A multiadjuvant polysaccharide-amino acid-lipid (PAL) subunit nanovaccine generates robust systemic and lung-specific mucosal immune responses against SARS-CoV-2 in mice.

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, which are essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters could overcome the shortcomings of parenteral vaccines and enhance pre- existing systemic immunity. Here we present a new protein subunit nanovaccine using multiadjuvanted (e.g. RIG-I: PUUC, TLR9: CpG) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL- NPs, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lung, and showed robust systemic humoral immunity. Interestingly, as a purely intranasal vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. Our data suggest that PUUC+CpG PAL-NP subunit vaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

Systemic Immune Modulation Alters Local Bone Regeneration in a Delayed Treatment Composite Model of Non-Union Extremity Trauma.

Bone non-unions resulting from severe traumatic injuries pose significant clinical challenges, and the biological factors that drive progression towards and healing from these injuries are still not well understood. Recently, a dysregulated systemic immune response following musculoskeletal trauma has been identified as a contributing factor for poor outcomes and complications such as infections. In particular, myeloid-derived suppressor cells (MDSCs), immunosuppressive myeloid-lineage cells that expand in response to traumatic injury, have been highlighted as a potential therapeutic target to restore systemic immune homeostasis and ultimately improve functional bone regeneration. Previously, we have developed a novel immunomodulatory therapeutic strategy to deplete MDSCs using Janus gold nanoparticles that mimic the structure and function of antibodies. Here, in a preclinical delayed treatment composite injury model of bone and muscle trauma, we investigate the effects of these nanoparticles on circulating MDSCs, systemic immune profiles, and functional bone regeneration. Unexpectedly, treatment with the nanoparticles resulted in depletion of the high side scatter subset of MDSCs and an increase in the low side scatter subset of MDSCs, resulting in an overall increase in total MDSCs. This overall increase correlated with a decrease in bone volume (P = 0.057) at 6 weeks post-treatment and a significant decrease in mechanical strength at 12 weeks post-treatment compared to untreated rats. Furthermore, MDSCs correlated negatively with endpoint bone healing at multiple timepoints. Single cell RNA sequencing of circulating immune cells revealed differing gene expression of the SNAb target molecule S100A8/A9 in MDSC sub-populations, highlighting a potential need for more targeted approaches to MDSC immunomodulatory treatment following trauma. These results provide further insights on the role of systemic immune dysregulation for severe trauma outcomes in the case of non-unions and composite injuries and suggest the need for additional studies on targeted immunomodulatory interventions to enhance healing.

Failure Mode Identification of Insulin Drug Products - Impact of Relevant Stress Conditions on the Quality of the Drug.

Fast-acting insulin drug products (DPs) are carried and administered by diabetic patients to maintain their blood glucose level throughout the day, exposing the DPs to stress conditions. Apidra, Novolog, and Humalog insulin DPs were tested under various stress conditions. Dynamic light scattering (DLS), and size exclusion chromatography (SEC) were used to monitor the stability and aggregation. Thermal stress alone did not influence the stability. However, 24 hr exposure to vigorous mechanical stress shifted the DLS size peaks of Novolog and Humalog from 5 ± 1 nm to > 50.9 ± 25.6 nm, and the SEC native protein peak areas decreased 52% for Novolog and 18.4% for Humalog. Combined stress accelerated protein aggregation more drastically. Novolog and Humalog size shifted (>75 nm) after 3 hr and the peak area decreased > 97.9% after 6 hr exposure, indicating that high temperature accelerated the aggregation triggered by agitation. Soluble aggregates were captured by DLS early on compared to SEC. Apidra was comparably stable indicating DP formulation plays a critical role in stability. Our study provides a greater understanding of potential failure modes patients and care givers may encounter while handling insulin DPs.

A bilayered skin substitute developed using an eggshell membrane crosslinked gelatin-chitosan cryogel.

Commercially available allografts and xenografts pose problems such as high cost, risk of infection transmission and immune rejection of grafts. Thus, bioengineered skin substitutes fabricated from natural biomaterials or synthetic polymers are currently the focus of skin tissue engineering. In this study, eggshell membrane (ESM) powder was used to crosslink a gelatin-chitosan cryogel thereby replacing glutaraldehyde, a known cytotoxic chemical crosslinker. The resultant ESM-crosslinked macroporous cryogel with a pore size ranging between 10 and 350 µm has improved flexibility, biodegradability and biocompatibility compared to a glutaraldehyde-crosslinked cryogel. For healing of large and deep wounds, bilayered scaffolds which exhibit key aspects of skin physiology are being explored. Hence, we fabricated a bilayered substitute by coupling the ESM-crosslinked cryogel (dermal equivalent) to a non-porous, physically-crosslinked gelatin-chitosan film (epidermal equivalent). The epidermal layer provides the requisite barrier properties while the dermal layer facilitates cell attachment and migration for optimal wound healing. Further, chitosan confers antibacterial properties to the cryogel with almost 50% reduction in bacterial viability. Animal studies confirm that the developed bilayered skin substitute is non-allergic, aids wound healing by improving re-epithelialization within 14 days and supports the formation of skin appendages. This system presents a new and alternative treatment option for burn and chronic wounds.

Coupling Self-Assembly Mechanisms to Fabricate Molecularly and Electrically Responsive Films.

Biomacromolecules often possess information to self-assemble through low energy competing interactions which can make self-assembly responsive to environmental cues and can also confer dynamic properties. Here, we coupled self-assembling systems to create biofunctional multilayer films that can be cued to disassemble through either molecular or electrical signals. To create functional multilayers, we: (i) electrodeposited the pH-responsive self-assembling aminopolysaccharide chitosan, (ii) allowed the lectin Concanavalin A (ConA) to bind to the chitosan-coated electrode (presumably through electrostatic interactions), (iii) performed layer-by-layer self-assembly by sequential contacting with glycogen and ConA, and (iv) conferred biological (i.e., enzymatic) function by assembling glycoprotein (i.e., enzymes) to the ConA-terminated multilayer. Because the ConA tetramer dissociates at low pH, this multilayer can be triggered to disassemble by acidification. We demonstrate two approaches to induce acidification: (i) glucose oxidase can induce multilayer disassembly in response to molecular cues, and (ii) anodic reactions can induce multilayer disassembly in response to electrical cues.