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

Squalene, a triterpene and a member of the large terpenoid family, is a crucial component of many vaccines, serving as a potent adjuvant. Its high concentration in shark liver oil has traditionally made it a readily available source. However, overfishing has drastically reduced shark populations, raising concerns about squalene's long-term availability. This is particularly critical given its use in hundreds of millions of influenza vaccine doses and its role in numerous COVID-19 and other vaccine candidates. The mechanisms of squalene's adjuvant action, while not fully understood, involve increased antigen uptake, enhanced immune cell recruitment and activation, and the production of danger-associated molecular patterns. The need for a sustainable, renewable alternative with comparable or superior adjuvant properties is paramount. Synthetic biology offers a solution, exemplified by the successful industrial-scale production of artemisinin and β-farnesene using metabolically engineered yeast. β-farnesene, derived from sugarcane fermentation, serves as a readily available and sustainable precursor for squalene synthesis. This study leverages β-farnesene to create a library of squalene analogues, exploring the structure-activity relationship to identify superior alternatives.

The literature extensively documents the use of squalene as a vaccine adjuvant, highlighting its safety profile and dose-sparing capabilities. Studies have shown its effectiveness in enhancing immune responses, increasing antigen uptake, and promoting immune cell activation. However, the exact mechanisms by which squalene exerts its adjuvant effects remain incompletely understood. The overfishing crisis and the consequent decline in shark populations have prompted research into sustainable alternatives. Previous studies have explored other terpenoids and investigated various structural modifications to optimize adjuvant activity. This research builds upon these efforts, aiming to provide a more comprehensive understanding of the structure-activity relationships of squalene-like molecules and to identify suitable sustainable replacements.

This study involved the chemical synthesis of over 20 squalene analogues from β-farnesene, a sustainably produced precursor derived from sugarcane fermentation. The synthesis methods, compound characterization (purity, structural properties using GC-FID, HPLC, NMR, and MS), and IUPAC names are detailed in the Supplementary Information. The synthesized terpenoids were formulated into oil-in-water nanoemulsions using two different excipient compositions: a stable emulsion (SE) and a MF59-like composition. High-pressure homogenization was employed to ensure consistent emulsion properties. Emulsion physicochemical stability (droplet size, polydispersity index, zeta potential) was assessed over time under various storage conditions (5°C, 25°C, 40°C). For biological activity evaluation, stable emulsions were tested for innate immune stimulation using human whole blood, measuring cytokine production (IL-8, MCP-1, MIP-1β, IL-6). Adaptive immune responses were assessed in mice immunized with a split, inactivated influenza vaccine, measuring antigen-specific serum antibody titers (IgG1, IgG2c, total IgG), functional hemagglutination inhibition (HAI) titers, and long-lived antibody-secreting plasma cells. A desirability function approach, incorporating multiple immunological readouts, was used to rank the compounds relative to shark squalene. The weighting system for the desirability function is provided in the paper, allowing for user modification to adjust the relative importance of the different immunological readouts.

The study successfully synthesized and characterized a diverse library of squalene analogues with purities ≥90%. Emulsion droplet sizes and polydispersity indices were generally consistent across the different terpenoids, comparable to shark squalene emulsions. Several terpenoid emulsions exhibited excellent physical stability, particularly at lower temperatures. In vitro testing revealed differential innate immune stimulation profiles. While some analogues showed comparable or enhanced cytokine production compared to shark squalene, others elicited minimal responses. In vivo studies in mice demonstrated that several terpenoid emulsions elicited significantly enhanced antigen-specific serum IgG, HAI titers, and long-lived antibody-secreting plasma cells compared to the vaccine antigen alone, with some exhibiting comparable or even superior activity to shark squalene. The desirability function analysis provided a quantitative ranking of the emulsions based on multiple immunological readouts, identifying several top-performing analogues. Correlations were identified between structural features (chain length, conformational restrictions, double bond saturation, charge) and adjuvant activity, indicating that the C11-C12 extended conformation is preferred for activity and that chain saturation significantly reduces activity. Notably, acids A SE achieved the highest composite desirability score, suggesting that negatively charged compounds could be particularly effective.

The study successfully demonstrates the feasibility of producing sustainable, non-shark-derived terpenoid-based vaccine adjuvants with comparable or even enhanced activity relative to shark squalene. The identification of several top-performing analogues, including acids A, farnesene thermal dimers, and solanesol, opens avenues for replacing shark squalene in vaccine formulations. The strong correlation between certain structural features and adjuvant activity provides valuable insights into structure-activity relationships and informs the design of future analogues. However, it's important to acknowledge that the in vitro and in vivo responses did not consistently correlate across all emulsions. The discrepancy may stem from differences in the complexity of the assays or potential confounding factors like emulsion particle size.

This research provides a significant step towards replacing shark squalene with sustainable alternatives in vaccine adjuvants. Several identified terpenoids exhibit promising adjuvant activity and stability. Future research should focus on optimizing emulsion formulations, evaluating protective efficacy in larger animal models, elucidating specific mechanisms of action, and scaling up the production of the most promising candidates. Further research into the impact of charge and exploring the use of additional adjuvant components could lead to even more potent and effective vaccine formulations.

The study's in vitro and in vivo findings did not always perfectly correlate, possibly due to the complexity of immune responses or confounding effects. Furthermore, the desirability function analysis relies on a subjective weighting scheme that might require adjustments depending on the specific immune response profile desired. While the current study identified multiple promising candidates, additional testing in larger animal models is necessary to confirm protective efficacy and safety. The study did not report a complete semi-synthetic process for generating squalene itself.