Needle-free jet-delivered mRNA-vaccine encoding influenza A(H1N1)pdm09 hemagglutinin protects mice from lethal virus infection

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Abstract

Seasonal influenza is an acute respiratory illness caused by influenza A and B viruses that circulate worldwide. Due to high variability, new strains of the virus emerge every year. Therefore, vaccine formulation has to be revised every year. The advantages of mRNA vaccines are that they can be produced quickly, and without preliminary adaptation of the vaccine strain to chicken embryos. Here, the results of developing and studying the mRNA-C3-H1 vaccine encoding the hemagglutinin (HA) of the influenza A(H1N1)pdm09 virus are presented. The design and production of a DNA-template for the synthesis of mature HA mRNA in one step were described. The obtained mRNA was purified from double-stranded RNA impurities using a method based on the use of cellulose powder. The efficacy of the vaccine was assessed on BALB/c mice. The mice were immunized with “naked” mRNA vaccine using a needle-free jet injector. According to ELISA results, the average antibody titer in the serum of immunized animals was 4.6 × 105. Sera of immunized animals neutralized the mouse-adapted influenza A/California/04/09 (H1N1) MA8 virus with an average titer of 6 × 102. As shown by the ELISpot, the developed mRNA vaccine induced a T-cell immune response in mice. After stimulation of splenocytes with specific peptides, the average number of T-lymphocytes secreting IFN-γ was 236 per 106 cells. Immunization with the mRNA vaccine was shown to protect mice from infection with a lethal dose of the influenza A/California/04/09 (H1N1) MA8 virus. Thus, the developed experimental mRNA-C3-H1 vaccine is immunogenic and prevents morbidity and mortality in mice after infection with a homologous strain of influenza virus.

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About the authors

S. V. Sharabrin

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Author for correspondence.
Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. A. Ilyichev

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

D. N. Kisakov

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

M. B. Borgoyakova

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

E. V. Starostina

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

L. A. Kisakova

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. A. Isaeva

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

D. N. Shcherbakov

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

S. I. Krasnikova

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. S. Gudymo

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

K. I. Ivanova

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

V. Y. Marchenko

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

V. A. Yakovlev

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

E. V. Tigeeva

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

T. N. Ilyicheva

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

N. B. Rudometova

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. A. Fando

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. P. Rudometov

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

A. A. Sergeev

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

L. I. Karpenko

Federal Budgetary Research Institution State Research Center of Virology and Biotechnology “Vector”, Rospotrebnadzor

Email: Sharabrin.sv@gmail.com
Russian Federation, Koltsovo, Novosibirsk Region, 630559

References

  1. Uyeki T.M., Hui D.S., Zambon M., Wentworth D.E., Monto A.S. (2022) Influenza. Lancet. 400(10353), 693–706. https://doi.org/10.1016/S0140-6736(22)00982-5
  2. Ohmit S.E., Thompson M.G., Petrie J.G., Thaker S.N., Jackson M.L., Belongia E.A., Zimmerman R.K., Gaglani M., Lamerato L., Spencer S.M., Jackson L., Meece J.K., Nowalk M.P., Song J., Zervos M., Cheng P.Y., Rinaldo C.R., Clipper L., Shay D.K., Piedra P., Monto A.S. (2014) Influenza vaccine effectiveness in the 2011–2012 season: protection against each circulating virus and the effect of prior vaccination on estimates. Clin. Infect. Dis. 58(3), 319‒327. https://doi.org/10.1093/cid/cit736
  3. Wei C.J., Crank M.C., Shiver J., Graham B.S., Mascola J.R., Nabel G.J. (2020) Next-generation influenza vaccines: opportunities and challenges. Nat. Rev. Drug Discov. 19(6), 239–252. https://doi.org/10.038/s4573-020-0066-8
  4. Scorza F.B., Pardi N. (2018) New kids on the block: RNA-based influenza virus vaccines. Vaccines (Basel). 6(2), 20. https://doi.org/10.3390/vaccines6020020
  5. Zhang C., Maruggi G., Shan H., Li J. (2019) Advances in mRNA vaccines for infectious diseases. Front. Immunol. 10, 594. https://doi.org/10.3389/fimmu.2019.00594
  6. Pardi N., Hogan M.J., Porter F.W., Weissman D. (2018) mRNA vaccines – a new era in vaccinology. Nat. Rev. Drug Discov. 7(4), 261‒279. https://doi.org/10.1038/nrd.2017.243
  7. Vogel A.B., Kanevsky I., Che Y., Swanson K.A., Muik A., Vormehr M., Kranz L.M., Walzer K.C., Hein S., Güler A., Loschko J., Maddur M.S., Ota-Setlik A., Tompkins K., Cole J., Lui B.G., Ziegenhals T., Plaschke A., Eisel D., Dany S.C., Fesser S., Erbar S., Bates F., Schneider D., Jesionek B., Sänger B., Wallisch A.K., Feuchter Y., Junginger H., Krumm S.A., Heinen A.P., Adams-Quack P., Schlereth J., Schille S., Kröner C., de la Caridad Güimil Garcia R., Hiller T., Fischer L., Sellers R.S., Choudhary S., Gonzalez O., Vascotto F., Gutman M.R., Fontenot J.A., Hall-Ursone S., Brasky K., Griffor M.C., Han S., Su A.A.H., Lees J.A., Nedoma N.L., Mashalidis E.H., Sahasrabudhe P.V., Tan C.Y., Pavliakova D., Singh G., Fontes-Garfias C., Pride M., Scully I.L., Ciolino T., Obregon J., Gazi M., Carrion R. Jr., Alfson K.J., Kalina W.V., Kaushal D., Shi P.Y., Klamp T., Rosenbaum C., Kuhn A.N., Türeci Ö., Dormitzer P.R., Jansen K.U., Sahin U. (2021) BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature. 592(7853), 283–289. https://doi.org/10.1038/s41586-021-03275-y
  8. Chan L., Alizadeh K., Alizadeh K., Fazel F., Kakish J.E., Karimi N., Knapp J.P., Mehrani Y., Minott J.A., Morovati S., Rghei A., Stegelmeier A.A., Vanderkamp S., Karimi K., Bridle B.W. (2021) Review of influenza virus vaccines: the qualitative nature of immune responses to infection and vaccination is a critical consideration. Vaccines (Basel). 9(9) 979. https://doi.org/10.3390/vaccines9090979
  9. Walsh E.E., Frenck R.W. Jr., Falsey A.R., Kitchin N., Absalon J., Gurtman A., Lockhart S., Neuzil K., Mulligan M.J., Bailey R., Swanson K.A., Li P., Koury K., Kalina W., Cooper D., Fontes-Garfias C., Shi P.Y., Türeci Ö., Tompkins K.R., Lyke K.E., Raabe V., Dormitzer P.R., Jansen K.U., Şahin U., Gruber W.C. (2020) Safety and immunogenicity of two RNA-based Covid-19 vaccine candidates. N. Engl. J. Med. 383(25), 2439–2450. https://doi.org/10.1056/NEJMoa2027906
  10. Teo S.P. (2022) Review of COVID-19 mRNA vaccines: BNT162b2 and mRNA-1273. J. Pharm. Pract. 35(6), 947–951. https://doi.org/10.1177/08971900211009650
  11. Freyn A.W., Pine M., Rosado V.C., Benz M., Muramatsu H., Beattie M., Tam Y.K., Krammer F., Palese P., Nachbagauer R., McMahon M., Pardi N. (2021) Antigen modifications improve nucleoside-modified mRNA-based influenza virus vaccines in mice. Mol. Ther. Methods Clin. Dev. 22, 84–95. https://doi.org/10.1016/j.omtm.2021.06.003
  12. Mazunina E.P., Gushchin V.A., Kleymenov D.A., Siniavin A.E., Burtseva E.I., Shmarov M.M., Mukasheva E.A., Bykonia E.N., Kozlova S.R., Evgrafova E.A., Zolotar A.N., Shidlovskaya E.V., Kirillova E.S., Krepkaia A.S., Usachev E.V., Kuznetsova N.A., Ivanov I.A., Dmitriev S.E., Ivanov R.A., Logunov D.Y., Gintsburg A.L. (2024) Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response. Front. Immunol. 15, 1381508. https://doi.org/10.3389/fimmu.2024.1381508
  13. Joe P.T., Christopoulou I., van Hoecke L., Schepens B., Ysenbaert T., Heirman C., Thielemans K., Saelens X., Aerts J.L. (2019) Intranodal administration of mRNA encoding nucleoprotein provides cross-strain immunity against influenza in mice. J. Transl. Med. 17(1), 242. https://doi.org/10.1186/s12967-019-1991-3
  14. Zhuang X., Qi Y., Wang M., Yu N., Nan F., Zhang H., Tian M., Li C., Lu H., Jin N. (2020) mRNA vaccines encoding the HA protein of influenza A H1N1 virus delivered by cationic lipid nanoparticles induce protective immune responses in mice. Vaccines (Basel). 8(1), 123. https://doi.org/10.3390/vaccines8010123
  15. Kackos C.M., DeBeauchamp J., Davitt C.J.H., Lonzaric J., Sealy R.E., Hurwitz J.L., Samsa M.M., Webby R.J. (2023) Seasonal quadrivalent mRNA vaccine prevents and mitigates influenza infection. NPJ Vaccines. 8(1), 157. https://doi.org/10.1038/s41541-023-00752-5
  16. Tian Y., Deng Z., Chuai Z., Li C., Chang L., Sun F., Cao R., Yu H., Xiao R., Lu S., Xu Y., Yang P. (2024) A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge. Virology. 596, 110125. https://doi.org/10.1016/j.virol.2024.110125
  17. Li Y., Wang X., Zeng X., Ren W., Liao P., Zhu B. (2023) Protective efficacy of a universal influenza mRNA vaccine against the challenge of H1 and H5 influenza A viruses in mice. mLife. 2(3), 308–316. https://doi.org/10.1002/mlf2.12085
  18. Nitika, Wei J., Hui A.M. (2022) The delivery of mRNA vaccines for therapeutics. Life (Basel). 12(8), 1254. https://doi.org/10.3390/life12081254
  19. Ramachandran S., Satapathy S.R., Dutta T. (2022) Delivery strategies for mRNA vaccines. Pharm. Med. 36(1), 11–20. https://doi.org/10.1007/s40290-021-00417-5
  20. Buschmann M.D., Carrasco M.J., Alishetty S., Paige M., Alameh M.G., Weissman D. (2021) Nanomaterial delivery systems for mRNA vaccines. Vaccines (Basel). 9(1), 65. https://doi.org/10.3390/vaccines9010065
  21. Kim B., Hosn R.R., Remba T., Yun D., Li N., Abraham W., Melo M.B., Cortes M., Li B., Zhang Y., Dong Y., Irvine D.J. (2023) Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines. J. Control. Release. 353, 241–253. https://doi.org/10.1016/j.jconrel.2022.11.022
  22. Tsilingiris D., Vallianou N.G., Karampela I., Liu J., Dalamaga M. (2022) Potential implications of lipid nanoparticles in the pathogenesis of myocarditis associated with the use of mRNA vaccines against SARS-CoV-2. Metabol. Open. 13, 100159. https://doi.org/10.1016/j.metop.2021.100159
  23. Wilczewska A.Z., Niemirowicz K., Markiewicz K.H., Car H. (2012) Nanoparticles as drug delivery systems. Pharmacol. Rep. 64(5), 1020–1037. https://doi.org/10.1016/s1734-1140(12)70901-5
  24. Eygeris Y., Gupta M., Kim J., Sahay G. (2022) Chemistry of lipid nanoparticles for RNA delivery. Acc. Chem. Res. 55(1), 2–12. https://doi.org/10.1021/acs.accounts.1c00544
  25. Fraiman J., Erviti J., Jones M., Greenland S., Whelan P., Kaplan R.M., Doshi P. (2022) Serious adverse events of special interest following mRNA COVID-19 vaccination in randomized trials in adults. Vaccine. 40(40), 5798–5805. https://doi.org/10.1016/j.vaccine.2022.08.036
  26. Giannotta G., Murrone A., Giannotta N. (2023) COVID-19 mRNA vaccines: the molecular basis of some adverse events. Vaccines (Basel). 11(4), 747. https://doi.org/10.3390/vaccines11040747
  27. Parés-Badell O., Martínez-Gómez X., Pinós L., Borras-Bermejo B., Uriona S., Otero-Romero S., Rodrigo-Pendás J.Á., Cossio-Gil Y., Agustí A., Aguilera C., Campins M. (2021) Local and systemic adverse reactions to mRNA COVID-19 vaccines comparing two vaccine types and occurrence of previous COVID-19 infection. Vaccines (Basel). 9(12), 463. https://doi.org/10.3390/vaccines9121463
  28. Dey A., Chozhavel Rajanathan T.M., Chandra H., Pericherla H.P.R., Kumar S., Choonia H.S., Bajpai M., Singh A.K., Sinha A., Saini G., Dalal P., Vandriwala S., Raheem M.A., Divate R.D., Navlani N.L., Sharma V., Parikh A., Prasath S., Sankar Rao M., Maithal K. (2021) Immunogenic potential of DNA vaccine candidate, ZyCoV-D against SARS-CoV-2 in animal models. Vaccine. 39(30), 4108–4116. https://doi.org/10.1016/j.vaccine.2021.05.098
  29. Kwilas S., Kishimori J.M., Josleyn M., Jerke K., Ballantyne J., Royals M., Hooper J.W. (2014) A hantavirus pulmonary syndrome (HPS) DNA vaccine delivered using a spring-powered jet injector elicits a potent neutralizing antibody response in rabbits and nonhuman primates. Curr. Gene Ther. 14(3), 200–210. https://doi.org/10.2174/1566523214666140522122633
  30. Hu J., Shi H., Zhao C., Li X., Wang Y., Cheng Q., Goswami R., Zhen Q., Mei M., Song Y., Yang S., Li Q. (2016) Lispro administered by the QS-M Needle-Free Jet Injector generates an earlier insulin exposure. Expert Opin. Drug Deliv. 13(9), 1203–1207. https://doi.org/10.1080/17425247.2016.1198772
  31. Kwon T.R., Seok J., Jang J.H., Kwon M.K., Oh C.T., Choi E.J., Hong H.K., Choi Y.S., Bae J., Kim B.J. (2016) Needle-free jet injection of hyaluronic acid improves skin remodeling in a mouse model. Eur. J. Pharm. Biopharm. 105, 69–74. https://doi.org/10.1016/j.ejpb.2016.05.014
  32. Ravi A.D., Sadhna D., Nagpaal D., Chawla L. (2015) Needle free injection technology: a complete insight. Int. J. Pharm. Investig. 5(4), 192–199. https://doi.org/10.4103/2230-973X.167662
  33. Scheib N., Tiemann J., Becker C., Probst H.C., Raker V.K., Steinbrink K. (2022) The dendritic cell dilemma in the skin: between tolerance and immunity. Front. Immunol. 13, 929000. https://doi.org/10.3389/fimmu.2022.929000
  34. Brocato R.L., Kwilas S.A., Kim R.K., Zeng X., Principe L.M., Smith J.M., Hooper J.W. (2021) Protective efficacy of a SARS-CoV-2 DNA vaccine in wild-type and immunosuppressed Syrian hamsters. NPJ Vaccines. 6(1), 16. https://doi.org/10.1038/s41541-020-00279-z
  35. Alamri S.S., Alluhaybi K.A., Alhabbab R.Y., Basabrain M., Algaissi A., Almahboub S., Alfaleh M.A., Abujamel T.S., Abdulaal W.H., ElAssouli M.Z., Alharbi R.H., Hassanain M., Hashem A.M. (2021) Synthetic SARS-CoV-2 spike-based DNA vaccine elicits robust and long-lasting Th1 humoral and cellular immunity in mice. Front. Microbiol. 12, 727455. https://doi.org/10.3389/fmicb.2021.727455
  36. Abbasi S., Matsui-Masai M., Yasui F., Hayashi A., Tockary T.A., Mochida Y., Akinaga S., Kohara M., Kataoka K., Uchida S. (2024) Carrier-free mRNA vaccine induces robust immunity against SARS-CoV-2 in mice and non-human primates without systemic reactogenicity. Mol. Ther. 32(5), 1266–1283. https://doi.org/10.1016/j.ymthe.2024.03.022
  37. Кисаков Д.Н., Кисакова Л.А., Шарабрин С.В., Яковлев В.А., Тигеева Е.В., Боргоякова М.Б., Старостина Е.В., Зайковская А.В., Рудометов А.П., Рудометова Н.Б., Карпенко Л.И., Ильичев А.А. (2023) Доставка экспериментальной мРНК-вакцины, кодирующей RBD SARS-CoV-2 с помощью струйной инжекции. Бюллетень экспериментальной биологии и медицины. 176(12), 751–756. https://doi.org/10.47056/0365-9615-2023-176-12-751-756
  38. Шарабрин С.В., Бондарь А.А., Старостин E.B., Кисаков Д.Н. Кисакова Л.А., Задорожный А.М, Рудометов А.П., Ильичев А.А., Карпенко Л.И. (2023) Удаление примесной дцРНК из препарата синтезированной матричным синтезом мРНК. Бюллетень экспериментальной биологии и медицины. 176(12), 723–728. https://doi.org/10.47056/0365-9615-2023-176-12-723-728
  39. Karpenko L.I., Rudometov A.P., Sharabrin S.V., Shcherbakov D.N., Borgoyakova M.B., Bazhan S.I., Volosnikova E.A., Rudometova N.B., Orlova L.A., Pyshnaya I.A., Zaitsev B.N., Volkova N.V., Azaev M.S., Zaykovskaya A.V., Pyankov O.V., Ilyichev A.A. (2021) Delivery of mRNA vaccine against SARS-CoV-2 using a polyglucin: spermidine conjugate. Vaccines (Basel). 9(2), 76. https://doi.org/10.3390/vaccines9020076
  40. Gross F.L., Bai Y., Jefferson S., Holiday C., Levine M.Z. (2017) Measuring influenza neutralizing antibody responses to A(H3N2) viruses in human sera by microneutralization assays using MDCK-SIAT1 cells. J. Vis. Exp. 129, 56448. https://doi.org/10.3791/56448
  41. Portal M.M., Pavet V., Erb C., Gronemeyer H. (2015) Human cells contain natural double-stranded RNAs with potential regulatory functions. Nat. Struct. Mol. Biol. 22, 89–97.
  42. Weissman D., Pardi N., Muramatsu H., Karikó K. (2013) HPLC purification of in vitro transcribed long RNA. Methods Mol. Biol. 969, 43–54. https://doi.org/10.1007/978-1-62703-260-5_3
  43. Chen Y., Lin J., Zhao Y., Ma X., Yi H. (2021) Toll-like receptor 3 (TLR3) regulation mechanisms and roles in antiviral innate immune responses. J. Zhejiang Univ. Sci. B. 22(8), 609–632. https://doi.org/10.1631/jzus.B2000808
  44. Karikó K., Muramatsu H., Welsh F.A., Ludwig J., Kato H., Akira S., Weissman D. (2008) Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16(11), 1833–1840. https://doi.org/10.1038/mt.2008.200
  45. Adibzadeh S., Fardaei M., Takhshid M.A., Miri M.R., Rafiei Dehbidi G., Farhadi A., Ranjbaran R., Alavi P., Nikouyan N., Seyyedi N., Naderi S., Eskandari A., Behzad-Behbahani A. (2019) Enhancing stability of destabilized green fluorescent protein using chimeric mRNA containing human beta-globin 5ꞌ and 3ꞌ untranslated regions. Avicenna J. Med. Biotechnol. 11(1), 112–117.
  46. Cao J., Novoa E.M., Zhang Z., Chen W.C.W., Liu D., Choi G.C.G., Wong A.S.L., Wehrspaun C., Kellis M., Lu T.K. (2021) High-throughput 5’ UTR engineering for enhanced protein production in non-viral gene therapies. Nat. Commun. 12(1), 4138. https://doi.org/10.1038/s41467-021-24436-7
  47. Yu J., Russell J.E. (2001) Structural and functional analysis of an mRNP complex that mediates the high stability of human beta-globin mRNA. Mol. Cell Biol. 21(17), 5879–5888. https://doi.org/10.1128/MCB.21.17.5879-5888.2001
  48. Ferizi M., Aneja M.K., Balmayor E.R., Badieyan Z.S., Mykhaylyk O., Rudolph C., Plank C. (2016) Human cellular CYBA UTR sequences increase mRNA translation without affecting the half-life of recombinant RNA transcripts. Sci. Rep. 6, 39149. https://doi.org/10.1038/srep39149
  49. Keshavarz M., Mirzaei H., Salemi M., Momeni F., Mousavi M.J., Sadeghalvad M., Arjeini Y., Solaymani-Mohammadi F., Sadri Nahand J., Namdari H., Mokhtari-Azad T., Rezaei F. (2019) Influenza vaccine: where are we and where do we go? Rev. Med. Virol. 29(1), e2014. https://doi.org/10.1002/rmv.2014
  50. Armbruster N., Jasny E., Petsch B. (2019) Advances in RNA vaccines for preventive indications: a case study of a vaccine against rabies. Vaccines (Basel). 7(4), 132. https://doi.org/10.3390/vaccines7040132
  51. Sonoda J., Mizoguchi I., Inoue S., Watanabe A., Sekine A., Yamagishi M., Miyakawa S., Yamaguchi N., Horio E., Katahira Y., Hasegawa H., Hasegawa T., Yamashita K., Yoshimoto T. (2023) A promising needle-free pyro-drive jet injector for augmentation of immunity by intradermal injection as a physical adjuvant. Int. J. Mol. Sci. 24(10), 9094. https://doi.org/10.3390/ijms24109094
  52. Хромова Е.А., Ахматова Н.К., Костинов М.П., Сходова С.А., Столпникова В.Н., Власенко А.Е., Полищук В.Б., Шмитько А.Д. (2023) Влияние иммуноадъювантной и безадъювантных вакцин против гриппа на иммунофенотип лимфоцитов in vitro. Инфекция и иммунитет. 13(3), 430–438. https://doi.org/10.15789/2220-7619-TIO-1250

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2. Fig. 1. Schematic diagram of the plasmid encoding influenza A(H1N1) virus HA mRNA and analysis of the expressed target product. a – Map of the pVAX-C3-H1-PolyA plasmid. The HA gene fragment (1608 bp) and the DNA fragment corresponding to the encoded mRNA transcript (1920 bp) are shown. b – Electrophoretic separation of C3-H1 mRNA in 2% agarose gel: 1 – mRNA before purification, 2 – ssRNA Ladder marker (New England Biolabs, USA), 3 – mRNA after purification. c – Induction of IFN-α by purified and non-purified dsRNA mRNA in BALB/c mice (n = 5). The background level of IFN-α in the blood serum of the control group is shown by the dotted line. Activation of CD4+ (d) and CD8+ (e) T lymphocytes in mice after stimulation with different fractions of the target mRNA compared to the control (intact mice). The results are presented as the percentage of CD3+CD4+ and CD3+CD8+ T lymphocytes, respectively, carrying the early activation marker CD69+. The data were obtained by flow cytometry. The results are expressed as the median with range. The data were analyzed using the nonparametric Kruskal–Wallis test (*p < 0.01). e – Dot blot immunoassay with antibodies to dsRNA: mRNA before purification (1), mRNA after purification (2), dsRNA (3, positive control). g – Immunoblot analysis of the culture medium of HEK293 cells: 1 – untransfected cells (negative control), 2 – cells transfected with mRNA-C3-H1, 3 – recombinant protein HA H1 (positive control), 4 – molecular weight marker of proteins Precision Plus Protein Dual Color Standards (“Bio-Rad”, USA).

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3. Fig. 2. Design of the mouse immunization experiment. ELISA – enzyme-linked immunosorbent assay, VNA – virus-neutralizing activity assay.

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4. Fig. 3. Analysis of the humoral immune response in vaccinated BALB/c mice. a – Titers of HA-specific IgG antibodies in mouse sera (n = 10 in each group). ELISA was performed using the recombinant HA protein of the H1N1 influenza virus as an antigen. K‒ is a negative control. b – Titers of Flu-M-specific IgG antibodies in mouse sera (n = 10 in each group). ELISA was performed using the Flu-M vaccine as an antigen. Reliability was calculated using the nonparametric Mann–Whitney analysis. c – Virus-neutralizing activity of sera from mice immunized with the indicated preparations (n = 8 in each group). All results are expressed as median with range. Reliability was calculated using the nonparametric Kruskal–Wallis test. Statistical analysis was performed using GraphPad Prism 8.0 software. *p < 0.0001, **p < 0.001.

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5. Fig. 4. Analysis of T-cell immune response in vaccinated BALB/c mice (n = 5 in each group) by ELISpot. Results are presented as spot-forming units (SFU) per 106 cells. Significance was calculated using nonparametric Mann–Whitney analysis. Statistical analysis was performed using GraphPad Prism 8.0 software. *p < 0.01.

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6. Fig. 5. Analysis of the protective efficacy of the mRNA vaccine. a – Survival of BALB/c mice (n = 10 in each group) immunized with mRNA-C3-H1 or Flu-M after infection with the influenza virus A/California/04/09 (H1N1) MA8. Control – non-immunized animals. Modeling of the survival function was performed using the Kaplan–Meier multiplier estimate, comparison with survival in the control group – using the Mantel–Cox test. b – Dynamics of changes in mouse weight during the experiment.

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