(W1030-09-48) Comparison of Plant and Animal Sources of Squalene as Adjuvants for Use in Vaccine Applications

Purpose: Squalene is a natural organic compound that is used in many applications such as cancer treatments, detoxifiers, supplements, and vaccine adjuvants. As a vaccine adjuvant, squalene emulsions are approved for use in influenza vaccines in over 30 countries [1-3]. The squalene used in such formulations is animal based, derived from shark liver oil. Given the current industry push for high-purity, sustainable and non-animal derived squalene sources , plant-derived squalene offers vaccine formulators a better alternative. Such plant-derived squalene is even higher quality than its animal-derived counterpart because there are no low levels of impurities such as mercury, which are often associated with marine animals. The objective of this work was to compare different sources of squalene, formulated as an emulsion with standard commercial adjuvants. We aimed to demonstrate the suitability of plant-derived squalene as an effective sustainable source for use in a vaccine adjuvant. Our assessment used physico-chemical and in-vitro toxicity tests.

Methods: Plant-derived squalene (PhytoSquene®) and animal-derived squalene were separately formulated as an oil-in-water emulsion containing 4.3% squalene along with non-ionic emulsifiers, 0.5% Tween 80 and 0.5% Span 85. Citrate buffer was used as aqueous media [1]. The compositions were evaluated for Z-average particle size and Poly dispersibility index (PDI) using dynamic light scattering, pH, osmotic pressure and viscosity. The formulations were also compared with standard formulations available on the market for research purpose. Furthermore, an MTS assay and cell uptake measurements using Jurkat cells were done to compare the in-vitro toxicity of the two adjuvant formulations along with the commercial product.

Results: This study compared and demonstrated the similarity of adjuvants prepared with either plant-derived or shark-derived squalene. The results indicated that the sizes and zeta potential of the two adjuvants were similar and reproducible and compared well with the commercial product (Figure 1). Furthermore, the emulsion properties such as appearance, osmotic pressure, viscosity and pH were also similar and compared well with the commercial product (Table 1). The results of the in-vitro study indicated that both non-animal-derived (PhytoSquene®) and shark-derived squalene adjuvant formulations were not toxic to cells and were taken up by the cells equally (Figure 2A and 2B). The threshold for cytotoxicity (reduction of cell viability) was set at more than 30% compared to the control. However, all the formulation and excipient showed much lower than 30% threshold in reduction of cell viability confirming non-cytotoxic effect of the formulation components.

Conclusion: This study demonstrated that the different sources of squalene had similar formulation and toxicity properties. Plant-derived squalene, PhytoSquene®, is a sustainable, pure material that can be used to replace shark-derived squalene as an adjuvant in vaccines.

References: 1. Eun-Ju Koa et al., Immunology and efficacy of MF59-adjuvanted vaccines/ Human Vaccines & Immunotherapeutics, 2018, 14 (12), 3041–3045.
2. O’hagan, D. T., et al., The mechanism of action of MF59–an innately attractive adjuvant formulation." Vaccine 2012, 30 (29), 4341-4348.
3. Seubert, A., et al., The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractant and enhance monocyte differentiation toward dendritic cells. The journal of Immunology, 2008, 180(8), 5402-5412.

Figure 1 - Particle size and polydispersity index (PDI) of various squalene emulsions using dynamic light scattering (DLS)

Table 1 - Squalene emulsion physical properties

Figure 2: Non-cytotoxic impact on the in-vitro cell viability of (A) Emulsions (ratios reported in v/v) and (B) Individual excipients.