Immunogenicity evaluation of an Alum-adjuvanted recombinant prefusion RBD-Fd SARS-CoV-2 protein subunit produced in Glycoengineered Pichia pastoris

Andri Wardiana,  Hariyatun Hariyatun,  Dian Fitria Agustiyanti,  Yana Rubiyana,  Alfi Taufik Fathurahman,  Herjuno Ari Nugroho,  Endah Puji Septisetyani,  Popi Hadi Wisnuwardhani,  Sugiyono Saputra,  A’liyatur Rosyidah,  Syaiful Rizal,  Hastuti Handayani S. Purba,  Kartika Sari Dewi,  Nissa Arifa,  Ratih Asmana Ningrum,  Wien Kusharyoto   

Andri Wardiana1, Hariyatun Hariyatun1, Dian Fitria Agustiyanti1, Yana Rubiyana1, Alfi Taufik Fathurahman1, Herjuno Ari Nugroho2, Endah Puji Septisetyani1, Popi Hadi Wisnuwardhani1, Sugiyono Saputra2, A’liyatur Rosyidah3, Syaiful Rizal4, Hastuti Handayani S. Purba1, Kartika Sari Dewi1, Nissa Arifa1, Ratih Asmana Ningrum1, Wien Kusharyoto1

1Research Centre for Genetic Engineering, National Research and Innovation Agency, KST Soekarno BRIN, Bogor, Indonesia.

2Research Centre for Applied Microbiology, National Research and Innovation Agency, KST Soekarno BRIN, Bogor, Indonesia.

3Research Centre for Vaccine and Drugs, National Research and Innovation Agency, KST Soekarno BRIN, Bogor, Indonesia.

4Research Centre for Applied Zoology, National Research and Innovation Agency, KST Soekarno BRIN, Bogor, Indonesia.

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Published:  Oct 13, 2025

DOI: 10.7324/JAPS.2026.270912
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Abstract

The global coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has accelerated vaccine development worldwide. While whole inactivated virus vaccines have reduced the incidence of severe disease and mortality, their effectiveness against emerging variants is limited. mRNA vaccines offer broader protection but face challenges in cost, production, and storage. Protein subunit vaccines targeting the viral spike (S) protein present a promising alternative due to their safety and scalability. In this study, we developed a recombinant protein subunit using the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, fused with a foldon domain (RBD-Fd) to promote trimer formation. The protein was expressed in Pichia pastoris GlycoSwitch® to achieve human-like glycosylation and was formulated with aluminium hydroxide (Alhydrogel®/Alum) to enhance immunogenicity. The resulting prototype protein subunit was evaluated in mice via subcutaneous injection at doses of 5 μg or 10 μg. Results showed that the alum-adjuvanted RBD-Fd formulation induced a strong antibody response following two doses at both concentrations. However, it generated only a partial T cell response with CD8? T cell activation but no corresponding CD4? response. These findings highlight the potential of prefusion RBD-based protein subunit and support further optimization to enhance cellular immunity.


Keyword:     RBD-Fd protein subunit Pichia pastoris Glycoswitch Aluminium hydroxide COVID-19 T cell activation

Citation:

Wardiana A, Hariyatun H, Agustiyanti DF, Rubiyana Y, Fathurahman AT, Nugroho HA, Septisetyani EP, Wisnuwardhani PH, Saputra S, Rosyidah A, Rizal S, Purba HHS, Dewi KS, Arifa N, Ningrum RA, Kusharyoto W. Immunogenicity evaluation of an Alum-adjuvanted recombinant prefusion RBD-Fd SARS-CoV-2 protein subunit produced in Glycoengineered Pichia pastoris. J Appl Pharm Sci. 2025. Article in Press. http://doi.org/10.7324/JAPS.2026.270912

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Reference

1. Naimi A, Yashmi I, Jebeleh R, Imani Mofrad M, Azimian Abhar S, Jannesar Y, et al. Comorbidities and mortality rate in COVID-19 patients with hematological malignancies: a systematic review and meta-analysis. J Clin Lab Anal. 2022;36(5):e24387. doi: https://doi.org/10.1002/jcla.24387

2. Koupaei M, Naimi A, Moafi N, Mohammadi P, Tabatabaei FS, Ghazizadeh S, et al. Clinical characteristics, diagnosis, treatment, and mortality rate of TB/COVID-19 coinfected patients: a systematic review. Front Med. 2021;8:740593. doi: https://doi.org/10.3389/fmed.2021.740593

3. Hotez PJ, Bottazzi ME. Whole inactivated virus and protein-based COVID-19 vaccines. Annu Rev Med. 2022;73:55–64. doi: https://doi.org/10.1146/annurev-med-042420-113212

4. Kowalzik F, Schreiner D, Jensen C, Teschner D, Gehring S, Zepp F. MRNA-based vaccines. Vaccines. 2021;9(4):390. doi: https://doi.org/10.3390/vaccines9040390

5. Gote V, Bolla PK, Kommineni N, Butreddy A, Nukala PK, Palakurthi SS, et al. A comprehensive review of mRNA vaccines. Int J Mol Sci. 2023;24(3):2700. doi: https://doi.org/10.3390/ijms24032700

6. Leong KY, Tham SK, Poh CL. Revolutionizing immunization: a comprehensive review of mRNA vaccine technology and applications. Virol J.2025;22(1):71. doi: https://doi.org/10.1186/s12985-025-02645-6

7. Ota N, Itani M, Aoki T, Sakurai A, Fujisawa T, Okada Y, et al. Expression of SARS-CoV-2 spike protein in cerebral arteries: implications for hemorrhagic stroke Post-mRNA vaccination. J Clin Neurosci. 2025;136:111223. doi: https://doi.org/10.1016/j.jocn.2025.111223

8. Heidary M, Kaviar VH, Shirani M, Ghanavati R, Motahar M, Sholeh M, et al. A comprehensive review of the protein subunit vaccines against COVID-19. Front Microbiol. 2022;13:927306. doi: https://doi.org/10.3389/fmicb.2022.927306

9. Martínez-Flores D, Zepeda-Cervantes J, Cruz-Reséndiz A, Aguirre- Sampieri S, Sampieri A, Vaca L. SARS-CoV-2 vaccines based on the spike glycoprotein and implications of new viral variants. Front Immunol. 2021;12:701501. doi: https://doi.org/10.3389/fimmu.2021.701501

10. Huang B, Dai L, Wang H, Hu Z, Yang X, Tan W, et al. Serum sample neutralisation of BBIBP-CorV and ZF2001 vaccines to SARS-CoV-2 501Y.V2. Lancet Microbe. 2021;2(7):285. doi: https://doi.org/10.1016/S2666-5247(21)00082-3

11. Li H, Sethuraman N, Stadheim TA, Zha D, Prinz B, Ballew N, et al. Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat Biotechnol. 2006;24(2):210–5. doi: https://doi.org/10.1038/nbt1178

12. Choi BK, Actor JK, Rios S, D’Anjou M, Stadheim TA, Warburton S, et al. Recombinant human lactoferrin expressed in glycoengineered Pichia pastoris: effect of terminal N-acetylneuraminic acid on in vitro secondary humoral immune response. Glycoconj J.2008;25(6):581– 93. doi: https://doi.org/10.1007/s10719-008-9123-y

13. Liu B, Yin Y, Liu Y, Wang T, Sun P, Ou Y, et al. A vaccine based on the receptor-binding domain of the spike protein expressed in glycoengineered pichia pastoris targeting SARS-CoV-2 stimulates neutralizing and protective antibody responses. Eng (Beijing). 2022;13:107–15. doi: https://doi.org/10.1016/j.eng.2021.06.012

14. Pallesen J, Wang N, Corbett KS, Wrapp D, Kirchdoerfer RN, Turner HL, et al. Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc Natl Acad Sci U S A. 2017;114(35):E7348–57. doi: https://doi.org/10.1073/pnas.1707304114

15. Hsieh CL, Goldsmith JA, Schaub JM, DiVenere AM, Kuo HC, Javanmardi K, et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science. 2020;369(6510):1501–5. doi: https://doi.org/10.1126/science.abd0826

16. Watterson D, Wijesundara DK, Modhiran N, Mordant FL, Li Z, Avumegah MS, et al. Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2. Clin Transl Immunol. 2021;10(4):1269. doi: https://doi.org/10.1002/cti2.1269

17. Zhao T, Cai Y, Jiang Y, He X, Wei Y, Yu Y, et al. Vaccine adjuvants: mechanisms and platforms. Signal Transduct Target Ther. 2023;8(1):283. doi: https://doi.org/10.1038/s41392-023-01557-7

18. 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: https://doi.org/10.3390/vaccines10050819

19. HogenEsch H, O’Hagan DT, Fox CB. Optimizing the utilization of aluminum adjuvants in vaccines: you might just get what you want. Vaccines. 2018;3(1):51. doi: https://doi.org/10.1038/s41541-018- 0089-x

20. Rukmana A, Supardi LA, Sjatha F, Nurfadilah M. Responses of humoral and cellular immune mediators in BALB/c Mice to LipX (PE11) as seed tuberculosis vaccine candidates. Genes (Basel). 2022;13(11):1954. doi: https://doi.org/10.3390/genes13111954

21. Tai W, Zhao G, Sun S, Guo Y, Wang Y, Tao X, et al. A recombinant receptor-binding domain of MERS-CoV in trimeric form protects human dipeptidyl peptidase 4 (hDPP4) transgenic mice from MERS-CoV infection. Virology. 2016;499:375–82. doi: https://doi.org/10.1016/j.virol.2016.10.005

22. Papanikolopoulou K, Forge V, Goeltz P, Mitraki A. Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage T4 fibritin. J Biol Chem. 2004;279(10):8991–8. doi: https://doi.org/10.1074/jbc. M311791200

23. Knetsch TGJ, Ubbink M. Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: insights from multi-angle light scattering. Protein Expr Purif. 2025;227:106641. doi: https://doi.org/10.1016/j.pep.2024.106641

24. Yang MC, Wang CC, Tang WC, Chen KM, Chen CY, Lin HH, et al. Immunogenicity of a spike protein subunit-based COVID-19 vaccine with broad protection against various SARS-CoV-2 variants in animal studies. PLoS One. 2023;18(3):283473. doi: https://doi.org/10.1371/journal.pone.0283473

25. Mettu RR, Charles T, Landry SJ.CD4+ T-cell epitope prediction using antigen processing constraints. J Immunol Methods. 2016;432:72–81. doi: https://doi.org/10.1016/j.jim.2016.02.013

26. Rapaka RR. How do adjuvants enhance immune responses? eLife. 2024;13:e101259. doi: https://doi.org/10.7554/eLife.101259

27. . Rood JE, Yoon SK, Heard MK, Carro SD, Hedgepeth EJ, O’Mara ME, et al. Endogenous antigen processing promotes mRNA vaccine CD4+ T cell responses. bioRxiv. 2025; doi: https://doi.org/10.1101/2025.03.11.642674

28. Laidlaw BJ, Craft JE, Kaech SM. The multifaceted role of CD4(+) T cells in CD8(+) T cell memory. Nat Rev Immunol. 2016;16(2):102– 11. doi: https://doi.org/10.1038/nri.2015.10

29. Painter MM, Mathew D, Goel RR, Apostolidis SA, Pattekar A, Kuthuru O, et al. Rapid induction of antigen-specific CD4(+) T cells is associated with coordinated humoral and cellular immunity to SARS-CoV-2 mRNA vaccination. Immunity. 2021;54(9):2133–42. doi: https://doi.org/10.1016/j.immuni.2021.08.001

30. Topchyan P, Lin S, Cui W. The role of CD4 T cell help in CD8 T cell differentiation and function during chronic infection and cancer. Immune Netw. 2023;23(5):41. doi: https://doi.org/10.4110/in.2023.23.e41

31. Xie L, Fang J, Yu J, Zhang W, He Z, Ye L, et al. The role of CD4(+) T cells in tumor and chronic viral immune responses. MedComm. 2023;4(5):390. doi: https://doi.org/10.1002/mco2.390

32. Su R, Shi Z, Li E, Zhu M, Li D, Liu X, et al. A Trim-RBD-GEM vaccine candidate protects mice from SARS-CoV-2. Virology. 2023;585:145–54. doi: https://doi.org/10.1016/j.virol.2023.06.005

33. Flórez L, Echeverri-De la Hoz D, Calderón A, Serrano-Coll H, Martinez C, Guzmán C, et al. Preclinical evaluation of the RBD-Trimeric vaccine: a novel approach to strengthening biotechnological sovereignty in developing countries against SARS-CoV-2 variants. Travel Med Infect Dis. 2025;64:102820. doi: https://doi.org/10.1016/j.tmaid.2025.102820

34. Kim E, Khan MS, Ferrari A, Huang S, Sammartino JC, Percivalle E, et al. SARS-CoV-2 S1 subunit booster vaccination elicits robust humoral immune responses in aged mice. Microbiol Spectr. 2023;11(3):436322. doi: https://doi.org/10.1128/spectrum.04363-22

35. Korosec CS, Dick DW, Moyles IR, Watmough J.SARS-CoV-2 booster vaccine dose significantly extends humoral immune response half-life beyond the primary series. Sci Rep. 2024;14(1):8426. doi: https://doi.org/10.1038/s41598-024-58811-3

36. Gebauer M, Hürlimann HC, Behrens M, Wolff T, Behrens SE. Subunit vaccines based on recombinant yeast protect against influenza A virus in a one-shot vaccination scheme. Vaccine. 2019;37(37):5578– 87. doi: https://doi.org/10.1016/j.vaccine.2019.07.094

37. Lang Q, Huang N, Li L, Liu K, Chen H, Liu X, et al. Novel and efficient yeast-based strategies for subunit vaccine delivery against COVID-19. Int J Biol Macromol. 2025;294:139254. doi: https://doi.org/10.1016/j.ijbiomac.2024.139254

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