(s) with the potential to retolerize the immune response. Soluble antigen arrays (SAgAs) are composed of hyaluronic acid (HA) cografted with disease-specific autoantigen (proteolipid protein peptide) and an ICAM-1 inhibitor peptide (LABL). SAgAs were designed as an antigen-SIT that codeliver peptides to suppress experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Codelivery of antigen and cell adhesion inhibitor (LABL) conjugated to HA was essential for SAgA treatment of EAE. Individual SAgA components or mixtures thereof reduced proinflammatory cytokines in cultured splenocytes from EAE mice; however, these treatments showed minimal to no in vivo therapeutic effect in EAE mice. Thus, carriers that codeliver antigen and a secondary “context” signal (e.g., LABL) in vivo may be an important design criteria to consider when designing antigen-SIT for autoimmune therapy

To leave or not to leave: American Association for the Surgery of Trauma (AAST) panel discussion on personal, parental, and family leave

Navigating planned and emergent leave during medical practice is very confusing to most physicians. This is especially challenging to the trauma and acute care surgeon, whose practice is unique due to overnight in-hospital call, alternating coverage of different services, and trauma center’s staffing challenges. This is further compounded by a surgical culture that promotes the image of a ‘tough’ surgeon and forgoing one’s personal needs on behalf of patients and colleagues. Frequently, surgeons find themselves having to make a choice at the crossroads of personal and family needs with work obligations: to leave or not to leave. Often, surgeons prioritize their professional commitment over personal wellness and family support. Extensive research has been conducted on the topic of maternity leave and inequality towards female surgeons, primarily focused on trainees. The value of paternity leave has been increasingly recognized recently. Consequently, significant policy changes have been implemented to support trainees. Practicing surgeon, however, often lack such policy support, and thus may default to local culture or contractual agreement. A panel session at the American Association for the Surgery of Trauma 2022 annual meeting was held to discuss the current status of planned or unanticipated leave for practicing surgeons. Experiences, perspectives, and propositions for change were discussed, and are presented here