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

Abstract

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and β-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 β-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

“…The animal experiment was performed in accordance with national and institutional guidelines for animal care of laboratory animals and was approved by the Bloodworks Northwest Research Institute’s Institutional Animal Care and Use Committee. The results from the control groups were previously reported as part of a concurrent study …”

“…Importantly, semisynthetic squalene formed emulsions of ∼100 nm diameter in a manner indistinguishable from shark squalene and exhibited emulsion stability for at least one year, again indistinguishable from shark squalene. Immunological characterization of an emulsion formulated with semisynthetic squalene in an established mouse model for vaccine adjuvanticity with split, inactivated H5N1 influenza virus 12 demonstrated indistinguishable humoral and cellular adjuvant activities compared to emulsion formulated with shark squalene in immunized mice.…”

“…Emulsions were mixed with split inactivated H5N1 vaccine antigen immediately before intramuscular immunization of C57BL/6 mice (n = 8) with 0.01 μg antigen and 2% v/v oil. 12 A control group of mice was administered antigen without emulsion. Mice were immunized twice, 3 weeks apart.…”

“…9 Among other products, trans-β-farnesene is used for the commercial semisynthesis of squalane, 11 which is used as an emollient; it has also been used as the precursor for a series of squalene analogues, some of which display vaccine adjuvant properties equivalent or superior to squalene when appropriately formulated in o/w emulsions. 12 these analogues have not undergone human testing and would require extensive clinical trials before they could be considered as replacements for squalene in adjuvanted vaccines. As the need to replace shark squalene in o/w vaccine adjuvants remains pressing, we have developed a kilo-scale semisynthesis of GMP vaccine-grade squalene with sustainable trans-βfarnesene as the feedstock, the details of which are reported herein.…”

“…A more recent example of industrial metabolic engineering is the development of yeast to produce the sesquiterpene trans -β-farnesene, which is now produced commercially by fermentation of sustainably grown sugar cane . Among other products, trans -β-farnesene is used for the commercial semisynthesis of squalane, which is used as an emollient; it has also been used as the precursor for a series of squalene analogues, some of which display vaccine adjuvant properties equivalent or superior to squalene when appropriately formulated in o/w emulsions . While of great interest for the elucidation of squalene structure–function relationships relating to its vaccine adjuvant properties in o/w emulsions, these analogues have not undergone human testing and would require extensive clinical trials before they could be considered as replacements for squalene in adjuvanted vaccines.…”