Squalene-based adjuvant ASV24: the effectiveness of enhancing the humoral immune response against various viral antigens

Background. Squalene represents a promising basis for the development of novel adjuvant compositions with efficacy comparable to traditional adjuvants but with an improved safety profile.

Objective. To develop a novel squalene based adjuvant composition and evaluate its ability to enhance the humoral immune response against three different viral antigens — the receptor binding domain (RBDd) of the SARS CoV 2 Delta variant S protein, domain III (WDIII) of the West Nile virus E surface protein, and the surface glycoprotein (trGPM) of the Marburg virus.

Materials and methods. The adjuvant was prepared as an oil-in-water emulsion containing squalene, lecithin and Tween 80, followed by ultrasonic homogenization. BALB/c mice were immunized with two intramuscular injections 14 days apart. Specific IgG antibody titers were determined by ELISA. Statistical analysis was performed using the Mann–Whitney U-test.

Results. The ASV24 emulsion remained stable for 12 months. When immunized with the WDIII antigen produced in the prokaryotic E. coli system, the adjuvant activity of ASV24 was comparable to that of aluminum hydroxide. For the RBDd antigen produced in Chinese hamster ovary (CHO) cells, a trend toward higher titers was observed in the ASV24 group (3.6 × 10⁶) compared to aluminum hydroxide (9.6 × 10⁵), although no statistically significant differences were found. A pronounced advantage of ASV24 was demonstrated for trGPM, also produced in CHO cells: the mean reciprocal titer was 1,5 orders of magnitude higher than that in the aluminum hydroxide group.

Conclusion. The ASV24 adjuvant composition is stable, and its efficacy can surpass that of aluminum hydroxide when using complex multimeric antigens produced in eukaryotic expression systems. These findings suggest that ASV24 is a promising adjuvant for the development of subunit vaccines.

1. Apostolopoulos V, Chavda VP (eds.). Advanced vaccination technologies for infectious and chronic diseases: A Guide to vaccinology. Academic Press. 2024: 51–62. doi: 10.1016/B978-0-443-18564-9.00011-4

2. Xing J, Zhao X, Li X, Fang R, Sun M, Zhang Y, et al. The recent advances in vaccine adjuvants. Front Immunol. 2025; 16: 1557415. doi: 10.3389/fimmu.2025.1557415

3. Verma SK, Mahajan P, Singh NK, Gupta A, Aggarwal R, Rappuoli R, et al. New-age vaccine adjuvants, their development, and future perspective. Front Immunol. 2023; 14: 1043109. doi: 10.3389/fimmu.2023.1043109

4. Cui Y, Ho M, Hu Y, Shi Y. Vaccine adjuvants: current status, research and development, licensing, and future opportunities. J Mater Chem B. 2024; 12(17): 4118–4137. doi: 10.1039/d3tb02861e

5. Shi S, Zhu H, Xia X, Liang Z, Ma X, Sun B. Vaccine adjuvants: Understanding the structure and mechanism of adjuvanticity. Vaccine. 2019; 37(24): 3167–3178. doi: 10.1016/j.vaccine.2019.04.055

6. Facciolà A, Visalli G, Laganà A, Di Pietro A. An overview of vaccine adjuvants: current evidence and future perspectives. Vaccines. 2022; 10(5): 819. doi: 10.3390/vaccines10050819

7. Mendes A, Azevedo-Silva J, Fernandes JC. From sharks to yeasts: Squalene in the development of vaccine adjuvants. Pharmaceuticals (Basel). 2022; 15(3): 265. doi: 10.3390/ph15030265

8. Winokur PL, Hegmann TE, Keitel WA, Bernstein DI, Frey SE, Bryant C, et al. Safety and Immunogenicity of a monovalent inactivated influenza A/H5N8 virus vaccine given with and without AS03 or MF59 adjuvants in healthy adults. Clin Infect Dis. 2023: ciac983. doi: 10.1093/cid/ciac983

9. Volosnikova EA, Shcherbakov DN, Volkova NV, Esina TI, Zaikovskaya AV, Shimina GG, et al. Development of a vaccine adjuvant based on squalene and study of its adjuvant properties. Medical Immunology. 2023; 25(3): 685–690. (In Russ.). doi: 10.15789/1563-0625-DOA-2824

10. Kuznetsova TA, Persiyanova EV, Zaporozhets TS, Besednova NN. Adjuvants of influenza vaccines: new possibilities of using sulphated polysaccharides from marine brown algae. Problems of Virology. 2019; 64(1): 5–11. (In Russ.). doi: 10.18821/0507-4088-2019-64-1-5-11

11. Volosnikova EA, Volkova NV, Ermolaev VV, Borgoyakova MB, Nesmeyanova VS, Zaykovskaya AV, et al. Use of adjuvant compositions based on squalene ensures induction of neutralizing antibodies against SARS-COV-2. Bulletin of Experimental Biology and Medicine. 2024; 177(2): 221–224. (In Russ.) doi: 10.1007/s10517-024-06160-6

12. O’Hagan DT, Ott GS, Van Nest G, Rappuoli R, Giudice GD. The history of MF59(®) adjuvant: a phoenix that arose from the ashes. Expert Rev Vaccines. 2013; 12(1): 13– 30. doi: 10.1586/erv.12.140

13. Garçon N, Vaughn DW, Didierlaurent AM. Development and evaluation of AS03, an Adjuvant System containing α-tocopherol and squalene in an oil-in-water emulsion. Expert Rev Vaccines. 2012; 11(3): 349–366. doi: 10.1586/erv.11.192

14. Hogenesch H. Mechanism of immunopotentiation and safety of aluminum adjvants. Front Immunol. 2013; 3: 406. doi: 10.3389/fimmu.2012.00406

15. King LB, West BR, Schendel SL, Saphire EO. The structural basis for filovirus neutralization by monoclonal antibodies. Curr Opin Virol. 2018; 53: 196–202. doi: 10.1016/j.coi.2018.05.001

16. Kiesewetter A, Gupta A, Heinen-Kreuzig A, Greenhalgh T, Stein A. Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture. Biotechnol Prog. 2023; 39(6): e3375. doi: 10.1002/btpr.3375

17. Liang F, Lindgren G, Lin A, Thompson EA, Ols S, Röhss J, et al. Efficient targeting and activation of antigen-presenting cells in vivo after modified mRNA vaccine administration in Rhesus Macaques. Mol Ther. 2017; 25(12): 2635–2647. doi: 10.1016/j.ymthe.2017.08.006

18. O’Hagan DT, Lodaya RN, Lofano G. The continued advance of vaccine adjuvants – ‘we can work it out’. Semin Immunol. 2020; 50: 101426. doi: 10.1016/j.smim.2020.101426

19. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines – a new era in vaccinology. Nat Rev Drug Discov. 2018; 17(4): 261–279. doi: 10.1038/nrd.2017.243

20. Laera D, HogenEsch H, O’Hagan DT. Aluminum Adjuvants-’Back to the Future’. Pharmaceutics. 2023; 15(7): 1884. doi: 10.3390/pharmaceutics15071884

21. Wilkins AL, Kazmin D, Napolitani G, Clutterbuck EA, Pulendran B, Siegrist CA, et al. AS03- and MF59-adjuvanted influenza vaccines in children. Front Immunol. 2017; 8: 1760. doi: 10.3389/fimmu.2017.01760

22. de Bruyn G, Wang J, Purvis A, Ruiz MS, Adhikarla H, Alvi S, et al. Safety and immunogenicity of a variant-adapted SARS-CoV-2 recombinant protein vaccine with AS03 adjuvant as a booster in adults primed with authorized vaccines: a phase 3, parallel-group study. EClinicalMedicine. 2023; 62: 102109. doi: 10.1016/j.eclinm.2023.102109

23. Khurana S, King LR, Manischewitz J, Posadas O, Mishra AK, Liu D, et al. Licensed H5N1 vaccines generate cross-neutralizing antibodies against highly pathogenic H5N1 clade 2.3.4.4b influenza virus. Nat Med. 2024; 30(10): 2771–2776. doi: 10.1038/s41591-024-03189-y