The use of Squalene in Veterinary Vaccines

Squalene, a hydrocarbon that acts as a boosting agent, is an essential ingredient in the production of vaccines and is primarily obtained from shark liver oil for commercial purposes. The primary benefit of squalene is that it improves the immune system and makes vaccines more effective. Although all sharks have squalene, the deep-sea species have the highest oil concentrations due to their large livers. However, they are vulnerable to extinction, making non-animal sources like olive oil, sugar cane, wheat germ, bacteria, and yeast more viable options.

SqualEne is a linear hydrocarbon precursor of cholesterol found in many tissues, notably the livers of sharks (Squalus) and other fishes. SqualAne is prepared by hydrogenation of squalene and is fully saturated, which means that it is not subject to autoxidation. Small quantities of SqualAne are found in sebaceous secretions, so it is a natural product. When preparing vaccines, it is generally recommended to use SqualAne as an ingredient due to its superior stability. (3000 animals are required to produce 1 ton of squalene).

Besides its role in squalene is used in the cosmetics industry to soften skin, its use in vaccines has brought attention and controversy to this natural lipid. Nearly a decade ago, in 1997, Chiron used it for its influenza vaccine, followed by other major pharmaceutical companies like GSK and Novartis, which began to rely on squalene for their seasonal flu and swine flu vaccines.

Recently, there has been a growing concern about using animal-derived squalene in vaccine production. As a result, companies like Evonik and Croda have developed non-animal-derived squalene suitable for vaccines and other pharmaceutical applications. Evonik has launched a non-animal-derived squalene suitable for vaccines and other pharmaceutical applications derived from amaranth oil (Amaranthus caudatus) squalene as an alternative to animal-derived squalene and Croda has partnered with US companies Amyris and Botanical Solutions Inc (BSI) to source sustainable vaccine adjuvants.

Using squalene in vaccine formulations has gained significant attention during pandemics, as it has proven to be an effective adjuvant. Although several commercially available adjuvants, including GSK’s AS03, MF59, AF03, and ISA 35, have successfully enhanced immune response, this review will explore the use of these products for veterinary vaccines. With the world facing new and emerging threats, it is essential to develop alternative sources of squalene to ensure that vaccines remain practical and accessible. This is especially critical as it focuses on the One Health theme, which highlights the interconnectedness of human health, animal health, and our shared environment.

However, the use of squalene is intimately connected with the price of the products that uses it and the species that will be used on it. In a quick search we can see the price of squalene as high as 470 euros pre liter (Squalen >98%), almost 5x the price of mineral oils, with the vendors varying from multiple countries, nevertheless the use of such product has been show in several species with variable degrees of efficacy.

Adjuvants such as MF59, AS03 ((10) Vaccine Adjuvant Mode of Action | GSK – YouTube) and AF03 are often times used in horses, however other adjuvants developed by companies such Zoetis – *MetaStim® ((10) MetaStim® Adjuvant Advantage – YouTube) are founded in vaccines for equine influenza, West Nile and ISA 35® also funded in vaccines against influenza and herpes virus both with great efficiency.

MetaStim, the common name for the SP (Squalane, Pluronic®) an oil in water emulsions containing squalane. 0.4% (v/v), poloxamer 401. 0.2% (v/v), polysorbate 80, 0.032% (v/v), and provides a strong immune response, but there is minimal reactivity, and its effect is responsible for the long duration of immunity (DOI) of the vaccine.

The same adjuvant is also present in several other vaccines such as Fostera PCV – porcine circovirus type 2 (PCV2), Pyramid 3- bovine rhinotracheitis virus and bovine virus diarrhea (types 1 and 2), Pyramid® 5 + Presponse® SQ, Suvaxyn and other. The Metastim and the advantage of this adjuvant is the dual-phase formulation system composed of lipid droplets in an aqueous combined with a surfactant solution which helps to hide some of the viral antigens by concealing them within the vaccine droplet, giving the vaccine a “trojan horse effect”. The lipid-surfactant droplets are eventually broken down, releasing the virus antigen. The vaccine’s slow release allows for prolonged immune system exposure and effects a positive impact on immune system response.

The adjuvant is an ideal emulsion for animals like swine and equine thanks to its gentle properties. Our research has shown that they are sensitive to single-water emulsions and double expulsion, resulting in a heightened immune response with severe local reactions. These reactions include significant swelling, abscesses, fever, weight loss, among other undesired reactions.

Squalene has been successfully used in chicken and cattle for various infections, including bovine rhinotracheitis (IBR) and nanoemulsion preparations against several diseases, such as FMD. It has also been used with low local reaction in combination with Arlacel 80 and Tween 80 compared to mineral oil, although aluminum hydroxide is commonly used and accepted for rabies vaccines, squalene has been successfully used in academic settings.

The versatility of squalene is excellent, making it a serious candidate for developing any vaccine candidate. Its safety and efficacy in humans and its ability to reduce adverse reactions in species that can have one adverse reaction to normal water in water and mineral oil make it an asset in veterinary medicine.