Intranasal administration of unadjuvanted SARS-CoV-2 spike antigen boosts antigen-specific immune responses induced by parenteral protein subunit vaccine prime in mice and hamsters.

With the continued transmission of SARS-CoV-2 across widely vaccinated populations, it remains important to develop new vaccines and vaccination strategies capable of providing protective immunity and limiting the spread of disease. Heterologous prime-boost vaccination based on the selection of different vaccine formulations and administration routes for priming and booster doses presents a promising strategy for inducing broader immune responses in key systemic and respiratory mucosal compartments. Intranasal vaccination can induce mucosal immune responses at the site of SARS-CoV-2 infection; however, the lack of clinically approved mucosal adjuvants makes it difficult to induce robust immune responses with protein subunit vaccines. Herein, we evaluated the immunogenicity of heterologous prime-boost regimens in mice and hamsters based on a parenteral vaccination of the antigen in combination with sulfated lactosylarchaeol (SLA) archaeosomes, a liposome adjuvant comprised of a single semisynthetic archaeal lipid, followed by an intranasally administered unadjuvanted SARS-CoV-2 spike antigen. Intranasal administration of unadjuvanted spike to mice and hamsters increased serum spike-specific IgG titers and spike-neutralizing activity compared with nonboosted animals. Spike-specific IgA responses were also detected in the bronchoalveolar lavage fluid in the lungs of mice that received an intranasal boost. In hamsters, the intranasal boost showed high efficacy against SARS-CoV-2 infection by protecting from body weight loss and reducing viral titers in the lungs and nasal turbinate. Overall, our heterologous intramuscular prime-intranasal boost with SLA-adjuvanted and unadjuvanted spike, respectively, demonstrated the potential of protein subunit formulations to promote antigen-specific systemic and mucosal immune responses.

The Effects of High Pressure and High Temperature in Semidilute Aqueous Cellulose Nanocrystal Suspensions.

A semidilute cellulose nanocrystal suspension was tested for pressure, volume, temperature dependencies of its viscosity and density. The compression of a 2.0 wt % cellulose nanocrystal suspension under 5.0 MPa at room temperature resulted in morphological changes from istotropic to nematic form. However, at high temperature, high-pressure treatment caused desulfation and gelation. Those results have significant applications, not only as additives in drilling and fracturing fluids but also for the preparation of hydrogels.

Cationic poly(2-aminoethylmethacrylate) and poly(N-(2-aminoethylmethacrylamide) modified cellulose nanocrystals: synthesis, characterization, and cytotoxicity.

Cellulose nanocrystals (CNCs) continue to gain increasing attention in the materials community as sustainable nanoparticles with unique chemical and mechanical properties. Their nanoscale dimensions, biocompatibility, biodegradability, large surface area, and low toxicity make them promising materials for biomedical applications. Here, we disclose a facile synthesis of poly(2-aminoethylmethacrylate) (poly(AEM)) and poly(N-(2-aminoethylmethacrylamide) (poly(AEMA)) CNC brushes via the surface-initiated single-electron-transfer living radical polymerization technique. The resulting modified CNCs were characterized for their chemical and morphological features using a combination of analytical, spectroscopic, and microscopic techniques. Zeta potential measurements indicated a positive surface charge, and further proof of the cationic nature was confirmed by gold deposition as evidenced by electron microscopy. The cytotoxicity of these cationic modified CNCs was evaluated utilizing a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in two different cell lines, J774A1 (mouse monocyte cells) and MCF-7 (human breast adenocarcinoma cells). The results indicated that none of the cationic modified CNCs decreased cell viability at low concentrations, which could be suitable for biomedical applications.

Fabrication of thermo-responsive multicore microcapsules using a facile extrusion process.

A process employing extrusion was used to produce multicore microcapsules composed of multiple beads. The inner beads were made from κ-carrageenan (κ-c), a thermo-responsive linear sulphated polymer whose gelling temperature ranges at 40-60 °C, depending on the concentration of κ-c polymer and the amount of potassium chloride used for gelation. The resulting beads were then enveloped by chitosan through gelation with sodium triphosphate. The pesticide ammonium glufosinate was encapsulated in the κ-c/chitosan multicore microcapsules for demonstration of controlled release of the encapsulant. It was found that in response to an external stimulus, such as elevated temperature or solar simulation, the microcapsules exhibit the gradual release of encapsulated pesticide molecules from multicore microcapsules, compared with beads only. This process of making multicore microcapsules can be extended to other polymer pairs based on applications. This work is relevant to agriculture, where the controlled-release of the pesticides or fertilizers could be triggered by the sun and/or temperature changes, thus extending the residual period of the chemicals as well as decreasing the extent of pollution by leaching of abundant chemicals.

Recent advances in cellulose nanocrystals-based antimicrobial agents.

Over the past five years, there has been growing interest in the design of modified cellulose nanocrystals (CNCs) as nanoscale antimicrobial agents in potential end-user applications such as food preservation/packaging, additive manufacturing, biomedical and water purification. The interest of applying CNCs-based antimicrobial agents arise due to their abilities to be derived from renewable bioresources and their excellent physicochemical properties including rod-like morphologies, large specific surface area, low toxicity, biocompatibility, biodegradability and sustainability. The presence of ample surface hydroxyl groups further allows easy chemical surface modifications for the design of advanced functional CNCs-based antimicrobial materials. Furthermore, CNCs are used to support antimicrobial agents that are subjected to instability issues. The current review summarizes recent progress in CNC-inorganic hybrid-based materials (Ag and Zn nanoparticles, other metal/metal oxide) and CNC-organic hybrid-based materials (polymers, chitosan, simple organic molecules). It focuses on their design, syntheses and applications with a brief discussion on their probable modes of antimicrobial action whereby the roles of CNCs and/or the antimicrobial agents are highlighted.

Delivery of siRNA using cationic rosette nanotubes for gene silencing.

The quest for new therapeutic treatments for hereditary diseases has led to many advances in RNA interference (RNAi) and gene silencing. While this technique has the potential to address many problems, the key to its continued use is the development of effective delivery strategies that would reduce cellular toxicity and increase silencing efficiency. Rosette nanotubes (RNTs) are biomimetic supramolecular nanostructures formed through the self-assembly of hybrid guanine-cytosine (G∧C) DNA bases. Here, we used bioactive RNTs for siRNA delivery and gene silencing. Fifteen lysine-functionalized twin-G∧C motifs (KnT, n = 1 to 15) were synthesized using solid phase peptide synthesis to produce building blocks that self-assembled to produce cationic RNTs under physiological conditions. The intracellular uptake of siRNA delivered by the oligo-L-lysine RNTs was examined and it was found that the complexation of siRNA was affected by the cationic charges from the lysine residues and the length of RNTs formed, with the higher charged KnT RNTs delivering siRNA to the cells at a faster rate. In addition, by protecting siRNA from serum degradation, KnT RNTs were shown to deliver their cargo to the cells effectively via the endocytic pathway. A reduction in the expression (∼70%) of the target stat3 protein was observed during gene expression analysis in HCT116 and A549 cell lines.

Tuning the immune response: sulfated archaeal glycolipid archaeosomes as an effective vaccine adjuvant for induction of humoral and cell-mediated immunity towards the SARS-CoV-2 Omicron variant of concern.

Liposomes composed of sulfated lactosyl archaeol (SLA) have been shown to be a safe and effective vaccine adjuvant with a multitude of antigens in preclinical studies. In particular, SLA-adjuvanted SARS-CoV-2 subunit vaccines based on trimeric spike protein antigens were shown to be immunogenic and efficacious in mice and hamsters. With the continued emergence of SARS-CoV-2 variants, we sought to evaluate next-generation vaccine formulations with an updated antigenic identity. This was of particular interest for the widespread Omicron variant, given the abundance of mutations and structural changes observed within its spike protein compared to other variants. An updated version of our resistin-trimerized SmT1 corresponding to the B.1.1.529 variant was successfully generated in our Chinese Hamster Ovary (CHO) cell-based antigen production platform and characterized, revealing some differences in protein profile and ACE2 binding affinity as compared to reference strain-based SmT1. We next evaluated this Omicron-based spike antigen for its immunogenicity and ability to generate robust antigen-specific immune responses when paired with SLA liposomes or AddaS03 (a mimetic of the AS03 oil-in-water emulsion adjuvant system found in commercialized SARS-CoV-2 protein vaccines). Immunization of mice with vaccine formulations containing this updated antigen with either adjuvant stimulated neutralizing antibody responses favouring Omicron over the reference strain. Cell-mediated responses, which play an important role in the neutralization of intracellular infections, were induced to a much higher degree with the SLA adjuvant relative to the AddaS03-adjuvanted formulations. As such, updated vaccines that are better capable of targeting towards SARS-CoV-2 variants can be generated through an optimized combination of antigen and adjuvant components.

Sulfated Lactosyl Archaeol Archaeosome-Adjuvanted Vaccine Formulations Targeting Rabbit Hemorrhagic Disease Virus Are Immunogenic and Efficacious.

Vaccines play an important role in maintaining human and animal health worldwide. There is continued demand for effective and safe adjuvants capable of enhancing antigen-specific responses to a target pathogen. Rabbit hemorrhagic disease virus (RHDV) is a highly contagious calicivirus that often induces high mortality rates in rabbits. Herein, we evaluated the activity of an experimental sulfated lactosyl archaeol (SLA) archaeosome adjuvant when incorporated in subunit vaccine formulations targeting RHDV. The subunit antigens consisted of RHDV-CRM197 peptide conjugates or recombinant RHDV2 VP60. SLA was able to enhance antigen-specific antibody titers and cellular responses in mice and rabbits. Three weeks following immunization, antigen-specific antibody levels in rabbits vaccinated with RHDV2 VP60 + SLA were significantly higher than those immunized with antigen alone, with geomean titers of 7393 vs. 117. In addition, the SLA-adjuvanted VP60-based formulations were highly efficacious in a rabbit RHDV2 challenge model with up to 87.5% animals surviving the viral challenge. These findings demonstrate the potential utility of SLA adjuvants in veterinary applications and highlight its activity in different types of mammalian species.

Design of chitosan nanocrystals decorated with amino acids and peptides.

Antimicrobial peptides (AMPs) offer a great promise in designing new therapeutics due to their ability to interfere in bacterial growth by penetrating the cell wall. The overuse of antibiotics has resulted into antibiotic-resistant bacteria and AMPs could be an alternative to circumvent this resistance. Chitosan nanocrystals (ChsNCs) are rod-shaped polysaccharide-based nanomaterials, formed by deacetylation of seafood waste. They possess primary amino groups on the surface of the nanoparticles which can be as used a scaffold due to the built-in morphology and ease in functionalization. Here, we developed a new methodology to functionalize ChsNCs with amino acids and peptides by using fundamentals of solid phase peptide synthesis. The resulting functionalized rod-shaped nanomaterials were characterized using nuclear magnetic resonance (NMR), dynamic light scattering (DLS), zeta potential measurements and microscopy imaging. This synthetic strategy could be used in designing ChsNC-based nanomaterials to target specific cells by attaching bioactive peptides to the nanomaterial surface.

Effect of Chiral Purity on Adjuvanticity of Archaeol-Based Glycolipids.

Archaeosomes composed of sulfated lactosyl archaeol (SLA) glycolipids from stereoisomerically pure archaeol (1) are vaccine adjuvants that can boost immunogenicity and vaccine efficacy in preclinical models. Herein, we report a new synthesis of 2,3-bis((3,7,11,15-tetramethylhexadecyl)oxy) propan-1-ol (3) by treating (±)-3-benzyloxy-1,2-propanediol with a mesylated phytol derivative through a double nucleophilic substitution reaction, followed by reductive debenzylation. Three SLA archaeosomes from archaeols of different chiral purities were prepared, and the effect of stereochemistry on their adjuvanticity toward ovalbumin was investigated. It was found that all SLA archaeosomes induced strong humoral and cell-mediated antigen-specific immune responses following immunization of C57BL/6NCrl mice, with no significant differences, irrespective of the chiral purities. The responses were comparable or better than those obtained using mimetics of approved adjuvants. The performance of SLA archaeosomes during immunization and their lack of dependence on the stereochemistry of archaeol points toward a promising, safe, scalable, and economically viable vaccine adjuvant system.

Immunogenicity of SARS-CoV-2 spike antigens derived from Beta & Delta variants of concern.

Using our strongly immunogenic SmT1 SARS-CoV-2 spike antigen platform, we developed antigens based on the Beta & Delta variants of concern (VOC). These antigens elicited higher neutralizing antibody activity to the corresponding variant than comparable vaccine formulations based on the original reference strain, while a multivalent vaccine generated cross-neutralizing activity in all three variants. This suggests that while current vaccines may be effective at reducing severe disease to existing VOC, variant-specific antigens, whether in a mono- or multivalent vaccine, may be required to induce optimal immune responses and reduce infection against arising variants.

Preparation and Surface Functionalization of Carboxylated Cellulose Nanocrystals.

In recent years, cellulose nanocrystals (CNCs) have emerged as a leading biomass-based nanomaterial owing to their unique functional properties and sustainable resourcing. Sulfated cellulose nanocrystals (sCNCs), produced by sulfuric acid-assisted hydrolysis of cellulose, is currently the predominant form of this class of nanomaterial; its utilization leads the way in terms of CNC commercialization activities and industrial applications. The functional properties, including high crystallinity, colloidal stability, and uniform nanoscale dimensions, can also be attained through carboxylated cellulose nanocrystals (cCNCs). Herein, we review recent progress in methods and feedstock materials for producing cCNCs, describe their functional properties, and discuss the initial successes in their applications. Comparisons are made to sCNCs to highlight some of the inherent advantages that cCNCs may possess in similar applications.

The Synergistic Effects of Sulfated Lactosyl Archaeol Archaeosomes When Combined with Different Adjuvants in a Murine Model.

Archaeosomes, composed of sulfated lactosyl archaeol (SLA) glycolipids, have been proven to be an effective vaccine adjuvant in multiple preclinical models of infectious disease or cancer. SLA archaeosomes are a promising adjuvant candidate due to their ability to strongly stimulate both humoral and cytotoxic immune responses when simply admixed with an antigen. In the present study, we evaluated whether the adjuvant effects of SLA archaeosomes could be further enhanced when combined with other adjuvants. SLA archaeosomes were co-administered with five different Toll-like Receptor (TLR) agonists or the saponin QS-21 using ovalbumin as a model antigen in mice. Both humoral and cellular immune responses were greatly enhanced compared to either adjuvant alone when SLA archaeosomes were combined with either the TLR3 agonist poly(I:C) or the TLR9 agonist CpG. These results were also confirmed in a separate study using Hepatitis B surface antigen (HBsAg) and support the further evaluation of these adjuvant combinations.

Sulfated Lactosyl Archaeol Archaeosomes Synergize with Poly(I:C) to Enhance the Immunogenicity and Efficacy of a Synthetic Long Peptide-Based Vaccine in a Melanoma Tumor Model.

Cancer remains a leading cause of morbidity and mortality worldwide. While novel treatments have improved survival outcomes for some patients, new treatment modalities/platforms are needed to combat a wider variety of tumor types. Cancer vaccines harness the power of the immune system to generate targeted tumor-specific immune responses. Liposomes composed of glycolipids derived from archaea (i.e., archaeosomes) have been shown to be potent adjuvants, inducing robust, long-lasting humoral and cell-mediated immune responses to a variety of antigens. Herein, we evaluated the ability of archaeosomes composed of sulfated lactosyl archaeol (SLA), a semi-synthetic archaeal glycolipid, to enhance the immunogenicity of a synthetic long peptide-based vaccine formulation containing the dominant CD8+ T cell epitope, SIINFEKL, from the weakly immunogenic model antigen ovalbumin. One advantage of immunizing with long peptides is the ability to include multiple epitopes, for example, the long peptide antigen was also designed to include the immediately adjacent CD4+ epitope, TEWTSSNVMEER. SLA archaeosomes were tested alone or in combination with the toll-like receptor 3 (TLR3) agonist Poly(I:C). Overall, SLA archaeosomes synergized strongly with Poly(I:C) to induce robust antigen-specific CD8+ T cell responses, which were highly functional in an in vivo cytolytic assay. Furthermore, immunization with this vaccine formulation suppressed tumor growth and extended mouse survival in a mouse melanoma tumor model. Overall, the combination of SLA archaeosomes and Poly(I:C) appears to be a promising adjuvant system when used along with long peptide-based antigens targeting cancer.

Immunogenic and efficacious SARS-CoV-2 vaccine based on resistin-trimerized spike antigen SmT1 and SLA archaeosome adjuvant.

The huge worldwide demand for vaccines targeting SARS-CoV-2 has necessitated the continued development of novel improved formulations capable of reducing the burden of the COVID-19 pandemic. Herein, we evaluated novel protein subunit vaccine formulations containing a resistin-trimerized spike antigen, SmT1. When combined with sulfated lactosyl archaeol (SLA) archaeosome adjuvant, formulations induced robust antigen-specific humoral and cellular immune responses in mice. Antibodies had strong neutralizing activity, preventing viral spike binding and viral infection. In addition, the formulations were highly efficacious in a hamster challenge model reducing viral load and body weight loss even after a single vaccination. The antigen-specific antibodies generated by our vaccine formulations had stronger neutralizing activity than human convalescent plasma, neutralizing the spike proteins of the B.1.1.7 and B.1.351 variants of concern. As such, our SmT1 antigen along with SLA archaeosome adjuvant comprise a promising platform for the development of efficacious protein subunit vaccine formulations for SARS-CoV-2.

Cellular Delivery of Plasmid DNA into Wheat Microspores Using Rosette Nanotubes.

Plant genetic engineering offers promising solutions to the increasing demand for efficient, sustainable, and high-yielding crop production as well as changing environmental conditions. The main challenge for gene delivery in plants is the presence of a cell wall that limits the transportation of genes within the cells. Microspores are plant cells that are, under the right conditions, capable of generating embryos, leading to the formation of haploid plants. Here, we designed cationic and fluorescent rosette nanotubes (RNTs) that penetrate the cell walls of viable wheat microspores under mild conditions and in the absence of an external force. These nanomaterials can capture plasmid DNA to form RNT-DNA complexes and transport their DNA cargo into live microspores. The nanomaterials and the complexes formed were nontoxic to the microspores.

Mechanistic insight into the induction of cellular immune responses by encapsulated and admixed archaeosome-based vaccine formulations.

Archaeosomes are liposomes formulated using total polar lipids (TPLs) or semi-synthetic glycolipids derived from archaea. Conventional archaeosomes with entrapped antigen exhibit robust adjuvant activity as demonstrated by increased antigen-specific humoral and cell-mediated responses and enhanced protective immunity in various murine infection and cancer models. However, antigen entrapment efficiency can vary greatly resulting in antigen loss during formulation and variable antigen:lipid ratios. In order to circumvent this, we recently developed an admixed archaeosome formulation composed of a single semi-synthetic archaeal lipid (SLA, sulfated lactosylarchaeol) which can induce similarly robust adjuvant activity as an encapsulated formulation. Herein, we evaluate and compare the mechanisms involved in the induction of early innate and antigen-specific responses by both admixed (Adm) and encapsulated (Enc) SLA archaeosomes. We demonstrate that both archaeosome formulations result in increased immune cell infiltration, enhanced antigen retention at injection site and increased antigen uptake by antigen-presenting cells and other immune cell types, including neutrophils and monocytes following intramuscular injection to mice using ovalbumin as a model antigen. In vitro studies demonstrate SLA in either formulation is preferentially taken up by macrophages. Although the encapsulated formulation was better able to induce antigen-specific CD8+ T cell activation by dendritic cells in vitro, both encapsulated and admixed formulations gave equivalently enhanced protection from tumor challenge when tested in vivo using a B16-OVA melanoma model. Despite some differences in the immunostimulatory profile relative to the SLA (Enc) formulation, SLA (Adm) induces strong in vivo immunogenicity and efficacy, while offering an ease of formulation.

Mechanisms of the immune response cause by cationic and anionic surface functionalized cellulose nanocrystals using cell-based assays.

The interest in functionalized cellulose nanocrystals (CNCs) for multiple biomedical application has been increasing in recent years. CNCs are suitable to functionalization with an array of polymers, generating chemically related nanomaterials with different morphologies, surface charges that can affect bioreactivity, including immune response. In this study, we sought to understand the mechanistic differences regarding immunological responses evoked by functionalized CNCs and whether surface charges play a role in this effect. We investigated the effect of a cationic, CNCs-poly(APMA), and an anionic, CNCs-poly(NIPAAm) derivatives on the secretion of inflammatory cytokines, mitochondria-derived ROS and mitochondrial function and antioxidant response as well as on endoplasmic reticulum (ER) stress, in human and murine inflammatory cells. The cationic CNCs-poly(APMA) evoked a more robust immunological response in murine cell line, while the anionic CNCs-poly(NIPAAm) showed a significant NLRP3 inflammasome-dependent and independent immunological response in human monocytes. Moreover, CNCs-poly(NIPAAm) induced greater formation of acidic vesicular organelles, mitochondrial ROS in non-stimulated cells while CNCs-poly(APMA) mainly affected mitochondrial function by decreasing the intracellular ATP. The differences on the biological responses may be related to the surface charges of CNCs, and their likely interactions with intra and extracellular biomolecules.

Effect of surface organic coatings of cellulose nanocrystals on the viability of mammalian cell lines.

Cellulose nanocrystals (CNCs) have emerged as promising candidates for a number of bio-applications. Surface modification of CNCs continues to gain significant research interest as it imparts new properties to the surface of the nanocrystals for the design of multifunctional CNCs-based materials. A small chemical surface modification can potentially lead to drastic behavioral changes of cell-material interactions thereby affecting the intended bio-application. In this work, unmodified CNCs were covalently decorated with four different organic moieties such as a diaminobutane fragment, a cyclic oligosaccharide (ß-cyclodextrin), a thermoresponsive polymer (poly[N-isopropylacrylamide]), and a cationic aminomethacrylamide-based polymer using different synthetic covalent methods. The effect of surface coatings of CNCs and the respective dose-response of the above organic moieties on the cell viability were evaluated on mammalian cell cultures (J774A.1 and MFC-7), using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide and lactate dehydrogenase assays. Overall, the results indicated that cells exposed to surface-coated CNCs for 24 h did not display major changes in cell viability, membrane permeability as well as cell morphology. However, with longer exposure, all these parameters were somewhat affected, which appears not to be correlated with either anionic or cationic surface coatings of CNCs used in this study.

Cellulose nanocrystals: a versatile nanoplatform for emerging biomedical applications.

INTRODUCTION: Cellulose nanocrystals (CNCs) are bio-based nanomaterials typically derived from the acid hydrolysis of the most abundant natural polymer, cellulose. These nanomaterials have garnered significant interest due to their unique properties, such as uniform rod-like shape, high surface area, high strength, liquid crystalline behavior, tailored surface chemistry, biocompatibility, biodegradability, sustainability and non-toxic carbohydrate-based nature. AREAS COVERED: The recent developments in the use of unmodified and modified CNCs as versatile nanoplatforms for emerging biomedical applications such as drug delivery systems, enzyme/protein immobilization scaffolds, bioimaging, biosensing and tissue engineering are highlighted. A brief discussion of the biological and toxicity properties of CNCs is also presented. EXPERT OPINION: While a number of recent studies have indicated that CNCs are promising nanomaterials for biomedical applications, there is a substantial amount of work that still remains to be done before realizing the full therapeutic potential of CNCs. Major effort should be focused on detailed in vitro and in vivo studies of modified CNCs constructs in order to better understand the integration of CNCs in the biological systems.