Identification of film-based formulations that move mRNA lipid nanoparticles out of the freezer.

COVID-19 vaccines consisting of mRNA lipid nanoparticles (LNPs) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein antigen protected millions of people from severe disease; however, they must be stored frozen prior to use. The objective of this study was to evaluate the compatibility and stability of mRNA LNPs within a polymer-based film matrix. An optimized formulation of polymer base, glycerol, surfactants, and PEGylated lipid that prevents damage to the LNP due to physical changes during the film-forming process (osmotic stress, surface tension, spatial stress, and water loss) was identified. Surfactants added to LNP stock prior to mixing with other film components contributed to this effect. Formulations prepared at pH ≥ 8.5 extended transfection efficiency beyond 4 weeks at 4°C when combined with known nucleic acid stabilizers. mRNA LNPs were most stable in films when manufactured in an environment of ∼50% relative humidity. The optimized formulation offers 16-week stability at 4°C.

Atomic-Layer Deposition Processes Applied to Phage λ and a Phage-like Particle Platform Yield Thermostable, Single-Shot Vaccines.

Especially in developing countries, the impact of vaccines can be limited by logistical obstacles associated with multiple dose regimens, pathogen variants, and challenges imposed by requirements for maintaining vaccines at low temperatures during shipping and storage. Thus, there is a need for vaccines that can be flexibly modified to address evolving pathogen landscapes, are stable outside of narrow "cold-chain" temperatures and require administration of only single doses. Here we demonstrate in proof-of-concept studies a vaccine platform that addresses these impediments to more widespread use of vaccines. The platform relies on bacteriophage-derived phage-like-particles (PLPs) that utilize a "plug-and-play" antigen delivery system that allows for fast, easy alteration of antigens on the surface of the PLPs. Thermostability of PLP-based vaccines can be achieved by embedding the PLPs within glassy particles produced by spray drying, and nanoscopic aluminum oxide layers applied using atomic layer deposition (ALD) can serve to control release of antigen in vivo, yielding vaccine formulations that elicit strong immune responses after administration of single doses. Bacteriophage λ was stabilized by spray drying to form powders that were incubated at 37 °C for up to a year without loss of infectious activity. PLPs derived from bacteriophage λ were expressed and purified from E. coli cultures, and an in vitro conjugation strategy was used to decorate specific PLP surface sites with T4-lysozyme, a model vaccine antigen. The resulting T4-lysozyme:PLP complexes (Lys-PLPs) were embedded in glassy dry powders formed by spray drying and coated with nanometer-thick layers of alumina deposited by ALD in a fluidized bed reactor. Alumina-coated Lys-PLP vaccines were stable for a least a month at 50 °C, and single doses of the alumina-coated vaccines elicited immune responses that were indistinguishable from responses generated by conventional two-dose, prime-and-boost dosing regimens of alum-adjuvanted Lys-PLP vaccines.

Evaluation of intermolecular interactions required for thermostability of a recombinant adenovirus within a film matrix.

Thermostability of vaccines and biologic drugs are key to increasing global access to a variety of life-saving agents. In this report, we characterize interactions between a novel zwitterionic surfactant and adenovirus serotype 5 which allow the virus to remain stable at room temperature in a thin film matrix. Complexity of the adenovirus capsid and the polydispersity of the surfactant required use of a variety of techniques to achieve this goal. The CMC of the surfactant in Tris buffer (pH 6.5) was estimated to be 0.7-1.17 × 10-4 M by the pyrene 1:3 ratio method. TEM images depict micelle formation around virus capsids. An estimated Kd of the virus-surfactant interaction of 2.25 × 10-9 M was determined by isothermal titration calorimetry. Associated data suggest that this interaction may be thermodynamically favorable and entropically driven. A competitive saturation study and TEM images indicate that the surfactant also binds to hexon proteins on the virus capsid. Taken together, these data support the working hypothesis that the surfactant is capable of forming micelles in the solid and liquid state and that it forms a protective coating around the virus by binding to hexon proteins on the virus capsid during the film forming process.

The effect of inherent and incidental constraints on bimanual and social coordination.

The current investigation was designed to examine the influence of inherent and incidental constraints on the stability characteristics associated with bimanual and social coordination. Individual participants (N = 9) and pairs of participants (N = 18, 9 pairs) were required to rhythmically coordinate patterns of isometric forces in 1:1 in-phase and 1:2 multi-frequency patterns by exerting force with their right and left limbs. Lissajous information was provided to guide performance. Participants performed 13 practice trials and 1 test trial per pattern. On the test trial, muscle activity from the triceps brachii muscles of each arm was recorded. EMG-EMG coherence between the two EMG signals was calculated using wavelet coherence. The behavioral data indicated that individual participants performed the 1:1 in-phase pattern more accurately and with less variability than paired participants. The EMG coherence analysis indicated significantly higher coherence for individual participants than for the paired participants during the 1:1 in-phase pattern, whereas no differences were observed between groups for the 1:2 coordination pattern. The results of the current investigation support the notion that neural crosstalk can stabilize 1:1 in-phase coordination when contralateral and ipsilateral signals are integrated via the neuromuscular linkage between two effectors.

The Influence of Altered-Gravity on Bimanual Coordination: Retention and Transfer.

Many of the activities associated with spaceflight require individuals to coordinate actions between the limbs (e.g., controlling a rover, landing a spacecraft). However, research investigating the influence of gravity on bimanual coordination has been limited. The current experiment was designed to determine an individual's ability to adapt to altered-gravity when performing a complex bimanual force coordination task, and to identify constraints that influence coordination dynamics in altered-gravity. A tilt table was used to simulate gravity on Earth [90° head-up tilt (HUT)] and microgravity [6° head-down tilt (HDT)]. Right limb dominant participants (N = 12) were required to produce 1:1 in-phase and 1:2 multi-frequency force patterns. Lissajous information was provided to guide performance. Participants performed 14, 20 s trials at 90° HUT (Earth). Following a 30-min rest period, participants performed, for each coordination pattern, two retention trials (Earth) followed by two transfer trials in simulated microgravity (6° HDT). Results indicated that participants were able to transfer their training performance during the Earth condition to the microgravity condition with no additional training. No differences between gravity conditions for measures associated with timing (interpeak interval ratio, phase angle slope ratio) were observed. However, despite the effective timing of the force pulses, there were differences in measures associated with force production (peak force, STD of peak force mean force). The results of this study suggest that Lissajous displays may help counteract manual control decrements observed during microgravity. Future work should continue to explore constraints that can facilitate or interfere with bimanual control performance in altered-gravity environments.