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

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

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

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\u27s staffing challenges. This is further compounded by a surgical culture that promotes the image of a \u27tough\u27 surgeon and forgoing one\u27s 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

Antigen-Specific Binding of Multivalent Soluble Antigen Arrays Induces Receptor Clustering and Impedes B Cell Receptor Mediated Signaling

A pressing need exists for autoimmune disease therapies that act in an antigen-specific manner while avoiding global immunosuppression. Multivalent soluble antigen arrays (SAgA_PLP:LABL), designed to induce tolerance to a specific multiple sclerosis autoantigen, consist of a flexible hyaluronic acid (HA) polymer backbone cografted with multiple copies of autoantigen peptide (PLP) and cell adhesion inhibitor peptide (LABL). Previous in vivo studies revealed copresentation of both signals on HA was necessary for therapeutic efficacy. To elucidate therapeutic cellular mechanisms, in vitro studies were performed in a model B cell system to evaluate binding and specificity. Compared to HA and HA arrays containing only grafted PLP or LABL, SAgA_PLP:LABL displaying both PLP and LABL exhibited greatly enhanced B cell binding. Furthermore, the binding avidity of SAgA_PLP:LABL was primarily driven by the PLP antigen, determined via flow cytometry competitive dissociation studies. Fluorescence microscopy showed SAgA_PLP:LABL induced mature receptor clustering that was faster than other HA arrays with only one type of grafted peptide. SAgA_PLP:LABL molecules also reduced and inhibited IgM-stimulated signaling as discerned by a calcium flux assay. The molecular mechanisms of enhanced antigen-specific binding, mature receptor clustering, and dampened signaling observed in B cells may contribute to SAgA_PLP:LABL therapeutic efficacy

Molecular Dynamics of Multivalent Soluble Antigen Arrays Support a Two-Signal Co-delivery Mechanism in the Treatment of Experimental Autoimmune Encephalomyelitis

Many current therapies for autoimmune diseases such as multiple sclerosis (MS) result in global immunosuppression, rendering insufficient efficacy with increased risk of adverse side effects. Multivalent soluble antigen arrays, nanomaterials presenting both autoantigen and secondary inhibitory signals on a flexible polymer backbone, are hypothesized to shift the immune response toward selective autoantigenic tolerance to repress autoimmune disease. Two-signal co-delivery of both autoantigen and secondary signal were deemed necessary for therapeutic efficacy against experimental autoimmune encephalomyelitis, a murine model of MS. Dynamic light scattering and in silico molecular dynamics simulations complemented these studies to illuminate the role of two-signal co-delivery in determining therapeutic potential. Physicochemical characteristics such as particle size and molecular affinity for intermolecular interactions and chain entanglement likely facilitated cotransport of two signals to produce efficacy. These findings elucidate potential mechanisms whereby soluble antigen arrays enact their therapeutic effect and help to guide the development of future multivalent antigen-specific immunotherapies

Combined PET and whole-tissue imaging of lymphatic-targeting vaccines in non-human primates

Antigen accumulation in lymph nodes (LNs) is critical for vaccine efficacy, but understanding of vaccine biodistribution in humans or large animals remains limited. Using the rhesus macaque model, we employed a combination of positron emission tomography (PET) and fluorescence imaging to characterize the whole-animal to tissue-level biodistribution of a subunit vaccine comprised of an HIV envelope trimer protein nanoparticle (trimer-NP) and lipid-conjugated CpG adjuvant (amph-CpG). Following immunization in the thigh, PET imaging revealed vaccine uptake primarily in inguinal and iliac LNs, reaching distances up to 17 cm away from the injection site. Within LNs, trimer-NPs exhibited striking accumulation on the periphery of follicular dendritic cell (FDC) networks in B cell follicles. Comparative imaging of soluble Env trimers (not presented on nanoparticles) in naïve or previously-immunized animals revealed diffuse deposition of trimer antigens in LNs following primary immunization, but concentration on FDCs in pre-immunized animals with high levels of trimer-specific IgG. These data demonstrate the capacity of nanoparticle or "albumin hitchhiking" technologies to concentrate vaccines in genitourinary tract-draining LNs, which may be valuable for promoting mucosal immunity