Biology subjects

Grosskopf, A. K.

Publications and source records attributed to Grosskopf, A. K..

At least 19 recordsLinked to original sources

bioRxiv2025

Eph Receptor Tyrosine Kinases Are Functional Entry Receptors for Murine Gammaherpesvirus 68

Interactions between viral glycoproteins and cellular receptors determine virus tropism and represent promising targets for vaccines. Eph receptor tyrosine kinases are conserved receptors for the human oncogenic gammaherpesviruses, Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr virus (EBV), and mediate entry into target cells by interaction with the viral gH/gL glycoprotein complex. To evaluate the use of murine gammaherpesvirus 68 (MHV68), a natural pathogen of rodents, as an in vivo model system for early events in gammaherpesvirus infection, we characterized the interaction of the MHV68 gH/gL complex with Eph receptors. We demonstrate a direct interaction of MHV68 gH/gL with EphA4 and EphB3, that is conserved between human and murine receptors. Pre-incubation of MHV68 inocula with soluble decoy receptors decreased infection of permissive fibroblasts. Ectopic expression of EphA4 and EphB3 enabled MHV68 to infect otherwise non-permissive human B cells, demonstrating EphA4 and EphB3 receptor function. Targeted mutations informed by protein structure predictions demonstrate that the MHV68 gH/gL-Eph interaction is determined by domain I (D-I) and follows structural motifs previously described in the KSHV gH/gL-EphA2 complex. The importance of gH D-I is further highlighted by the analysis of gH-targeting neutralizing antibodies. Antibody adsorption via the full gH ectodomain or gH D-I led to comparable reductions in neutralization capacity of serum from WT infected mice, indicating the Eph-binding domain is a major target for gH/gL-directed neutralizing antibodies. Our study characterizes Eph receptors as novel interaction partners and entry receptors for MHV68. Conservation of entry mechanisms provides the basis for future in vivo analyses of the contribution of Eph receptors to cell-type dependent MHV68 infection, as well as targeted strategies to prevent transmission and diseases associated with chronic infection. Author SummaryVirus-specific intervention and prevention is needed to fight cancers and morbidity caused by the oncoviruses Kaposi sarcoma herpesvirus (KSHV) and Epstein-Barr Virus (EBV). The targeted design of such strategies requires a detailed knowledge of early events in the viral life cycle and the contribution of cellular receptors to infection and spread in vivo. Here, we characterize the role of Eph receptors in murine gammaherpesvirus 68 (MHV68) infection, a well-established mouse model for gammaherpesvirus (GHV) pathogenesis and preclinical therapeutic evaluations. As described for human GHVs, the interaction of Eph receptors with the gH/gL complex is mediated by a conserved motif consistent of the N-terminal domain of glycoprotein gH co-folded with gL and contributes to variable levels to infection of different target cells. We demonstrate that antibodies directed against the N-terminal domain of the gH/gL glycoprotein complex are generated upon MHV68 infection in mice and neutralize MHV68 infection in cell culture. Our data enables the analysis of the contribution of Eph receptor engagement to GHV infection in vivo and provides a promising target for subunit vaccination approaches.

microbiology↗

bioRxiv2024

Glycoprotein K8.1 is Critical for Liver and Bone Marrow Tropism of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) in the Marmoset Infection Model

Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus with significant disease burden, particularly in Sub-Saharan Africa, where Kaposis sarcoma is prevalent. KSHV disproportionately affects immunocompromised individuals and men who have sex with men. A suitable animal model is critical for the development of a preventive vaccine, as it must replicate viral spread and immune interactions. The common marmoset (Callithrix jacchus) had previously been established as conducive to KSHV infection with non-BAC-derived virus via the intravenous (i.v.) route. We used this animal model to establish the ability to infect marmosets with bacterial artificial chromosome (BAC)-derived KSHV and to establish the role of individual glycoproteins (GPs) in the colonization of specific host tissues. We infected three groups of four animals each with BAC16-derived KSHV wild type (wt), KSHV gHASAELAAN, or KSHV{Delta}K8.1. KSHV gHASAELAAN contains mutations in gH that interfere with binding to the EphA2 receptor, which normally forms a high-affinity complex with gH/gL, whereas KSHV{Delta}K8.1 does not express GP K8.1. The KSHV gHASAELAAN mutant was used at a relatively higher concentration to overcome its intrinsic defect and to allow for analysis of tissue tropism. While seroconversion to the KSHV LANA antigen was variable, all animals exhibited clearly detectable viral DNA load in many tissues. Highest loads were detected in spleen, liver, heart and bone marrow. Viral DNA loads in these four tissues did not significantly differ between groups infected with KSHV wt or with the gHASAELAAN mutant. However, KSHV{Delta}K8.1-infected animals exhibited significantly lower levels of viral DNA in liver and bone marrow, but not in spleen or heart. Analysis of immune cells demonstrated significant activation of CD4+ and CD8+ T-cells in KSHV wt-infected animals. In wt and gHASAELAAN-infected animals, different B cell subpopulations were found to expand after infection, which implies that the gH/gL glycoprotein complex may shape the host response to KSHV in unexpected ways. In summary, our findings demonstrate that neither the interaction with Eph family receptors nor GP K8.1 is essential for infection via the i.v. route but K8.1 plays a critical role for KSHV tissue tropism in vivo.

microbiology↗

bioRxiv2024

Dynamic Hydrogel Crosslink Strength Controls Yielding Characteristics and Cell Viability During Injection

Yielding of dynamically crosslinked hydrogels, or transition between a solid-like and liquid-like state, allows facile injection and utility in translational biomedical applications including delivery of therapeutic cells. Unfortunately, characterizing the time-varying nature of the transition has not been attempted, nor are there design rules for understanding the effects of yielding on encapsulated cells. Here, we unveil underlying molecular mechanisms governing the yielding transition of dynamically crosslinked gels currently being researched for use in cell therapy. We demonstrate through nonlinear rheological characterization that the network dynamics of the dynamic hydrogels dictate the speed and character of their yielding transition. Rheological testing of these materials reveals unexpected elastic strain stiffening during yielding, as well as characterizing the rapidity of the yielding transition. A slower yielding speed explains enhanced protection of directly injected cells from shear forces, highlighting the importance of mechanical characterization of all phases of yield-stress biomaterials. Significance StatementMany direct-injection cell therapies suffer from poor viability of injected cells due to membrane disruption by shear stresses during injection. Injectable hydrogels with dynamic crosslinks can increase viability during injection, utilizing the ability of these materials to undergo a reversible solid-to-liquid shear-rate dependent yielding transition. Much effort has been applied to understanding the yielding transition of these materials, yet design rules for connecting the character of yielding to viability of injected cells has not been established. Here, we apply rheological testing to show that faster network dynamics result in a more gradual yielding transition, affording greater protection to cells during injection. This work introduces a new rheological protocol for the development of injectable hydrogels for cell therapy.

bioengineering↗

bioRxiv2023

Kaposi's sarcoma-associated herpesvirus (KSHV) gB dictates a low-pH endocytotic entry pathway as revealed by a dual-fluorescent virus system and a rhesus monkey rhadinovirus expressing KSHV gB

Interaction with host cell receptors initiates internalization of Kaposis sarcoma-associated herpesvirus (KSHV) particles via endocytosis or macropinocytosis. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of gH, gL, and gB, that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed single- and dual-fluorescent recombinant KSHV strains that incorporate tagged glycoproteins, capsid proteins or a combination thereof. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. Our data demonstrate lytic reactivation and infectivity of dual-fluorescent KSHV and incorporation of fluorescently-tagged proteins into viral particles. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. By using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established the KSHV fusion kinetics upon infection of different target cells. We measured marked differences in the "time-to-fusion" between different cell types. Inhibition of vesicle acidification prevented virus-cell fusion, implicating low vesicle pH as a requirement for the KSHV fusion step. These findings were corroborated by comparison of RRV-YFP reporter virus with wildtype gB and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB, these two novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion.

microbiology↗

bioRxiv2023

Tim-1 and Tim-4 mediate entry of the human Kaposi's sarcoma-associated herpesvirus and the related rhesus monkey rhadinovirus

Kaposis sarcoma-associated herpesvirus (KSHV) is a human tumor virus. It is associated with Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. KSHV is known to interact with several different receptors, among them heparan sulfate proteoglycans, Eph family receptors, and integrins. We mutated the closely related rhesus monkey rhadinovirus in the known receptor interaction sites for Eph family and Plexin domain containing proteins and found it to still replicate on certain cells. This lytic virus was then used as a selection agent in a genome-wide CRISPR knockout screen, which identified TIM1 and NRP1 as host dependency factors. NRP1 is also host factor for the related Epstein-Barr virus and was recently reported to promote KSHV infection, which we confirm even if it functions with low efficiency on most cells and became functional only after ablation of the Eph receptor interaction. Further analysis through overexpression demonstrated that Tim-1 and the related Tim-4 are strong mediators of RRV and KSHV infection, in particular in the absence of other receptor interactions and even more pronounced for a KSHV mutant deleted in glycoprotein K8.1. Both Tim-1 and Tim-4 are heavily O-glycosylated phosphatidylserine (PS) receptors. For KSHV in particular, experiments with mutated Tim-1 and comparison to Ebola virus glycoprotein-driven entry indicate that the interaction with Tim-1 occurs through PS-binding by Tim-1 and suggest additional interaction in a PS-independent manner. The mucin-like domain of Tim-1 is required for optimal receptor function. The use of Tim proteins for entry is a novelty for herpesviruses and underscores the unique biology of KSHV and RRV.

microbiology↗

bioRxiv2023

Label-Free Composition Analysis of Supramolecular Polymer - Nanoparticle Hydrogels by Reversed-Phase Liquid Chromatography Coupled with a Charged Aerosol Detector

Supramolecular hydrogels formed through polymer-nanoparticle interactions are promising biocompatible materials for translational medicines. This class of hydrogels exhibits shear-thinning behavior and rapid recovery of mechanical properties following applied stresses, providing desirable attributes for formulating sprayable and injectable therapeutics. Characterization of hydrogel composition and loading of encapsulated drugs is critical to achieving desired rheological behavior as well as tunable in vitro and in vivo payload release kinetics. However, quantitation of hydrogel compositions is challenging due to material complexity, heterogeneity, high molecular weight, and the lack of chromophores. Here, we present a label-free approach to simultaneously determine hydrogel polymeric components and encapsulated payloads by coupling a reversed phase liquid chromatographic method with a charged aerosol detector (RPLC-CAD). The hydrogel studied consists of modified hydroxypropylmethylcellulose, self-assembled PEG-b-PLA nanoparticles, and a therapeutic compound, Bimatoprost. The three components were resolved and quantitated using the RPLC-CAD method with a C4 stationary phase. The method demonstrated robust performance, applicability to alternative cargos (i.e. proteins), and was suitable for composition analysis as well as for evaluating in vitro release of cargos from the hydrogel. Moreover, this method can be used to monitor polymer degradation and material stability, which can be further elucidated by coupling the RPLC method with high resolution mass spectrometry and a Fourier-transform based deconvolution algorithm. To our knowledge, this is the first RPLC-CAD method for characterizing the critical quality attributes of supramolecular hydrogels. We envision this analytical strategy could be generalized to characterize other classes of supramolecular hydrogels, establish structure-property relationships, and provide rational design guidance in hydrogel drug product development.

biochemistry↗

bioRxiv2023

Kaposi's sarcoma-associated herpesvirus glycoprotein K8.1 is critical for infection in a cell-specific manner and functions at the attachment step on keratinocytes

Kaposis sarcoma-associated herpesvirus (KSHV) is associated with Kaposis sarcoma and several B cell malignancies. K8.1, the major antigenic component of the KSHV virion, has been reported to play a critical role in the infection of certain B cells, but otherwise its function remains enigmatic. We created a K8.1 knockout virus (KSHV{Delta}K8.1) in the BAC16 genetic background and analyzed its infectivity on a range of adherent cells. We observed a strong defect on several epithelial cells, e.g. the HaCaT keratinocyte model cell line, HEK 293T and A549 lung epithelial cells, but no such defect on other cells, among them e.g. lymphatic and blood endothelial cells. Mechanistically, we found that reduced infectivity of the K8.1 knockout virus correlated with reduced attachment to HaCaT cells. The defect in infectivity of KSHV{Delta}K8.1 could be rescued by complementation through expression of K8.1 in KSHV{Delta}K8.1 producing cells by means of a lentiviral vector. In a coculture infection model, KSHV{Delta}K8.1 was highly efficient at infecting the BJAB B cell line but was significantly impaired at infecting the MC116 B cell line, in line with a previous report. In fusion assays together with the gH/gL glycoprotein complex and gB, the components of the conserved herpesviral core fusion machinery, we did not observe activation of membrane fusion by K8.1 or its R8.1 homolog of the rhesus monkey rhadinovirus. In summary, we found K8.1 to function in a highly cell-specific manner during KSHV entry at the attachment step, playing an important role in the infection of epithelial cells.

microbiology↗

bioRxiv2022

Injectable polymer-nanoparticle hydrogel for the sustained intravitreal delivery of bimatoprost

Vision impairment resulting from chronic eye diseases, such as macular degeneration and glaucoma, severely impacts patients quality of life and poses an immense global financial burden. Current standard of care for such diseases includes daily eye drops or frequent intravitreal (ITV) injections, which are burdensome treatment modalities resulting in low patient compliance. There remains a growing need for easily administered long-acting delivery technologies for prolonging exposure of ocular therapeutics with each administration. Here, we deploy a supramolecular polymer-nanoparticle (PNP) hydrogel for ITV delivery of the glaucoma drug bimatoprost. PNP hydrogels are shear-thinning and self-healing, key properties for injectability, and enable slow release of molecular cargo in vitreous humor (VH) mimics. An in vivo study in New Zealand white rabbits demonstrated intravitreally injected PNP hydrogels form depots that degrade slowly over time, maintaining detectable levels of bimatoprost in the VH up to eight weeks following injection. Ophthalmic examinations and histopathology identified a mild foreign body response (FBR) to the hydrogel, characterized by rare clusters of foamy macrophages and giant cells associated with minimal, patchy fibroplasia. This work shows that PNP hydrogels exhibit numerous desirable traits for sustained drug delivery and further work will be necessary to optimize tolerability in the eye.

bioengineering↗

bioRxiv2022

Stable High-Concentration Monoclonal Antibody Formulations Enabled by an Amphiphilic Copolymer Excipient

Monoclonal antibodies are a staple in modern pharmacotherapy. Unfortunately, these biopharmaceuticals are limited by their tendency to aggregate in formulation, resulting in poor stability and often requiring low concentration drug formulations. Moreover, existing excipients designed to stabilize these formulations are often limited by their toxicity and tendency to form particles such as micelles. Here, we demonstrate the ability of a simple "drop-in", amphiphilic copolymer excipient to enhance the stability of high concentration formulations of clinically-relevant monoclonal antibodies without altering their pharmacokinetics or injectability. Through interfacial rheology and surface tension measurements, we demonstrate that the copolymer excipient competitively adsorbs to formulation interfaces. Further, through determination of monomeric composition and retained bioactivity through stressed aging, we show that this excipient confers a significant stability benefit to high concentration antibody formulations. Finally, we demonstrate that the excipient behaves as an inactive ingredient, having no significant impact on the pharmacokinetic profile of a clinically relevant antibody in mice. This amphiphilic copolymer excipient demonstrates promise as a simple formulation additive to create stable, high concentration antibody formulations, thereby enabling improved treatment options such as a route-of-administration switch from low concentration intravenous (IV) to high concentration subcutaneous (SC) delivery while reducing dependence on the cold chain.

bioengineering↗

bioRxiv2022

Yield Stress and Creep Control Depot Formation and Persistence of Injectable Hydrogels Following Subcutaneous Administration

Hydrogels that can be injected into the body using standard needles or catheters enable a minimally invasive strategy to prolong local delivery of therapeutic drug and cellular cargo. In particular, physically crosslinked hydrogels exhibit shear-thinning and self-healing behaviors enabling facile injectability and depot formation upon administration. While prior efforts to characterize these systems have focused on injectability and cargo release behaviors, prediction of cargo release in the body often assumes the materials form a depot rather than spreading out upon administration. Here, we evaluate how hydrogel rheology correlates with depot formation and persistence following subcutaneous administration in mice with two physicochemically-distinct, physically crosslinked hydrogel systems. We evaluate calcium-alginate and polymer-nanoparticle hydrogel systems exhibiting variable mechanical behaviors across several rheological properties (stiffness, viscoelasticity, yield stress, and creep). By relating measured rheological properties to depot formation and persistence time following subcutaneous administration, we identify that yield stress is predictive of initial depot formation while creep is predictive of depot persistence. Indeed, only materials with yield stresses greater than 25 Pa form robust depots and reduced creep correlates with longer depot persistence. These findings provide predictive insights into design considerations for hydrogel technologies capable of extended controlled release of therapeutic cargo.

bioengineering↗

bioRxiv2021

Delivery of CAR-T Cells in a Transient Injectable Stimulatory Hydrogel Niche Improves Treatment of Solid Tumors

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers such as B cell malignancies, but traditional approaches to ACT are poorly effective in treating the multifarious solid tumors observed clinically. Locoregional cell delivery methods have shown promising results in treating solid tumors compared to standard intravenous delivery methods, but the approaches that have been described to date have several critical drawbacks ranging from complex manufacturing and poor modularity to challenging adminstration. In this work, we develop a simple-to-implement self-assembled and injectable hydrogel material for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improves treatment of solid tumors. We evaluate a range of hydrogel formulations to optimize the creation of a transient inflammatory niche that affords sustained exposure of CAR-T cells and cytokines. This facile approach yields increased CAR-T cell expansion, induces a more tumor-reactive CAR-T phenotype, and improves efficacy in treating solid tumors in mice.

bioengineering↗

bioRxiv2021

Injectable Liposome Based Supramolecular Hydrogels for the Programmable Release of Multiple Protein Drugs

Directing and manipulating biological functions is at the heart of next-generation biomedical initiatives such as tissue and immuno-engineering. Yet, the ambitious goal of engineering complex biological networks requires the ability to precisely perturb specific signaling pathways at distinct times and places. Using lipid nanotechnology and the principles of supramolecular self-assembly, we have developed an injectable liposomal nanocomposite hydrogel platform to precisely control drug presentation through programing of the co-release of multiple protein drugs. These liposomal hydrogels exhibited robust shear-thinning and self-healing behaviors enabling facile injectability for local drug delivery applications. By integrating modular lipid nanotechnology into this hydrogel platform, we introduced multiple mechanisms of protein release based on liposome surface chemistry. When injected into immuno-competent mice, these liposomal hydrogels exhibited formulation-dependent rates of dissolution and excellent biocompatibility. To fully validate the utility of this system for multi-protein delivery, we demonstrated the synchronized, sustained, and localized release of IgG antibody and IL-12 cytokine in vivo, despite the significant size differences between these two proteins. Overall, these liposomal nanocomposite hydrogels are a highly modular platform technology with the ability the mediate orthogonal modes of protein release and the potential to precisely coordinate biological cues both in vitro and in vivo.

bioengineering↗

bioRxiv2021

Interferon-Induced Transmembrane Proteins Inhibit Infection by the Kaposis Sarcoma-Associated Herpesvirus and the Related Rhesus Monkey Rhadinovirus in a Cell Type-Specific Manner.

The interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral proteins that inhibit the entry of enveloped viruses. We analyzed the effect of IFITMs on the gamma2-herpesviruses Kaposis sarcoma-associated herpesvirus (KSHV) and the closely related rhesus monkey rhadinovirus (RRV). We used CRISPR/Cas9-mediated gene knockout to generate A549, human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC) with combined IFITM1/2/3 knockout and identified IFITMs as cell type-dependent inhibitors of KSHV and RRV infection in A549 and HFF but not HUVEC. IFITM overexpression revealed IFITM1 as the relevant IFITM that inhibits KSHV and RRV infection. Fluorescent KSHV particles did not pronouncedly colocalize with IFITM-positive compartments. However, we found that KSHV and RRV glycoprotein-mediated cell-cell fusion is enhanced upon IFITM1/2/3 knockout. Taken together, we identified IFITM1 as a cell type-dependent restriction factor of KSHV and RRV that acts at the level of membrane fusion. Strikingly, we observed that the endotheliotropic KSHV circumvents IFITM-mediated restriction in HUVEC despite high IFITM expression, while influenza A virus (IAV) glycoprotein-driven entry into HUVEC is potently restricted by IFITMs even in the absence of interferon. IMPORTANCEIFITM proteins are the first line of defense against infection by many pathogens, which may also have therapeutic importance, as they, among other effectors, mediate the antiviral effect of interferons. Neither their function against herpesviruses nor their mechanism of action are well understood. We report here that in some cells, but not in, for example, primary umbilical vein endothelial cells, IFITM1 restricts KSHV and RRV, and that, mechanistically, this is likely effected by reducing the fusogenicity of the cell membrane. Further, we demonstrate potent inhibition of IAV glycoprotein-driven infection of cells of extrapulmonary origin by high constitutive IFITM expression.

microbiology↗

bioRxiv2021

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, we explore the ability of a slow-releasing injectable hydrogel depot to reduce the problematic dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining their potent anti-cancer efficacy. We leverage a previously described polymer-nanoparticle (PNP) hydrogel system that exhibits shear-thinning and yield-stress properties that we hypothesized would improve locoregional delivery of the CD40a immunotherapy. Using PET imaging, we demonstrate that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

bioengineering↗

bioRxiv2021

Consistent Tumorigenesis with Self-Assembled Hydrogels Enables High-powered Murine Cancer Studies

Preclinical cancer research is heavily dependent on allograft and xenograft models, but current approaches to tumor inoculation yield inconsistent tumor formation and growth, ultimately wasting valuable resources (e.g., animals, time, and money) and limiting experimental progress. Here we demonstrate a method for tumor inoculation using self-assembled hydrogels to reliably generate tumors with low variance in growth. The observed reduction in model variance enables smaller animal cohorts, improved effect observation and higher powered studies.

bioengineering↗

bioRxiv2021

Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

bioengineering↗

bioRxiv2020

SARS-CoV-2 differs from SARS-CoV in the requirements for receptor expression and proteolytic activation to trigger cell-cell fusion and is not inhibited by Bromhexine

The severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infects cells through interaction of its spike protein (SARS2-S) with Angiotensin-converting enzyme 2 (ACE2) and activation by proteases, in particular transmembrane protease serine 2 (TMPRSS2). Viruses can also spread through fusion of infected with uninfected cells. We compared the requirements of ACE2 expression, proteolytic activation, and the sensitivity to inhibitors for SARS2-S-mediated and SARS-CoV-S(SARS1-S)-mediated cell-cell fusion. SARS2-S-driven fusion was moderately increased by TMPRSS2 and strongly by ACE2, while SARS1-S-driven fusion was strongly increased by TMPRSS2 and less so by ACE2 expression. In contrast to SARS1-S, SARS2-S-mediated cell-cell fusion was efficiently activated by Batimastat-sensitive metalloproteases. Mutation of the S1/S2 proteolytic cleavage site reduced effector-target-cell fusion when ACE2 or TMPRSS2 were limiting and rendered SARS2-S-driven cell-cell fusion more dependent on TMPRSS2. When both ACE2 and TMPRSS2 were abundant, initial target-effector-cell fusion was unaltered compared to wt SARS2-S, but syncytia remained smaller. Mutation of the S2 site specifically abrogated activation by TMPRSS2 for both cell-cell fusion and SARS2-S-driven pseudoparticle entry but still allowed for activation by metalloproteases for cell-cell fusion and by cathepsins for particle entry. Finally, we found that the TMPRSS2 inhibitor Bromhexine was unable to reduce TMPRSS2-activated cell-cell fusion by SARS1-S and SARS2-S as opposed to the inhibitor Camostat. Paradoxically, Bromhexine enhanced cell-cell fusion in the presence of TMPRSS2, while its metabolite Ambroxol exhibited inhibitory activity in some conditions. On Calu-3 lung cells, Ambroxol weakly inhibited SARS2-S-driven lentiviral pseudoparticle entry, and both substances exhibited a dose-dependent trend towards weak inhibition of authentic SARS-CoV-2. IMPORTANCECell-cell fusion allows the virus to infect neighboring cells without the need to produce free virus and contributes to tissue damage by creating virus-infected syncytia. Our results demonstrate that the S2 cleavage site is essential for activation by TMPRSS2 and unravel important differences between SARS-CoV and SARS-CoV-2, among those greater dependence of SARS-CoV-2 on ACE2 expression and activation by metalloproteases for cell-cell fusion. Bromhexine, reportedly an inhibitor of TMPRSS2, is currently tested in clinical trials against coronavirus disease 2019. Our results indicate that Bromhexine enhances fusion in some conditions. We therefore caution against use of Bromhexine in higher dosage until its effects on SARS-CoV-2 spike activation are better understood. The related compound Ambroxol, which similarly to Bromhexine is clinically used as an expectorant, did not exhibit activating effects on cell-cell fusion. Both compounds exhibited weak inhibitory activity against SARS-CoV-2 infection at high concentrations, which might be clinically attainable for Ambroxol.

microbiology↗

bioRxiv2020

Plxdc family members are novel receptors for the rhesus monkey rhadinovirus (RRV)

The rhesus monkey rhadinovirus (RRV), a {gamma}2-herpesvirus of rhesus macaques, shares many biological features with the human pathogenic Kaposis sarcoma-associated herpesvirus (KSHV). Both viruses, as well as the more distantly related Epstein-Barr virus, engage cellular receptors from the Eph family of receptor tyrosine kinases (Ephs). However, the importance of the Eph interaction for RRV entry varies between cell types suggesting the existence of Eph-independent entry pathways. We therefore aimed to identify additional cellular receptors for RRV by affinity enrichment and mass spectrometry. We identified an additional receptor family, the Plexin domain containing proteins 1 and 2 (Plxdc1/2) that bind the RRV gH/gL glycoprotein complex. Preincubation of RRV with soluble Plxdc2 decoy receptor reduced infection by approx. 60%, while overexpression of Plxdc1 and 2 dramatically enhanced RRV susceptibility of otherwise marginally permissive Raji cells. While the Plxdc2 interaction is conserved between two RRV strains, 26-95 and 17577, Plxdc1 specifically interacts with RRV 26-95 gH. The Plxdc interaction is mediated by a short motif at the N-terminus of RRV gH that is partially conserved between isolate 26-95 and isolate 17577, but absent in KSHV gH. Mutation of this motif abrogated the interaction with Plxdc1/2 in in vitro assays and reduced RRV infection in a cell-type specific manner. Taken together, our findings characterize Plxdc1/2 as novel interaction partners and entry receptors for RRV and support the concept of the N-terminal domain of the gammaherpesviral gH/gL complex as a multifunctional receptor-binding domain. Further, Plxdc1/2 usage defines an important biological difference between KSHV and RRV. AUTHORS SUMMARYKSHV is the causative agent of a group of malignancies which account for a substantial disease burden in particular in sub-Saharan Africa. RRV, a related virus of rhesus macaques, has shown promise as model system for KSHV and for the development of immunization strategies. To exploit the full potential of the RRV animal model system, detailed knowledge of commonalities and differences between KSHV and RRV is key. Here, we describe the Plexin domain containing proteins 1 and 2 as a novel receptor family which mediates entry of RRV, but not of KSHV. Infection experiments using RRV mutants deleted of the Plxdc interaction motif suggest a cell type-specific contribution of Plxdc receptors to RRV infection. As information on Plxdc1/2 and its biological function is still sparse, analysis of the RRV-Plxdc interaction will help to characterize the physiological and pathophysiological role of this receptor family.

microbiology↗