Safety and Immunogenicity of Influenza A/H5N8 Virus Vaccine in Healthy Adults: Durability and Cross-reactivity of Antibody Responses.

BACKGROUND: Influenza A/H5N8 viruses infect poultry and wild birds in many countries. In 2021, the first human A/H5N8 cases were reported. METHODS: We conducted a phase I, cohort-randomized, double-blind, controlled trial of inactivated influenza A/H5N8 vaccine (clade 2.3.4.4c) administered with or without adjuvant. Cohort 1 subjects received either two doses of AS03-adjuvanted vaccine containing 3.75 µg or 15 µg hemagglutinin (HA); two doses of 15 µg HA unadjuvanted vaccine; or one dose of AS03-adjuvanted vaccine (3.75 µg or 15 µg HA), followed by one dose of non-adjuvanted vaccine (same HA content). Cohort 2 subjects received two doses of MF59-adjuvanted vaccine containing 3.75 µg or 15 µg HA, or 15 µg HA of non-adjuvanted vaccine. Subjects were followed for 13 months for safety and immunogenicity. RESULTS: We enrolled 386 adult subjects in good health. Solicited adverse events were generally mild and more common among subjects who received adjuvanted vaccines. Antibody responses (hemagglutination inhibition or microneutralization assays) were highest in the two-dose AS03 group, followed by the one-dose AS03 group, the MF59 groups, and the non-adjuvanted groups. Antibody levels returned to baseline 12 months after the second vaccination in all groups except the 15 µg AS03-adjuvanted group. Cross-reactive antibodies to clade 2.3.4.4b strains isolated from recent human cases were demonstrated in a subset of both 15 µg adjuvanted groups. CONCLUSIONS: Two doses of influenza A/H5N8 vaccine were well-tolerated. Immunogenicity improved with receipt of two doses of adjuvanted vaccine and higher antigen content. (Funded by the National Institute of Allergy and Infectious Diseases.

Osmotic damage as a predictor of motility loss during convective desiccation of bovine sperm.

Current state-of-the art technologies are lagging in the application of desiccation storage to mammalian cells using nonreducing sugars. For bovine sperm, motility is irreversibly lost before reaching a sufficiently low moisture content necessary for preservation. It is hypothesized that much of the damage during drying is related to the osmotic stress encountered due to increased osmolarity of the extracellular environment. To test this hypothesis, we subjected sperm to liquid hyperosmotic environments for varying time-periods and measured their motility. We then extracted parameters for two models for motility loss based on these experiments: a first-order rate injury model (Fast or Slow) and a multi-modal (MM) injury model. The MM injury model incorporated an additional function accounting for damage induced by a time-independent osmotic change. Based on these models, we predicted sperm motility loss measured from natural and forced convective desiccation experiments. The MM injury model was able to closely bracket motility loss for desiccation as an osmotic change event with time-independent and time-dependent components. While the mechanistic basis of osmotic damage requires further exploration, the model can serve as a bracketing tool for predicting motility loss during desiccation based on excipients designed to minimize osmotic damage.

A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm.

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode's buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of "intracellular+extracellular" trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.

Desiccation tolerance in bovine sperm: a study of the effect of intracellular sugars and the supplemental roles of an antioxidant and a chelator.

Desiccation preservation holds promise as a simplified alternative to cryopreservation for the long term storage of cells. We report a study on the protective effects of intracellular and extracellular sugars during bovine sperm desiccation and the supplemental effects of the addition of an antioxidant (catalase) or a chelator (desferal). The goal of the study was to preserve mammalian sperm in a partially or completely desiccated state. Sperm loaded intracellularly with two different types of sugars, trehalose or sucrose, were dried with and without catalase and desferal and evaluated for motility and membrane integrity immediately after rehydration. Intracellular sugars were loaded using ATP induced poration. Drying was performed in desiccator boxes maintained at 11% relative humidity (RH). Results indicated that sperm exhibited improved desiccation tolerance if they were loaded with either intracellular trehalose or sucrose. Survival was further enhanced by the addition of 1mM desferal to the desiccation buffer. Though sperm motility after drying to low dry basis water fractions (DBWF) did not show significant improvement under any of the tested conditions, there was an increase in the sperm membrane integrity that could be retained after partial desiccation through the use of intracellular sugars and desferal.

Developing a predictive tool for reactive oxygen species damage during bovine sperm storage at ambient temperature.

Preservation of sperm at ambient storage temperature (25°C) provides a simplified alternative for short-term storage applications. The goals of the study were (i) to improve the storage ability of bovine sperm in liquid suspension by scavenging the reactive oxygen species (ROS) with the help of antioxidants and chelators, (ii) to develop a predictive tool to determine sperm motility as a function of storage temperature. Two types of antioxidants (catalase and superoxide dismutase, SOD) and 2 chelators (Desferal and ethylenediaminetetraacetic acid, EDTA) were used. Different concentrations of the supplements were added in egg yolk Tris (EYT) extender and stored at 4°C, room temperature (25°C), and 37°C, respectively. Motility and membrane integrity (MI) were assessed on days 1, 4, 7, 10, and 14 for samples stored at 4°C and 25°C. Results did not suggest the beneficial effect of SOD and EDTA. Catalase caused a slight improvement in motility at 4°C storage and its effectiveness was found to increase at higher storage temperature. The presence of Desferal resulted in a much higher increase in motility and membrane integrity at both 4°C and 25°C. Similarly, the fitting of Arrhenius damage model to the motility data provided a useful tool to predict motility degradation time as a function of storage temperature.

Moisture sorption characteristics and thermophysical properties of trehalose-PBS mixtures.

The goal of the study was to quantify the thermophysical properties and the moisture sorption characteristics of the trehalose-PBS (phosphate-buffered saline) from the desiccation preservation perspective. A moisture sorption study was undertaken to determine the desorption isotherms of the trehalose-PBS mixtures. The Brunauer, Emmett, and Teller (BET)-equation and the Guggenheim, Anderson, and de Boer equation were used to quantify the desorption data. The glass transition temperature of the mixtures of trehalose-PBS, equilibrated at different relative humidities was studied using a differential scanning calorimeter. Fourier transform infrared spectroscopy was used to study the molecular interaction between the trehalose and PBS mixtures. The results showed that the addition of PBS to the trehalose mixture causes a shift from the type II isotherm to a type III isotherm (characterized by BET equation) which may have detrimental effect on cell desiccation. The results showed that an increase in PBS mass fraction in the trehalose-PBS mixture causes a decrease in the glass transition temperature (Tg) of the mixture and also a decrease in the hydrogen bonding capacity of the trehalose glasses. The addition of PBS to trehalose posed some challenges and should be subject to further optimization to use it in desiccation preservation of biologics.