Screening of Oligomeric (Meth)acrylate Vaccine Adjuvants Synthesized via Catalytic Chain Transfer Polymerization.

This report details the first systematic screening of free-radical-produced methacrylate oligomer reaction mixtures as alternative vaccine adjuvant components to replace the current benchmark compound squalene, which is unsustainably sourced from shark livers. Homo-/co-oligomer mixtures of methyl, butyl, lauryl, and stearyl methacrylate were successfully synthesized using catalytic chain transfer control, where the use of microwave heating was shown to promote propagation over chain transfer. Controlling the mixture material properties allowed the correct viscosity to be achieved, enabling the mixtures to be effectively used in vaccine formulations. Emulsions of selected oligomers stimulated comparable cytokine levels to squalene emulsion when incubated with human whole blood and elicited an antigen-specific cellular immune response when administered with an inactivated influenza vaccine, indicating the potential utility of the compounds as vaccine adjuvant components. Furthermore, the oligomers' molecular sizes were demonstrated to be large enough to enable greater emulsion stability than squalene, especially at high temperatures, but are predicted to be small enough to allow for rapid clearance from the body.

Physicochemical and immunological effects of adjuvant formulations with snake venom antigens for immunization of horses for antivenom production.

Enhancement of antivenom immune responses in horses through adjuvant technology improves antivenom production efficiency, but substantial local reactogenicity associated with some traditional veterinary adjuvants limits their usability. To explore modern adjuvant systems suitable for generating antivenom responses in horses, we first assessed their physicochemical compatibility with Bothrops asper snake venom. Liposome and nanoparticle aluminum adjuvants exhibited changes in particle size and phospholipid content after mixing with venom, whereas squalene emulsion-based adjuvants remained stable. Next, we evaluated serum antibody response magnitude and neutralization capacity in horses immunized with adjuvant-containing Echis ocellatus, Bitis arietans, Naja nigricollis, and Dendroaspis polylepis venom preparations. Whereas all tested adjuvants elicited significant neutralization capacity against the viperid venoms, the greatest antibody responses were generated by a squalene-in-water emulsion, thus representing a promising novel alternative for antivenom production.

Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene.

Emulsions of the triterpene squalene ((6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of trans-ß-farnesene ((6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process. The scalability of the proposed route was verified by a kilo-scale GMP synthesis. We also report data demonstrating the synthesized semi-synthetic squalene's physical stability and biological activity when used in a vaccine adjuvant formulation.

Intranasal delivery of a synthetic Entamoeba histolytica vaccine containing adjuvant (LecA + GLA-3 M-052 liposomes): In vitro characterization.

Amebiasis, a disease caused by the parasite Entamoeba histolytica, is estimated to cause millions of infections and at least 55,000 deaths globally each year. With no vaccine currently available, there is an urgent need for an accessible means of stimulating protective mucosal immunity. The objective of this study was to characterize the nasal spray of a novel amebiasis vaccine candidate from a syringe-based liquid atomization device, the Teleflex MAD Nasal™, in both adult and infant nasal airways. Human ergonomic testing was completed to determine realistic actuation parameters. Spray pattern, plume geometry, and droplet size distribution were measured to evaluate reproducibility of free plume characteristics. The Alberta Idealized Nasal Inlet (AINI) and three realistic infant nasal airways were used to determine the in vitro deposition profile in adult and infant airways, respectively. Collectively, in vitro results demonstrated the feasibility of delivering the vaccine candidate to target sites within the nasal airways. Penetration through the nasal airways that could lead to deposition in the lungs was below the limit of quantification for both adult and infant geometries, indicating a low likelihood of adverse events due to lung exposure. These results support continued investigation of intranasal delivery of the synthetic Entamoeba histolytica vaccine.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Nanobeam electron diffraction and high resolution imaging analysis of InN films grown on sapphire.

A JEOL JEM-3000F field emission, analytical, high-resolution transmission electron microscope (HRTEM) was used to study InN films grown on sapphire substrates. It was found that, while the InN films maintained the hexagonal (wurtzite) structure, InN nanodomains with a cubic (zincblende) structure were also formed in the films. Nanobeam electron diffraction techniques were applied for identification of the cubic phase. The identification of the cubic InN was confirmed by HRTEM structural imaging. The cubic InN nanodomains are 3-10 nm in diameter, and are orientated in two different orientations with their [110](cubic) and [110](cubic) axes parallel to each other and their (111)(cubic) planes parallel to the (0001)(hex) plane of the hexagonal InN.