Carbohydrate-Lectin Interactions Reprogram Dendritic Cells to Promote Type 1 Anti-Tumor Immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) is thought to shape immunity by directing T cell function. Controlling downstream responses, however, remains a major challenge. A critical goal in advancing vaccine development involves the identification of receptors that drive type 1 cellular immunity. The immune system monitors cells for aberrant glycosylation (a sign of a foreign entity), but potent activation occurs when a second signal, such as single-stranded RNA or lipopolysaccharide, is present to activate TLR signaling. To exploit dual signaling, we engineered a glycan-costumed virus-like particle (VLP) vaccine that displays a DC-SIGN-selective aryl mannose ligand and encapsulates TLR7 agonists. These VLPs deliver programmable peptide antigens to induce robust DC activation and type 1 cellular immunity. In contrast, VLPs lacking this critical DC-SIGN ligand promoted DC-mediated humoral immunity, offering limited tumor control. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, significantly inhibiting tumor growth in a murine melanoma model. The tailored VLPs also afforded protection against the reintroduction of tumor cells. Thus, DC lectin-driven immune reprogramming, combined with the modular programmability of VLP platforms, provides a promising framework for directing cellular immunity to advance cancer immunotherapies and vaccines.

Transport of Molecular Cargo by Interaction with Virus-Like Particle RNA.

Virus-like particles (VLPs) derived from Leviviridae virions contain substantial amounts of cellular and plasmid-derived RNA. This encapsidated polynucleotide serves as a reservoir for the efficient binding of the intercalating dye thiazole orange (TO). Polyethylene glycol (PEG) molecules and oligopeptides of varying length, end-functionalized with TO, were loaded into VLPs up to approximately 50 % of the mass of the capsid protein (hundreds to thousands of cargo molecules per particle, depending on size). The kinetics of TO-PEG binding included a significant entropic cost for the reptation of long chains through the capsid pores. Cargo molecules were released over periods of 20-120 hours following simple reversible first-order kinetics in most cases. These observations define a simple general method for the noncovalent packaging, and subsequent release, of functional molecules inside nucleoprotein nanocages in a manner independent of modifications to the capsid protein.

Enveloped Virus Inactivation on Personal Protective Equipment by Exposure to Ozone.

Ozone is a highly oxidizing gas easily generated from atmospheric oxygen with inexpensive equipment and is commonly used for the disinfection of municipal water, foods, and surfaces. We report tests of the ability of ozone to inactivate enveloped respiratory viruses (influenza A virus and respiratory syncytial virus), chosen as more easily handled surrogates for SARS-CoV-2, on N95 respirators and other personal protective equipment (PPE) commonly used in hospitals. At 20 ppm, an ozone concentration easily achieved by standard commercial equipment, the viruses were inactivated with high efficiency as long as the relative humidity was above a threshold value of approximately 50%. In the absence of humidity control, disinfection is more variable and requires considerably longer exposure under relatively dry conditions. This report extends the observations of a previous publication (http://doi.org/10.1080/01919510902747969) to hospital-relevant materials and provides additional details about the relationship of humidity to the antiviral activity of ozone. Home CPAP disinfection devices using ozone can provide effective results for individuals. Ozone did not appear to degrade any of the materials tested except for elastic bands if strained during treatment (such as by the pressure exerted by stapled attachment to N95 respirators). The filtration efficiency of N95 respirator material was not compromised. Overall, we recommend exposures of at least 40 minutes to 20 ppm ozone and >70% relative humidity at ambient temperatures (21-24°C) for 4-log (99.99%) reduction of viral infectivity on a variety of PPE, including gowns, face shields, and respirators. Shorter exposure times are likely to be effective under these conditions, but at the risk of some variability for different materials. Higher ozone concentrations and higher humidity levels promoted faster inactivation of viruses. Our work suggests that ozone exposure can be a widely accessible method for disinfecting PPE, permitting safer re-use for healthcare workers and patients alike in times of shortage.

Neutrophil-Particle Interactions in Blood Circulation Drive Particle Clearance and Alter Neutrophil Responses in Acute Inflammation.

Although nano- and microparticle therapeutics have been studied for a range of drug delivery applications, the presence of these particles in blood flow may have considerable and understudied consequences to circulating leukocytes, especially neutrophils, which are the largest human leukocyte population. The objective of this work was to establish if particulate drug carriers in circulation interfere with normal neutrophil adhesion and migration. Circulating blood neutrophils in vivo were found to be capable of rapidly binding and sequestering injected carboxylate-modified particles of both 2 and 0.5 µm diameter within the bloodstream. These neutrophil-particle associations within the vasculature were found to suppress neutrophil interactions with an inflamed mesentery vascular wall and hindered neutrophil adhesion. Furthermore, in a model of acute lung injury, intravenously administered drug-free particles reduced normal neutrophil accumulation in the airways of C57BL/6 mice between 52% and 60% versus particle-free mice and between 93% and 98% in BALB/c mice. This suppressed neutrophil migration resulted from particle-induced neutrophil diversion to the liver. These data indicate a considerable acute interaction between injected particles and circulating neutrophils that can drive variations in neutrophil function during inflammation and implicate neutrophil involvement in the clearance process of intravenously injected particle therapeutics. Such an understanding will be critical toward both enhancing designs of drug delivery carriers and developing effective therapeutic interventions in diseases where neutrophils have been implicated.

Potent Immune Stimulation from Nanoparticle Carriers Relies on the Interplay of Adjuvant Surface Density and Adjuvant Mass Distribution.

Development of novel adjuvant delivery approaches which provide safe and effective immune stimulation are critical for prophylactic and therapeutic advances in a wide range of diseases. Toll-like receptor agonists (TLRas) have been identified as potent stimulators of antigen presenting cells (APCs) and are capable of inducing proinflammatory immune responses desirable for vaccine and immunostimulatory applications. Although TLRas have been successfully incorporated into nanoparticle platforms, minimal work has been done to evaluate the direct role of the adjuvant incorporation in these formulations in directing the immune response. Here, we developed a series of nanoparticle carriers with controlled surface densities of two TLRas, lipopolysaccharide (LPS), corresponding to TLR-4, and CpG oligodeoxynucleotide, corresponding to TLR-9. The proinflammatory cytokine production and expression of costimulatory molecules on APCs were evaluated following a 24 h particle incubation period in vitro using bone marrow derived macrophages and in vivo following particle instillation in the airway of mice. Results demonstrate that proinflammatory cytokine production is predominantly driven by the distribution of the adjuvant dose to a maximal number of cells, whereas the upregulation of costimulatory molecules needed to drive APC maturation and promote adaptive responses indicate the requirement of an optimal density of TLRa on the particle surface. These results indicate that adjuvant surface density is an important parameter for tight control of immune stimulation and provide a foundation for pathogen mimicking particle (PMP) vaccines and immunostimulatory therapeutics.