Immunogenicity of various variants of Ebola and Marburg virus glycoprotein genes in recombinant adenoviral vectors

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.

AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).

MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID₅₀ assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.

RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.

CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

1. Dolzhikova IV, Zubkova OV, Tukhvatulin AI, Dzharullaeva AS, Tukhvatulina NM, Shcheblyakov DV, et al. Safety and immunogenicity of GamEvac-Combi, a heterologous VSVand Ad5-vectored Ebola vaccine: An open phase I/II trial in healthy adults in Russia. Hum Vaccin Immunother. 2017;13(3):613–20. https://doi.org/10.1080/21645515.2016.1238535

2. Zhang Z, Zhao Z, Wang Y, Wu S, Wang B, Zhang J, et al. Comparative immunogenicity analysis of intradermal versus intramuscular immunization with a recombinant human adenovirus type 5 vaccine against Ebola virus. Front Immunol. 2022;13:963049. https://doi.org/10.3389/fimmu.2022.963049

3. Ahmed I, Salsabil L, Hossain MJ, Shahriar M, Bhuiyan MA, Islam MR. The recent outbreaks of Marburg virus disease in African countries are indicating potential threat to the global public health: Future prediction from historical data. Health Sci Reports. 2023;6(7):e1395. https://doi.org/10.1002/hsr2.1395

4. Rojas M, Monsalve DM, Pacheco Y, Acosta-Ampudia Y, Ramírez-Santana C, Ansari AA, et al. Ebola virus disease: An emerging and re-emerging viral threat. J Autoimmun. 2020;106:102375. https://doi.org/10.1016/j.jaut.2019.102375

5. Jones SM, Feldmann H, Ströher U, Geisbert JB, Fernando L, Grolla A, et al. Live attenuated recombinant vaccine pro tects nonhuman primates against Ebola and Marburg vi ruses. Nat Med. 2005;11(7):786–90. https://doi.org/10.1038/nm1258

6. Swenson DL, Wang D, Luo M, Warfield KL, Woraratanadharm J, Holman DH, et al. Vaccine to confer to nonhuman primates complete protection against multistrain Ebola and Marburg virus infections. Clin Vaccine Immunol. 2008;15(3):460–7. https://doi.org/10.1128/CVI.00431-07

7. Afolabi MO, Ishola D, Manno D, Keshinro B, Bockstal V, Rogers B, et al. Safety and immunogenicity of the twodose heterologous Ad26.ZEBOV and MVA-BN-Filo Ebola vaccine regimen in children in Sierra Leone: a ran domised, double-blind, controlled trial. Lancet Infect Dis. 2022;22(1):110–22. https://doi.org/10.1016/S1473-3099(21)00128-6

8. Peng W, Rayaprolu V, Parvate AD, Pronker MF, Hui S, Parekh D, et al. Glycan shield of the ebolavirus envelope glycoprotein GP. Commun Biol. 2022;5(1):785. https://doi.org/10.1038/s42003-022-03767-1

9. Ahmad I, Fatemi SN, Ghaheri M, Rezvani A, Khezri DA, Natami M, et al. An overview of the role of Niemann– Pick C1 (NPC1) in viral infections and inhibition of viral infections through NPC1 inhibitor. Cell Commun Signal. 2023;21(1):352. https://doi.org/10.1186/s12964-023-01376-x

10. Murin CD, Gilchuk P, Crowe JE, Ward AB. Structural biology illuminates molecular determinants of broad ebolavirus neutralization by human antibodies for pan-ebolavirus therapeutic development. Front Immunol. 2022;12:808047. https://doi.org/10.3389/fimmu.2021.808047

11. Zubkova OV, Ozharovskaia TA, Dolzhikova IV, Popova O, Shcheblyakov DV, Grousova DM, et al. Expression vector for creating immunobiological agent for inducing specific immunity to virus of severe acute respiratory syndrome SARS-COV-2 (embodiments). Patent of the Russian Federa tion No. 2731356 C9; 2021 (In Russ.). EDN: KSWWVX

12. Lock M, Korn M, Wilson J, Sena-Esteves M, Gao G. Measuring the infectious titer of recombinant adenovirus using tissue culture infection dose 50% (TCID50) end-point dilution and quantitative polymerase chain reaction (qPCR). Cold Spring Harb Protoc. 2019;2019(8):pdb.prot095562. https://doi.org/10.1101/pdb.prot095562

13. Whitt MA. Generation of VSV pseudotypes using recombinant ΔG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines. J Virol Methods. 2010;169(2):365–74. https://doi.org/10.1016/j.jviromet.2010.08.006

14. Ozharovskaia TA, Popova O, Zubkova OV, Vavilova IV, Pochtovyy AA, Shcheblyakov DE, et al. Development and characterization of a vector system based on the simian adenovirus type 25. Bull Russ State Med Univ. 2023;(1):4-11 (In Russ.). https://doi.org/10.24075/vrgmu.2023.006

15. Carroll SA, Towner JS, Sealy TK, McMullan LK, Khris tova ML, Burt FJ, et al. Molecular evolution of viruses of the family Filoviridae based on 97 whole-genome sequences. J Virol. 2013;87(5):2608–16. https://doi.org/10.1128/JVI.03118-12

16. Yang ZY, Duckers HJ, Sullivan NJ, Sanchez A, Nabel EG, Nabel GJ. Identification of the Ebola virus glycoprotein as the main viral determinant of vascular cell cytotoxicity and injury. Nat Med. 2000;6(8):886–9. https://doi.org/10.1038/78645

17. Zhao Y, Ren J, Harlos K, Jones DM, Zeltina A, Bowden TA, et al. Toremifene interacts with and destabilizes the Ebola virus glycoprotein. Nature. 2016;535(7610):169–72. https://doi.org/10.1038/nature18615

18. Cottingham MG, Carroll F, Morris SJ, Turner AV, Vaughan AM, Kapulu MC, et al. Preventing spontaneous genetic rearrangements in the transgene cassettes of adenovirus vec tors. Biotechnol Bioeng. 2012;109(3):719–28. https://doi.org/10.1002/bit.24342

19. Gallais Y, Sierocki R, Lhomme G, Sivelle C, Kiseljak D, Wurm F, et al. Large-scale mapping of the Ebola NP and GP proteins reveals multiple immunoprevalent and conserved CD4 T-cell epitopes. Cell Mol Immunol. 2021;18(5):1323–5. https://doi.org/10.1038/s41423-020-0455-2

20. Bhatia B, Furuyama W, Hoenen T, Feldmann H, Marzi A. Ebola virus glycoprotein domains associated with protective efficacy. Vaccines. 2021;9(6):630. https://doi.org/10.3390/vaccines9060630

21. Meyer M, Yoshida A, Ramanathan P, Saphire EO, Collins PL, Crowe JE, et al. Antibody repertoires to the same Ebola vaccine antigen are differentially affected by vaccine vectors. Cell Rep. 2018;24(7):1816–29. https://doi.org/10.1016/j.celrep.2018.07.044

22. Kimble JB, Malherbe DC, Meyer M, Gunn BM, Karim MM, Ilinykh PA, et al. Antibody-mediated protective mecha nisms induced by a trivalent parainfluenza virus-vectored ebolavirus vaccine. J Virol. 2019;93(4):e01845–18. https://doi.org/10.1128/JVI.01845-18

23. Murin CD, Fusco ML, Bornholdt ZA, Qiu X, Olinger GG, Zeitlin L, et al. Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proc Natl Acad Sci USA. 2014;111(48):17182–7. https://doi.org/10.1073/pnas.1414164111

24. Saphire EO, Schendel SL, Fusco ML, Gangavarapu K, Gunn BM, Wec AZ, et al. Systematic analysis of monoclonal antibodies against Ebola virus GP defines features that contribute to protection. Cell. 2018;174(4):938–952.e13. https://doi.org/10.1016/j.cell.2018.07.033

25. Bornholdt ZA, Turner HL, Murin CD, Li W, Sok D, Souders CA, et al. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak. Science. 2016;351(6277):1078–83. https://doi.org/10.1126/science.aad5788

26. Towner JS, Sealy TK, Khristova ML, Albariño CG, Conlan S, Reeder SA, et al. Newly discovered Ebola virus associated with hemorrhagic fever outbreak in Uganda. PLoS Pathog. 2008;4(11):e1000212. https://doi.org/10.1371/journal.ppat.1000212

27. Hensley LE, Mulangu S, Asiedu C, Johnson J, Honko AN, Stanley D, et al. Demonstration of cross-protective vaccine immunity against an emerging pathogenic Ebolavirus species. PLoS Pathog. 2010;6(5):e1000904. https://doi.org/10.1371/journal.ppat.1000904

28. Hevey M, Negley D, Pushko P, Smith J, Schmaljohn A. Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology. 1998;251(1):28–37. https://doi.org/10.1006/viro.1998.9367

29. Zhu F-C, Guan X-H, Li Y-H, Huang J-Y, Jiang T, Hou L-H, et al. Immunogenicity and safety of a recombinant adenovirus type-5-vectored COVID-19 vaccine in healthy adults aged 18 years or older: a randomised, double-blind, placebocontrolled, phase 2 trial. Lancet. 2020;396(10249):479–88. https://doi.org/10.1016/S0140-6736(20)31605-6

30. Flickinger JC, Singh J, Carlson R, Leong E, Baybutt TR, Barton J, et al. Chimeric Ad5.F35 vector evades anti-adenovirus serotype 5 neutralization opposing GUCY2C-targeted antitumor immunity. J Immunother Cancer. 2020;8(2):e001046. https://doi.org/10.1136/jitc-2020-001046

31. Flickinger JC, Staudt RE, Singh J, Carlson RD, Barton JR, Baybutt TR, et al. Chimeric adenoviral (Ad5.F35) and listeria vector prime-boost immunization is safe and effective for cancer immunotherapy. NPJ Vaccines. 2022;7(1):61. https://doi.org/10.1038/s41541-022-00483-z

32. Mitchell DAJ, Dupuy LC, Sanchez-Lockhart M, Palacios G, Back JW, Shimanovskaya K, et al. Epitope mapping of Ebola virus dominant and subdominant glycoprotein epitopes facilitates construction of an epitope-based DNA vaccine able to focus the antibody response in mice. Hum Vaccin Immunother. 2017;13(12):2883–93. https://doi.org/10.1080/21645515.2017.1347740