Promotion of Th17 Polarized Immunity via Co-Delivery of Mincle Agonist and Tuberculosis Antigen Using Silica Nanoparticles.

Adjuvants and immunomodulators that effectively drive a Th17-skewed immune response are not part of the standard vaccine toolkit. Vaccine adjuvants and delivery technologies that can induce Th17 or Th1/17 immunity and protection against bacterial pathogens, such as tuberculosis (TB), are urgently needed. Th17-polarized immune response can be induced using agonists that bind and activate C-type lectin receptors (CLRs) such as macrophage inducible C-type lectin (Mincle). A simple but effective strategy was developed for codelivering Mincle agonists with the recombinant Mycobacterium tuberculosis fusion antigen, M72, using tunable silica nanoparticles (SNP). Anionic bare SNP, hydrophobic phenyl-functionalized SNP (P-SNP), and cationic amine-functionalized SNP (A-SNP) of different sizes were coated with three synthetic Mincle agonists, UM-1024, UM-1052, and UM-1098, and evaluated for adjuvant activity in vitro and in vivo. The antigen and adjuvant were coadsorbed onto SNP via electrostatic and hydrophobic interactions, facilitating multivalent display and delivery to antigen presenting cells. The cationic A-SNP showed the highest coloading efficiency for the antigen and adjuvant. In addition, the UM-1098-adsorbed A-SNP formulation demonstrated slow-release kinetics in vitro, excellent stability over 12 months of storage, and strong IL-6 induction from human peripheral blood mononuclear cells. Co-adsorption of UM-1098 and M72 on A-SNP significantly improved antigen-specific humoral and Th17-polarized immune responses in vivo in BALB/c mice relative to the controls. Taken together, A-SNP is a promising platform for codelivery and proper presentation of adjuvants and antigens and provides the basis for their further development as a vaccine delivery platform for immunization against TB or other diseases for which Th17 immunity contributes to protection.

Understanding How Cationic Polymers' Properties Inform Toxic or Immunogenic Responses via Parametric Analysis.

Cationic polymers are widely used materials in diverse biotechnologies. Subtle variations in these polymers' properties can change them from exceptional delivery agents to toxic inflammatory hazards. Conventional screening strategies optimize for function in a specific application rather than observing how underlying polymer-cell interactions emerge from polymers' properties. An alternative approach is to map basic underlying responses, such as immunogenicity or toxicity, as a function of basic physicochemical parameters to inform the design of materials for a breadth of applications. To demonstrate the potential of this approach, we synthesized 107 polymers varied in charge, hydrophobicity, and molecular weight. We then screened this library for cytotoxic behavior and immunogenic responses to map how these physicochemical properties inform polymer-cell interactions. We identify three compositional regions of interest and use confocal microscopy to uncover the mechanisms behind the observed responses. Finally, immunogenic activity is confirmed in vivo. Highly cationic polymers disrupted the cellular plasma membrane to induce a toxic phenotype, while high molecular weight, hydrophobic polymers were uptaken by active transport to induce NLRP3 inflammasome activation, an immunogenic phenotype. Tertiary amine- and triethylene glycol-containing polymers did not invoke immunogenic or toxic responses. The framework described herein allows for the systematic characterization of new cationic materials with different physicochemical properties for applications ranging from drug and gene delivery to antimicrobial coatings and tissue scaffolds.

Development of Masitinib Derivatives with Enhanced Mpro Ligand Efficiency and Reduced Cytotoxicity.

Recently, a high-throughput screen of 1900 clinically used drugs identified masitinib, an orally bioavailable tyrosine kinase inhibitor, as a potential treatment for COVID-19. Masitinib acts as a broad-spectrum inhibitor for human coronaviruses, including SARS-CoV-2 and several of its variants. In this work, we rely on atomistic molecular dynamics simulations with advanced sampling methods to develop a deeper understanding of masitinib's mechanism of Mpro inhibition. To improve the inhibitory efficiency and to increase the ligand selectivity for the viral target, we determined the minimal portion of the molecule (fragment) that is responsible for most of the interactions that arise within the masitinib-Mpro complex. We found that masitinib forms highly stable and specific H-bond interactions with Mpro through its pyridine and aminothiazole rings. Importantly, the interaction with His163 is a key anchoring point of the inhibitor, and its perturbation leads to ligand unbinding within nanoseconds. Based on these observations, a small library of rationally designed masitinib derivatives (M1-M5) was proposed. Our results show increased inhibitory efficiency and highly reduced cytotoxicity for the M3 and M4 derivatives compared to masitinib.

Cell-targeted vaccines: implications for adaptive immunity.

Recent advancements in immunology and chemistry have facilitated advancements in targeted vaccine technology. Targeting specific cell types, tissue locations, or receptors can allow for modulation of the adaptive immune response to vaccines. This review provides an overview of cellular targets of vaccines, suggests methods of targeting and downstream effects on immune responses, and summarizes general trends in the literature. Understanding the relationships between vaccine targets and subsequent adaptive immune responses is critical for effective vaccine design. This knowledge could facilitate design of more effective, disease-specialized vaccines.

Size matters in non-canonical inflammasome activation and cell-mediated immunity.

Particulate adjuvants are key components of many approved vaccines, but their mechanism of adjuvanticity is debated. Muñoz-Wolf et al.1 find that 50-nm particles maximize cell-mediated immune responses by activating the caspase-11 inflammasome, providing mechanistic insight to particulate adjuvant technologies.

Immunostimulatory Polymers as Adjuvants, Immunotherapies, and Delivery Systems.

Activating innate immunity in a controlled manner is necessary for the development of next-generation therapeutics. Adjuvants, or molecules that modulate the immune response, are critical components of vaccines and immunotherapies. While small molecules and biologics dominate the adjuvant market, emerging evidence supports the use of immunostimulatory polymers in therapeutics. Such polymers can stabilize and deliver cargo while stimulating the immune system by functioning as pattern recognition receptor (PRR) agonists. At the same time, in designing polymers that engage the immune system, it is important to consider any unintended initiation of an immune response that results in adverse immune-related events. Here, we highlight biologically derived and synthetic polymer scaffolds, as well as polymer-adjuvant systems and stimuli-responsive polymers loaded with adjuvants, that can invoke an immune response. We present synthetic considerations for the design of such immunostimulatory polymers, outline methods to target their delivery, and discuss their application in therapeutics. Finally, we conclude with our opinions on the design of next-generation immunostimulatory polymers, new applications of immunostimulatory polymers, and the development of improved preclinical immunocompatibility tests for new polymers.

In Vitro and in Vivo Analyses of the Effects of Source, Length, and Charge on the Cytotoxicity and Immunocompatibility of Cellulose Nanocrystals.

Cellulose nanocrystals (CNCs) are an emergent, sustainable nanomaterial that are biosourced, abundant, and biodegradable. On account of their high aspect ratio, low density, and mechanical rigidity, they have been employed in numerous areas of biomedical research including as reinforcing materials for bone or tissue scaffolds or as carriers in drug delivery systems. Given the promise of these materials for such use, characterizing and understanding their interactions with biological systems is an important step to prevent toxicity or inflammation. Reported herein are studies aimed at exploring the in vitro and in vivo effects that the source, length, and charge of the CNCs have on cytotoxicity and immune response. CNCs from four different biosources (cotton, wood, Miscanthus x Giganteus, and sea tunicate) were prepared and functionalized with positive or negative charges to obtain a small library of CNCs with a range of dimensions and surface charge. A method to remove endotoxic or other impurities on the CNC surface leftover from the isolation process was developed, and the biocompatibility of the CNCs was subsequently assayed in vitro and in vivo. After subcutaneous injection, it was found that unfunctionalized (uncharged) CNCs form aggregates at the site of injection, inducing splenomegaly and neutrophil infiltration, while charged CNCs having surface carboxylates, sulfate half-esters, or primary amines were biologically inert. No effect of the particle source or length was observed in the in vitro and in vivo studies conducted. The lack of an in vitro or in vivo immune response toward charged CNCs in these experiments supports their use in future biological studies.

Site-specific antigen-adjuvant conjugation using cell-free protein synthesis enhances antigen presentation and CD8+ T-cell response.

Antigen-adjuvant conjugation is known to enhance antigen-specific T-cell production in vaccine models, but scalable methods are required to generate site-specific conjugation for clinical translation of this technique. We report the use of the cell-free protein synthesis (CFPS) platform as a rapid method to produce large quantities (> 100 mg/L) of a model antigen, ovalbumin (OVA), with site-specific incorporation of p-azidomethyl-L-phenylalanine (pAMF) at two solvent-exposed sites away from immunodominant epitopes. Using copper-free click chemistry, we conjugated CpG oligodeoxynucleotide toll-like receptor 9 (TLR9) agonists to the pAMF sites on the mutant OVA protein. The OVA-CpG conjugates demonstrate enhanced antigen presentation in vitro and increased antigen-specific CD8+ T-cell production in vivo. Moreover, OVA-CpG conjugation reduced the dose of CpG needed to invoke antigen-specific T-cell production tenfold. These results highlight how site-specific conjugation and CFPS technology can be implemented to produce large quantities of covalently-linked antigen-adjuvant conjugates for use in clinical vaccines.