A multi-epitope-based DNA vaccine confers cross-protection against influenza A H1N1 and H3N2 viruses
-
Khabat Barkhordari
,
Farhad Rezaei
,
Vahid Salimi
,
Kaveh Sadeghi
,
Mostafa Alavi Moghadam
,
Hossein Bagheri
,
Masoumeh Pourjabali
,
Maryam Karimi
,
Omid Fotouhi
,
Talat Mokhtari Azad
Abstract
Background and Objectives: Influenza A virus remains a major global health concern because antigenic drift reduces the long-term efficacy of current vaccines. This study evaluated a multi-epitope DNA vaccine targeting conserved regions of hemagglutinin (HA) and polymerase acidic (PA) proteins from influenza A/H1N1 and A/H3N2 viruses.
Materials and Methods: Conserved B-cell, CD4⁺ T-cell, and CD8⁺ T-cell epitopes were identified using immunoinformatic tools and linked to β-defensin-2 in the pcDNA3.1(+) vector. Protein expression was confirmed in HEK293 cells. BALB/c mice were immunized intramuscularly and challenged with lethal doses of H1N1 or H3N2 viruses. Humoral and cellular immune responses, lung viral loads, histopathology, and clinical outcomes were assessed.
Results: Vaccinated mice exhibited undetectable or markedly reduced lung viral loads (1,000-10,000-fold; p<0.01), significantly decreased pulmonary lesions, and milder clinical manifestations than controls. Vaccination induced significant CD4⁺ (p=0.041) and CD8⁺ (p=0.038) T-cell responses in bronchoalveolar lavage fluid, accompanied by increased interferon-γ and interleukin-4 production, indicating balanced cellular and humoral immunity. Complete protection against mortality was observed following viral challenge.
Conclusion: This multi-epitope DNA vaccine elicited cross-protective immunity against influenza A/H1N1 and A/H3N2 by targeting conserved HA and PA epitopes, supporting its potential as a candidate for universal influenza vaccine development.
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31. Jia JZ, Cohen CA, Gu H, McLean MR, Varadarajan R, Bhandari N, et al. Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children. Nat Commun 2024; 15: 3210.
2. Bedford T, Suchard MA, Lemey P, Dudas G, Gregory V, Hay AJ, et al. Integrating influenza antigenic dynamics with molecular evolution. Elife 2014; 3: e01914.
3. Lou J, Liang W, Cao L, Hu I, Zhao S, Chen Z, et al. Predictive evolutionary modelling for influenza virus by site-based dynamics of mutations. Nat Commun 2024; 15: 2546.
4. Shi W, Wohlwend J, Wu M, Barzilay R. Influenza vaccine strain selection with an AI-based evolutionary and antigenicity model. Nat Med 2025; 31: 3862-3870.
5. Yassine HM, Boyington JC, McTamney PM, Wei CJ, Kanekiyo M, Kong WP, et al. Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection. Nat Med 2015; 21: 1065-1070.
6. Krammer F, Palese P. Advances in the development of influenza virus vaccines. Nat Rev Drug Discov 2015; 14: 167-182.
7. Wei Y, Qiu T, Ai Y, Zhang Y, Xie J, Zhang D, et al. Advances of computational methods enhance the development of multi-epitope vaccines. Brief Bioinform 2024; 26(1): bbaf055.
8. Lu B, Lim JM, Yu B, Song S, Neeli P, Sobhani N, et al. The next-generation DNA vaccine platforms and delivery systems: advances, challenges and prospects. Front Immunol 2024; 15: 1332939.
9. Sidney J, Kim A-R, de Vries RD, Peters B, Meade PS, Krammer F, et al. Targets of influenza human T-cell response are mostly conserved in H5N1. mBio 2025; 16(2): e0347924.
10. Ooi A, Wong A, Esau L, Lemtiri-Chlieh F, Gehring C. A guide to transient expression of membrane proteins in HEK-293 cells for functional characterization. Front Physiol 2016; 7: 300.
11. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al. The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. Br J Pharmacol 2020; 177: 3617-3624.
12. Lequin RM. Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 2005; 51: 2415-2418.
13. Cuevas F, Kawabata H, Krammer F, Carreño JM. An In Vitro Microneutralization Assay for Influenza Virus Serology. Curr Protoc 2022; 2(7): e465.
14. Fischer AH, Jacobson KA, Rose J, Zeller R. Hematoxylin and eosin staining of tissue and cell sections. CSH Protoc 2008; 2008: pdb.prot4986.
15. Kostinov M, Chuchalin A, Chebykina A, Khrapunova I, Cherdantsev A, Solov’eva I, et al. Clinical status and cytokine profiles in patients with asthma or chronic obstructive pulmonary disease vaccinated against influenza. PLoS One 2025; 20(2): e0313539.
16. Tang W, Xie H, Ye Z, Eick-Cost AA, Scheckelhoff M, Gustin CE, et al. Post-vaccination serum cytokines levels correlate with breakthrough influenza infections. Sci Rep 2023; 13: 1174.
17. Sun J, Madan R, Karp CL, Braciale TJ. Effector T cells control lung inflammation during acute influenza virus infection by producing IL-10. Nat Med 2009; 15: 277-284.
18. Wang SM, Tsai MH, Lei HY, Wang JR, Liu CC. The regulatory T cells in anti-influenza antibody response post influenza vaccination. Hum Vaccin Immunother 2012; 8: 1243-1249.
19. Couper KN, Blount DG, Riley EM. IL-10: the master regulator of immunity to infection. J Immunol 2008; 180: 5771-5777.
20. Zheng MZ, Fritzlar S, Wang Z, Tan TK, Kedzierska K, Townsend A, et al. Cutting edge: high-dose live attenuated influenza vaccines elicit pulmonary tissue-resident memory CD8+ T cells in the face of pre-existing humoral immunity. J Immunol 2022; 209: 1832-1836.
21. Zheng MZ, Tan TK, Villalon-Letelier F, Lau H, Deng YM, Fritzlar S, et al. Single-cycle influenza virus vaccine generates lung CD8+ Trm that cross-react against viral variants and subvert virus escape mutants. Sci Adv 2023; 9(36): eadg3469.
22. Del Campo J, Bouley J, Chevandier M, Rousset C, Haller M, Indalecio A, et al. OVX836 heptameric nucleoprotein vaccine generates lung tissue-resident memory CD8+ T-cells for cross-protection against influenza. Front Immunol 2021; 12: 678483.
23. van de Ven K, Lanfermeijer J, van Dijken H, Muramatsu H, Vilas Boas de Melo C, Lenz S, et al. A universal influenza mRNA vaccine candidate boosts T cell responses and reduces zoonotic influenza virus disease in ferrets. Sci Adv 2022; 8(50): eadc9937.
24. McKinstry KK, Strutt TM, Swain SL. Hallmarks of CD4 T cell immunity against influenza. J Intern Med 2011; 269(5): 507-518.
25. Iwasaki A, Pillai PS. Innate immunity to influenza virus infection. Nat Rev Immunol 2014; 14: 315-328.
26. Hu J, Ma C, Liu X. PA-X: a key regulator of influenza A virus pathogenicity and host immune responses. Med Microbiol Immunol 2018; 207: 255-269.
27. Li X, Huang C, Rai KR, Xu Q. Innate immune role of IL-6 in influenza a virus pathogenesis. Front Cell Infect Microbiol 2025; 15: 1605446.
28. Rojas JM, Avia M, Martín V, Sevilla N. IL‐10: a multifunctional cytokine in viral infections. J Immunol Res 2017; 2017: 6104054.
29. Yuan L, Zhang S, Bi R, Liu X, Han Z, Li M, et al. A broad-spectrum multiepitope vaccine against seasonal influenza A and B viruses in mice. EBioMedicine 2024; 106: 105269.
30. Haupt R, Baracco L, Harberts EM, Loganathan M, Kerstetter LJ, Krammer F, et al. Enhancing the protection of influenza virus vaccines with BECC TLR4 adjuvant in aged mice. Sci Rep 2023; 13: 715.
31. Jia JZ, Cohen CA, Gu H, McLean MR, Varadarajan R, Bhandari N, et al. Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children. Nat Commun 2024; 15: 3210.
| Files | ||
| Issue | Vol 18 No 5 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i5.22875 | |
| Keywords | ||
| Influenza A virus H1N1 subtype H3N2 subtype Hemagglutinin DNA vaccine | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Barkhordari K, Rezaei F, Salimi V, Sadeghi K, Alavi Moghadam M, Bagheri H, Pourjabali M, Karimi M, Fotouhi O, Mokhtari Azad T. A multi-epitope-based DNA vaccine confers cross-protection against influenza A H1N1 and H3N2 viruses. Iran J Microbiol. 2026;18(5):841-860.
