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

The study addresses the need for sustainable, non-animal-derived replacements for shark-sourced squalene used in licensed vaccine emulsions. Shark squalene-based adjuvants have proven safety and efficacy and are widely used, but global shark populations are in steep decline due to overfishing, creating ethical and supply concerns. Advances in synthetic biology have enabled high-yield production of terpenoids such as β-farnesene via engineered yeast, offering a renewable feedstock for generating squalene-like triterpenoids. The research question is whether semi-synthetic terpenoid analogues derived from β-farnesene or other non-animal sources can be formulated into stable oil-in-water emulsions and match or surpass shark squalene’s adjuvant activity, and how structural features of these terpenoids relate to adjuvant function.

Background literature highlights squalene’s extensive use as an emulsion adjuvant component (e.g., MF59) with a strong safety record and dose-sparing capability, as well as its role in vaccines for influenza and other diseases. The logistical advantages of squalene-based emulsions (refrigerated storage) during pandemics are noted. Overfishing has reduced oceanic shark and ray abundance by about 71% since 1970, elevating the need for sustainable alternatives. Synthetic biology has previously enabled semi-synthetic production of artemisinin and commercial β-farnesene. β-Farnesene-derived squalane has displaced shark squalane in cosmetics. Mechanistic studies show squalene emulsions enhance antigen uptake, recruit and activate immune cells, and induce DAMPs leading to proinflammatory signaling, though precise structure–function relationships remain unclear. Prior work on squalene countertypes and Pinaceae oil emulsions suggested potential alternatives but lacked a systematic structure–activity map across a broad terpenoid series.

Compound generation and characterization: Over 20 squalene-like terpenoid analogues were generated primarily via semi-synthesis from fermentation-derived, isomerically pure β-farnesene; additional terpenoids were obtained from plants (e.g., solanesol) or synthetic chemistry. Purity targets were ≥90% by GC-FID or HPLC-MS (two exceptions at 82–85%). Structures were confirmed by GC-FID, HPLC, 1H/13C NMR, and MS. Plant-derived solanesol (≥93%) was sourced commercially.

Emulsion formulation: Oils were formulated into 4% v/v oil-in-water nanoemulsions by high-shear mixing and high-pressure homogenization in two systems: (1) SE: DMPC + poloxamer 188 emulsifiers, α-tocopherol antioxidant, glycerol tonicity agent, 25 mM ammonium phosphate buffer pH 5.8; (2) MF59-like: polysorbate 80 + sorbitan trioleate in 10 mM citrate pH 6.0. Some oils requiring viscosity or stability adjustments used alternative approaches (e.g., ether 6 mixed 1:1 with long-chain triglyceride; diols B and diesters B in MF59-like system). Negative control emulsions used grapeseed long-chain triglyceride oil previously shown non-immunostimulatory.

Physicochemical stability testing: Dynamic light scattering measured Z-average droplet diameter and polydispersity index, and zeta potential was recorded for stable formulations. Stability benchmarks required <20% particle growth over 3 months at 5 °C for progression to bioactivity testing. Emulsions were stored at 5 °C, 25 °C, and 40 °C and monitored up to 12 months. Selected oils were monitored for chemical stability by HPLC-CAD.

In vitro innate stimulation: Fresh heparinized whole blood from human donors (n=6–12; balanced sex) was incubated 18–24 h at 37 °C with emulsions at 0–0.4% v/v in saline. Supernatants were assayed by ELISA for IL-6, IL-8, MCP-1, and MIP-1β. Selected emulsions were evaluated for PBMC viability. Cytokines below standard curves were imputed at half the lowest standard.

In vivo immunogenicity: C57BL/6 mice (equal numbers male/female) were immunized intramuscularly on Day 0 and Day 21 with split, inactivated H5N1 A/Vietnam/1194/2004 antigen mixed immediately prior with 2% v/v emulsions (dose-finding supported 0.01 µg HA as most sensitive to adjuvant effects). Three separate experiments tested 17 emulsions plus controls. Outcomes at Day 21 and Day 42 included serum total IgG (and isotypes), HAI titers, bone marrow long-lived antibody-secreting plasma cells (ELISpot), and splenocyte cytokines (IFN-γ, IL-5 by ELISA/ELISpot). Antigen integrity post-mixing was assessed by SRID and droplet size by DLS.

Desirability function analysis: Log-transformed group means were ratioed to the same-experiment shark squalene control and normalized to 0–1 desirability. A weighted geometric mean composite score was computed with weights: HAI D42=5, LLPC D42=4, IgGT D42=4, IgG2c/IgG1 D42=3, HAI D21=2, IgGT D21=1. An editable spreadsheet allows alternative weightings.

Statistics: Normality by D’Agostino-Pearson or Shapiro-Wilk; comparisons by one-way ANOVA with Sidak or Kruskal–Wallis with Dunn; Pearson correlations where appropriate. GraphPad Prism 9.3.1 was used. Animal and human sample use complied with institutional approvals.

Formulation and stability: Many terpenoid oils formed nanoemulsions with droplet sizes similar to shark squalene SE (e.g., SE droplet diameters 71–126 nm; shark squalene SE 90.9 nm). MF59-like compositions produced larger droplets (151–199 nm vs shark squalene MF59-like 143.9 nm). Several oils were not formulatable or unstable in SE (e.g., acids C, diols B SE >200 nm PDI high; alcohols D unstable at 5 °C within 1 month; compounds 3 developed particulates ~7 months). Squalane SE and MF59-like and MF59-like formulations overall showed robust physical stability at 40 °C; shark squalene SE was moderately stable; some ethers and alcohols A SE were less stable at elevated temperatures. Zeta potentials were generally negative. Solanesol showed chemical stability comparable to shark squalene and squalane.

In vitro innate stimulation: Emulsions elicited diverse cytokine profiles. Some, such as farnesene thermal dimers, solanesol, and alcohols A, matched or exceeded shark squalene in dose-dependent IL-6, IL-8, MCP-1, and MIP-1β induction, while others (e.g., long-chain triglyceride) showed minimal activity. Certain emulsions (C20 dimer SE, compounds 3 SE, diols B MF59-like) induced high cytokines at low doses but little at higher doses. PBMC viability was minimally impacted by several emulsions (including shark squalene SE), but was reduced by C20 dimer SE, compounds 3 SE, diols B MF59-like, and acids A SE.

Mouse immunogenicity and desirability ranking: At 0.01 µg HA, many emulsions enhanced total IgG, HAI, and/or LLPC vs antigen alone. Composite desirability (relative to shark squalene SE) categorized performance:

• Better than shark squalene (>10% improvement): acids A SE (composite 0.730; +37%), farnesene thermal dimers SE (0.656; +23%), solanesol SE (0.644; +23%), alcohols A SE (0.629 and 0.599; +18% and +12%).
• Similar (≤10% change): aldehydes A SE (0.581; +9%), alcohols C SE (0.577; +8%), diols B MF59-like (0.576; +8%), DHIS SE (0.566; +6%), ether 4 SE (0.521; −2%), difarnesyl ether SE (0.509; −4%), ether 5 SE (0.504; −5%), C25 dimer SE (0.496; −7%), esters A SE (0.482; −10%), ether 3 SE (0.477; −10%).
• Worse (<−10%): ether 2 SE (0.401; −25%), C20 dimer SE (0.379; −29%), shark squalane SE (0.294; −45%), squalane SE (0.259; −51%), long-chain triglyceride SE (as low as 0.046; −91%), and antigen alone (0.254–0.403; −36 to −52%). Shark squalene controls across experiments scored 0.532.

Structure–function correlations:

• Chain length: For linear unsaturated terpenoids, adjuvant activity increased with chain length (cLogP/MW/carbons): C20 < C25 < C30 (squalene) ≈ C30 (DHIS) < C45 (solanesol), with solanesol markedly enhancing responses.
• Conformation: Conformationally restricted analogues resembling squalene’s extended C11–C12 conformation (acids A, alcohols A/C, aldehydes A, esters A) retained good to excellent activity, supporting the extended form as active; a folded conformation is not required.
• Unsaturation: Saturation reduced activity—squalane (saturated) performed substantially worse than unsaturated squalene; altering double-bond placement (DHIS) did not harm activity.
• Charge: Negatively charged acids A achieved the highest composite desirability, suggesting charge at the oil–water interface can enhance bioavailability and adjuvanticity. Other charged analogues were unstable and not bio-tested.
• Ring structures and linkages: Incorporation of central rings maintained/enhanced activity (e.g., cyclohexene farnesene thermal dimers > shark squalene; benzene-containing ether 5 similar to ether 4 and shark squalene). Linkage geometry mattered: ether 5 (1,2-linkage) outperformed ether 2 (1,3-linkage). For uncharged cyclohexene terpenoids, increasing side-chain MW inversely correlated with composite desirability.

Cross-assay correlations: Global correlation between in vitro cytokines and in vivo immunogenicity was weak. Within linear unsaturated terpenoids, in vitro MCP-1 correlated with in vivo composite scores. For SE formulations, in vitro cytokines inversely correlated with emulsion droplet size.

The study demonstrates that multiple non-animal terpenoid emulsions can match or exceed shark squalene’s adjuvant performance while being derived from renewable sources, directly addressing sustainability and supply risks. Structure–activity findings clarify that longer, unsaturated, and in some cases charged or conformationally restricted terpenoids can enhance humoral responses, while saturation (squalane) diminishes activity. Central ring systems and specific linkage geometries are compatible with or can improve adjuvanticity, revealing steric and geometrical determinants. Although innate in vitro cytokine responses did not consistently predict adaptive in vivo outcomes across all chemotypes, subset analyses identified meaningful correlations (e.g., MCP-1 for linear unsaturated series) and highlighted particle size as a confounder in vitro. The top in vivo performer, acids A, suggests interfacial charge or increased hydrophilicity may reorganize droplet morphology to elevate immunostimulatory bioavailability. Collectively, the results validate semi-synthetic terpenoids as sustainable adjuvant oils and provide a framework for rational design based on chain length, unsaturation, charge, and linkage geometry.

By integrating industrial synthetic biology, scalable semi-synthesis, formulation science, and immunology, the authors created and evaluated >20 terpenoid analogues as vaccine adjuvant oils. Several candidates (e.g., acids A, farnesene thermal dimers, solanesol, alcohols A, ether 4/5, diols B) delivered equal or superior immunogenicity versus shark squalene, with acceptable stability profiles. The work elucidates key structural determinants—chain length, unsaturation, charge, and ring/linkage features—and offers a desirability-based ranking to guide selection. These sustainable, non-animal alternatives could secure the future of squalene-like emulsion adjuvants for human and veterinary vaccines. Future work should optimize emulsifier systems tailored to top analogues to enhance stability and biocompatibility, investigate detailed mechanisms of action (especially the role of charge and interfacial organization), establish protective efficacy in larger animal models, and complete development of semi-synthetic squalene routes.

• Emulsifier systems (SE and MF59-like) were optimized historically for shark squalene; leading candidates like acids A showed reduced physical stability at elevated temperatures, indicating a need for formulation optimization.
• Several charged or more hydrophilic analogues could not be stably formulated and thus were not tested in vivo, limiting conclusions about charge effects.
• Limited correlation between in vitro cytokine readouts and in vivo immunogenicity across all chemotypes; particle size confounded in vitro results.
• Mechanisms of action were not fully elucidated; cellular responses were modest, and potential T cell responses may require additional adjuvant components.
• No protective efficacy studies in larger animal models were performed.
• A semi-synthetic route to squalene itself was not reported in this work.