Original Article

Evaluating the Immune Response of Recombinant H1N1 Hemagglutinin with MF59 Adjuvant in Animal Model as a Novel Alternative to the Influenza Vaccine


The H1N1 influenza virus is known as a serious pandemic threat across the globe. Vaccination is one of the most effective methods of protection against this virus and the way to reduce the seasonal pandemic risk. The commercial vaccine does not adequately respond to pandemic strains. This study examines the potential function of formulated H1N1 hemagglutinin with MF59 adjuvant against A/PR/8/34 (H1N1). To this end, a recombinant hemagglutinin (rHA) gene of influenza A virus was designed and expressed in SF9 cell by the Baculovirus expression system.
Four groups of mice were immunized by rHA in combination with MF59, Alum adjuvant, and virus split only. The immunized mice subsequently used for the humoral immune assay and the results compared with untreated mice (negative group). Besides, both treated and control mice groups were challenged with mouse-adapted influenza virus A/PR/8/34(H1N1) through the intranasal drop. Bodyweight, survival, temperature variation, and the medical conditions of the samples were assessed. Mice immunized with the recombinant protein demonstrated a humoral response to the influenza A virus.

Upon virus challenging, co-administration of rHA with MF59 adjuvant could lead to 92% survival of the vaccinated mice within 10 days. The MF59-treated group showed slight weight loss and high-temperature body two weeks after infection. This group also displayed a higher hemagglutination inhibition (HI) antibody titer as compared to the group vaccinated with virus split, and Alum adjuvant.
Altogether, the results showed that the recombinant protein with the MF59 adjuvant created better safety than the Alum adjuvant, thereby can be considered as a safe and reliable vaccine against the H1N1 virus for further investigations.

1. Noda T, Kawaoka Y. Classification and virion structure of influenza virus. Jpn Clin Med. 2006;64(10):1766-9.
2. Wright P, Neumann G, Kawaoka Y. Chapter 48 Orthomyxoviruses. Fields Virology Fifth Edition Philadelphia, PA, USA: Lippincott Williams & Wilkins, 2007:1691-740.
3. Cox NJ, Tamblyn SE, Tam T. Influenza pandemic planning. Vacc. 2003;21(16):1801-3.
4. Kilbourne ED. Influenza pandemics of the 20th century. Emerg Infect Dis. 2006;12(1):9.
5. Ford SM, Grabenstein JD. Pandemics, avian influenza A (H5N1), and a strategy for pharmacists. Pharmacother. 2006;26(3):312-22.
6. Gendon I. Influenza pandemic: Hypotheses and facts. Zh. Mikrobiol Epidemiol Immunobiol. 2008;5:109-18.
7. Kitikoon P, Vincent AL, Janke BH, Erickson B, Strait EL, Yu S, et al. Swine influenza matrix 2 (M2) protein contributes to protection against infection with different H1 swine influenza virus (SIV) isolates. Vaccine. 2009;28(2):523-31.
8. Zangwill KM, Belshe RB. Safety and efficacy of trivalent inactivated influenza vaccine in young children: a summary for the new era of routine vaccination. Pediatr Infect Dis J. 2004;23(3):189-97.
9. Frolov VG, Seid Jr RC, Odutayo O, Al Khalili M, Yu J, Frolova OY, et al. Transcutaneous delivery and thermostability of a dry trivalent inactivated influenza vaccine patch. Influenza Other Resp. 2008;2(2):53-60.
10. Watanabe T, Watanabe S, Kim JH, Hatta M, Kawaoka Y. Novel approach to the development of effective H5N1 influenza A virus vaccines: use of M2 cytoplasmic tail mutants. J Virol. 2008;82(5):2486-92.
11. Ebrahimi SM, Tebianian M, Toghyani H, Memarnejadian A, Attaran HR. Cloning, expression and purification of the influenza A (H9N2) virus M2e antigen and truncated Mycobacterium tuberculosis HSP70 as a fusion protein in Pichia pastoris. Protein Expr Purif. 2010;70(1):7-12.
12. Kawaoka Y. Influenza virology: current topics. Caister Academic Press; 2006.
13. Maeda Y, Horimoto T, Kawaoka Y. Classification and genome structure of influenza virus. Jpn Clin Med. 2003;61(11):1886-91.
14. Mair CM, Ludwig K, Herrmann A, Sieben C. Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection. Biochim Biophys Acta Biomembr. 2014;1838(4):1153-68.
15. Bobone S, Hilsch M, Storm J, Dunsing V, Herrmann A, Chiantia S. Phosphatidylserine lateral organization influences the interaction of Influenza virus Matrix Protein 1 with lipid membranes. J Virol. 2017;91(12):e00267-17.
16. Sedova E, Shcherbinin D, Migunov A, Iu LD, Shmarov M, Tsybalova L, et al. Recombinant influenza vaccines. Acta Nat. 2012;4(15):17-27.
17. Smits A, Hak E, Stalman W, Van Essen G, Hoes A, Verheij TJ. Clinical effectiveness of conventional influenza vaccination in asthmatic children. Epidemiol Infect. 2002;128(2):205-11.
18. Demoly P, Dhivert-Donnadieu H, Bousquet J. Vaccination with allergens in children. Allergy Immunol. 2000;32(10):397-401.
19. Perrie Y, Mohammed AR, Kirby DJ, McNeil SE, Bramwell VW. Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens. Int J Pharm Pharmacol. 2008;364(2):272-80.
20. Girard MP, Katz JM, Pervikov Y, Hombach J, Tam JS. Report of the 7th meeting on evaluation of pandemic influenza vaccines in clinical trials, World Health Organization, Geneva, 17–18 February 2011. Vaccine. 2011;29(44):7579-86.
21. Soleimanjahi H, Fotouhi F. Baculoviruses and insect cells as powerful tools for gene expression. 2009: Tehran: Jahad Daneshgahi Publication. Tehran, Iran. 5-45.
22. Hwang JH, Lee SW, Rha SJ. Dietary green tea extract improves growth performance, body composition, and stress recovery in the juvenile black rockfish, Sebastes schlegeli. Aquac Int. 2013;21(3):525-38.
23. Dormitzer PR, Suphaphiphat P, Gibson DG, Wentworth DE, Stockwell TB, Algire MA, et al. Synthetic generation of influenza vaccine viruses for rapid response to pandemics. Sci Transl Med. 2013;5(185):168-85.
24. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual, 3rd Edition (2001), Cold Spring Harbor Laboratory Press.
25. Zadeh SH, Fotouhi F, Kheiri MT, Razavi MR, Heydarchi B, Farahmand B, et al. Isolation and cloning of large subunit of Influenza virus A (H1N1) hemagglutinin gene into Bacmid vector to construct recombinant Baculovirus. In BMC Proc. 2011;5(1):1-1.
26. Smith LM, Hood LE, Hunkapiller MW, Hunkapiller TJ, inventors; California Institute of Technology, assignee. Automated DNA sequencing technique. United States patent US 5,171,534. 1992.
27. Abe R, Kudou M, Tanaka Y, Arakawa T, Tsumoto K. Immobilized metal affinity chromatography in the presence of arginine. Biochem Biophys Res Commun. 2009;381(3):306-10.
28. Kreijtz J, Fouchier R, Rimmelzwaan G. Immune responses to influenza virus infection. Virus Res. 2011;162(1-2):19-30.
29. Yang S, Lee JY, Lee JS, Mitchell WP, Oh HB, Kang C, et al. Influenza sequence and epitope database. Nucleic Acids Res. 2009;37(suppl_1):423-30.
30. Thakur R, Shankar J. Strategies for Gene Expression in Prokaryotic and Eukaryotic System. InMetabolic Engineering for Bioactive Compounds Springer, Singapore. 2017:223-47.
31. Wang K, Holtz KM, Anderson K, Chubet R, Mahmoud W, Cox MM. Expression and purification of an influenza hemagglutinin--one step closer to a recombinant protein-based influenza vaccine. Vaccine. 2006;24(12):2176-85.
32. Hu YC, Luo YL, Ji WT, Chulu JL, Chang PC, Shieh H, et al. Dual expression of the HA protein of H5N2 avian influenza virus in a Baculovirus system. J Virol Methods. 2006;135(1):43-8.
33. Choi Y, Kwon SY, Oh HJ, Shim S, Chang S, Chung HJ, et al. Application of recombinant hemagglutinin proteins as alternative antigen standards for pandemic influenza vaccines. Biotechnol Lett. 2017;39(9):1375-80.
34. Huang KYA, Chang SC, Huang YC, Chiu CH, Lin TY. Antibody responses to trivalent inactivated influenza vaccine in health care personnel previously vaccinated and vaccinated for the first time. Sci Rep. 2017;7(1):1-10.
35. Jafari M, Moghaddam Pour M, Taghizadeh M, Masoudi S, Bayat Z. Comparative assessment of humoral immune responses of aluminum hydroxide and oil-emulsion adjuvants in Influenza (H9N2) and Newcastle inactive vaccines to chickens. Artif Cells Nanomed Biotechnol. 2017;45(1):84-9.
36. Khurana S, Verma N, Yewdell JW, Hilbert AK, Castellino F, Lattanzi M, et al. MF59 adjuvant enhances diversity and affinity of antibody-mediated immune response to pandemic influenza vaccines. Sci Transl Med. 2011;3(85):48-85.
IssueVol 19 No 5 (2020) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v19i5.4465
Baculoviruses Hemagglutinin Humoral immunity H1N1 virus Sf9 cells

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How to Cite
Rashedi N, Taghizadeh M, Mohamadynejad P, Mahdavi M, Jalalirad R. Evaluating the Immune Response of Recombinant H1N1 Hemagglutinin with MF59 Adjuvant in Animal Model as a Novel Alternative to the Influenza Vaccine. Iran J Allergy Asthma Immunol. 19(5):497-508.