Development of a recombinant replication-deficient rabies virus-based bivalent-vaccine against MERS-CoV and rabies virus and its humoral immunogenicity in mice

Autoři: Hirofumi Kato aff001;  Mutsuyo Takayama-Ito aff001;  Itoe Iizuka-Shiota aff001;  Shuetsu Fukushi aff001;  Guillermo Posadas-Herrera aff001;  Madoka Horiya aff001;  Masaaki Satoh aff001;  Tomoki Yoshikawa aff001;  Souichi Yamada aff001;  Shizuko Harada aff001;  Hikaru Fujii aff001;  Miho Shibamura aff001;  Takuya Inagaki aff001;  Kinjiro Morimoto aff003;  Masayuki Saijo aff001;  Chang-Kweng Lim aff001
Působiště autorů: Department of Virology I, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan aff001;  Department of Life Science and Medical Bioscience, Waseda University, Shinjuku-ku, Tokyo, Japan aff002;  Department of Pharmacy, Yasuda Women’s University, Hiroshima, Hiroshima, Japan aff003
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223684


Middle East respiratory syndrome-coronavirus (MERS-CoV) is an emerging virus that causes severe disease with fatal outcomes; however, there are currently no approved vaccines or specific treatments against MERS-CoV. Here, we developed a novel bivalent vaccine against MERS-CoV and rabies virus (RV) using the replication-incompetent P-gene-deficient RV (RVΔP), which has been previously established as a promising and safe viral vector. MERS-CoV spike glycoprotein comprises S1 and S2 subunits, with the S1 subunit being a primary target of neutralizing antibodies. Recombinant RVΔP, which expresses S1 fused with transmembrane and cytoplasmic domains together with 14 amino acids from the ectodomains of the RV-glycoprotein (RV-G), was developed using a reverse genetics method and named RVΔP-MERS/S1. Following generation of RVΔP-MERS/S1 and RVΔP, our analysis revealed that they shared similar growth properties, with the expression of S1 in RVΔP-MERS/S1-infected cells confirmed by immunofluorescence and western blot, and the immunogenicity and pathogenicity evaluated using mouse infection experiments. We observed no rabies-associated signs or symptoms in mice inoculated with RVΔP-MERS/S1. Moreover, virus-specific neutralizing antibodies against both MERS-CoV and RV were induced in mice inoculated intraperitoneally with RVΔP-MERS/S1. These findings indicate that RVΔP-MERS/S1 is a promising and safe bivalent-vaccine candidate against both MERS-CoV and RV.

Klíčová slova:

Antibodies – Recombinant proteins – Recombinant vaccines – Vaccines – Viral vaccines – Coronaviruses – Vaccine development – Inoculation


1. Zumla A, Hui DS, Perlman S. Middle East Respiratory Syndrome. Lancet. 2015;386: 995–1007. doi: 10.1016/S0140-6736(15)60454-8 26049252

2. Chan JFW, Lau SKP, To KKW, Cheng VCC, Woo PCY, Yuen K-Y. Middle East Respiratory Syndrome coronavirus: Another zoonotic betacoronavirus causing SARS-like disease. Clin Microbiol Rev. 2015;28: 465–522. doi: 10.1128/CMR.00102-14 25810418

3. Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus ADME, Fouchier RAM. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012;367: 1814–1820. doi: 10.1056/NEJMoa1211721 23075143

4. World Health Organization. Middle East Respiratory Syndrome coronavirus (MERS-CoV). [cited 2019 May 9].

5. Kim Y, Lee S, Chu C, Choe S, Hong S, Shin Y. The characteristics of Middle Eastern Respiratory Syndrome coronavirus transmission dynamics in South Korea. Osong Public Health Res Perspect. 2016;7: 49–55. doi: 10.1016/j.phrp.2016.01.001 26981343

6. Modjarrad K. MERS-CoV vaccine candidates in development: The current landscape. Vaccine. 2016;34: 2982–2987. doi: 10.1016/j.vaccine.2016.03.104 27083424

7. Graham RL, Donaldson EF, Baric RS. A decade after SARS: strategies for controlling emerging coronaviruses. Nat Rev Microbiol. 2013;11: 836–848. doi: 10.1038/nrmicro3143 24217413

8. Raj VS, Mou H, Smits SL, Dekkers DHW, Müller MA, Dijkman R, et al. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013;495: 251–254. doi: 10.1038/nature12005 23486063

9. Li F. Receptor recognition mechanisms of coronaviruses: a decade of structural studies. J Virol. 2015;89:1954–1964. doi: 10.1128/JVI.02615-14 25428871

10. Lu G, Hu Y, Wang Q, Qi J, Gao F, Li Y, et al. Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26. Nature. 2013;500: 227–231. doi: 10.1038/nature12328 23831647

11. Du L, Zhao G, Kou Z, Ma C, Sun S, Poon VKM, et al. Identification of a receptor-binding domain in the S protein of the novel human coronavirus Middle East Respiratory Syndrome coronavirus as an essential target for vaccine development. J Virol. 2013;87: 9939–9942. doi: 10.1128/JVI.01048-13 23824801

12. Du L, Kou Z, Ma C, Tao X, Wang L, Zhao G, et al. A truncated receptor-binding domain of MERS-CoV spike protein potently inhibits MERS-CoV infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines. PLoS One. 2013;8: e81587. doi: 10.1371/journal.pone.0081587 24324708

13. Ma C, Li Y, Wang L, Zhao G, Tao X, Tseng C-TK, et al. Intranasal vaccination with recombinant receptor-binding domain of MERS-CoV spike protein induces much stronger local mucosal immune responses than subcutaneous immunization: Implication for designing novel mucosal MERS vaccines. Vaccine. 2014;32: 2100–2108. doi: 10.1016/j.vaccine.2014.02.004 24560617

14. Liu R, Wang J, Shao Y, Wang X, Zhang H, Shuai L, et al. A recombinant VSV-vectored MERS-CoV vaccine induces neutralizing antibody and T cell responses in rhesus monkeys after single dose immunization. Antiviral Res. 2018;150: 30–38. doi: 10.1016/j.antiviral.2017.12.007 29246504

15. Wirblich C, Coleman CM, Kurup D, Abraham TS, Bernbaum JG, Jahrling PB, et al. One-Health: a safe, efficient, dual-use vaccine for humans and animals against Middle East Respiratory Syndrome coronavirus and rabies virus. J Virol. 2017;91: e02040–16. doi: 10.1128/JVI.02040-16 27807241

16. Knipe DM, Howley PM. Fields Virology. 6th ed. Philadelphia: Lippincott-Raven Publishers; 2013.

17. Dacheux L, Delmas O, Bourhy H. Human rabies encephalitis prevention and treatment: progress since Pasteur’s discovery. Infect Disord Drug Targets. 2011;11: 251–299. 21488832

18. World Health Organization. Human rabies: 2016 updates and call for data. Wkly Epidemiol Rec. 2017;92: 77–86. 28211265

19. Gomme EA, Wanjalla CN, Wirblich C, Schnell MJ. Rabies virus as a research tool and viral vaccine vector. Adv Virus Res. 2011;79: 139–164. doi: 10.1016/B978-0-12-387040-7.00009-3 21601047

20. Chenik M, Schnell M, Conzelmann KK, Blondel D. Mapping the interacting domains between the rabies virus polymerase and phosphoprotein. J Virol. 1998;72: 1925–1930. 9499045

21. Schnell MJ, Conzelmann KK. Polymerase activity of in vitro mutated rabies virus L protein. Virology. 1995;214: 522–530. doi: 10.1006/viro.1995.0063 8553554

22. Morimoto K, Shoji Y, Inoue S. Characterization of P gene-deficient rabies virus: propagation, pathogenicity and antigenicity. Virus Res. 2005;111: 61–67. doi: 10.1016/j.virusres.2005.03.011 15896403

23. Mebatsion T, Weiland F, Conzelmann KK. Matrix protein of rabies virus is responsible for the assembly and budding of bullet-shaped particles and interacts with the transmembrane spike glycoprotein G. J Virol. 1999;73: 242–250. 9847327

24. Cenna J, Hunter M, Tan GS, Papaneri AB, Ribka EP, Schnell MJ, et al. Replication‐deficient rabies virus-based vaccines are safe and immunogenic in mice and nonhuman primates. J Infect Dis. 2009;200: 1251–1260. doi: 10.1086/605949 19764884

25. Ito N, Sugiyama M, Yamada K, Shimizu K, Takayama-Ito M, Hosokawa J, et al. Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain. Microbiol Immunol. 2005;49: 971–979. doi: 10.1111/j.1348-0421.2005.tb03692.x 16301807

26. Shoji Y, Inoue S, Nakamichi K, Kurane I, Sakai T, Morimoto K. Generation and characterization of P gene-deficient rabies virus. Virology. 2004;318: 295–305. doi: 10.1016/j.virol.2003.10.001 14972555

27. Schnell MJ, Mebatsion T, Conzelmann KK. Infectious rabies viruses from cloned cDNA. EMBO J. 1994;13: 4195–4203. 7925265

28. Mebatsion T, Schnell MJ, Cox JH, Finke S, Conzelmann KK. Highly stable expression of a foreign gene from rabies virus vectors. Proc Natl Acad Sci U S A. 1996;93: 7310–7314. doi: 10.1073/pnas.93.14.7310 8692989

29. Takayama-Ito M, Lim C-K, Yamaguchi Y, Posadas-Herrera G, Kato H, Iizuka-Shiota I, et al. Replication-incompetent rabies virus vector harboring Glycoprotein gene of Lymphocytic Choriomeningitis Virus (LCMV) protects mice from lethal LCMV challenge. PLoS Negl Trop Dis. 2018;16: e0006398.

30. Schnell MJ, Foley HD, Siler CA, McGettigan JP, Dietzschold B, Pomerantz RJ. Recombinant rabies virus as potential live-viral vaccines for HIV-1. Proc Natl Acad Sci U S A. 2000;97:3544–3549. doi: 10.1073/pnas.050589197 10706640

31. Faber M, Lamirande EW, Roberts A, Rice AB, Koprowski H, Dietzschold B, et al. A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies. J Gen Virol. 2005;86: 1435–1440. doi: 10.1099/vir.0.80844-0 15831955

32. Conzelmann KK, Cox JH, Schneider LG, Thiel HJ. Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B19. Virology. 1990;175: 485–499. doi: 10.1016/0042-6822(90)90433-r 2139267

33. Foley HD, McGettigan JP, Siler CA, Dietzschold B, Schnell MJ. A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection. Proc Natl Acad Sci U S A. 2000;97: 14680–14685. doi: 10.1073/pnas.011510698 11114165

34. McGettigan JP, Naper K, Orenstein J, Koser M, McKenna PM, Schnell MJ. Functional human immunodeficiency virus type 1 (HIV-1) Gag-Pol or HIV-1 Gag-Pol and env expressed from a single rhabdovirus-based vaccine vector genome. J Virol. 2003;77: 10889–10899. doi: 10.1128/JVI.77.20.10889-10899.2003 14512539

35. McGettigan JP, Sarma S, Orenstein JM, Pomerantz RJ, Schnell MJ. Expression and immunogenicity of human immunodeficiency virus type 1 Gag expressed by a replication-competent rhabdovirus-based vaccine vector. J Virol. 2001;75: 8724–8732. doi: 10.1128/JVI.75.18.8724-8732.2001 11507217

36. McGettigan JP, Pomerantz RJ, Siler CA, McKenna PM, Foley HD, Dietzschold B, et al. Second-generation rabies virus-based vaccine vectors expressing human immunodeficiency virus type 1 gag have greatly reduced pathogenicity but are highly immunogenic. J Virol. 2003;77: 237–244. doi: 10.1128/JVI.77.1.237-244.2003 12477829

37. Orciari LA, Niezgoda M, Hanlon CA, Shaddock JH, Sanderlin DW, Yager PA, et al. Rapid clearance of SAG-2 rabies virus from dogs after oral vaccination. Vaccine. 2001;19: 4511–4518. doi: 10.1016/s0264-410x(01)00186-4 11483278

38. Papaneri AB, Wirblich C, Cooper K, Jahrling PB, Schnell MJ, Blaney JE. Further characterization of the immune response in mice to inactivated and live rabies vaccines expressing Ebola virus glycoprotein. 2012 Sep 21;30: 6136–6141. [cited 2018 Oct 9]. Vaccine.

39. Koprowski H, Black J, Nelsen DJ. Studies on chick-embryo-adapted-rabies virus. VI. Further changes in pathogenic properties following prolonged cultivation in the developing chick embryo. J Immunol. 1954;72: 94–106. 13118196

40. Inoue K, Shoji Y, Kurane I, Iijima T, Sakai T, Morimoto K. An improved method for recovering rabies virus from cloned cDNA. J Virol Methods. 2003;107: 229–236. doi: 10.1016/s0166-0934(02)00249-5 12505638

41. Fukushi S, Fukuma A, Kurosu T, Watanabe S, Shimojima M, Shirato K, et al. Characterization of novel monoclonal antibodies against the MERS-coronavirus spike protein and their application in species-independent antibody detection by competitive ELISA. J Virol Methods. 2018;251: 22–29. doi: 10.1016/j.jviromet.2017.10.008 28993122

42. Luo TR, Minamoto N, Ito H, Goto H, Hiraga S, Ito N, et al. A virus-neutralizing epitope on the glycoprotein of rabies virus that contains Trp251 is a linear epitope. Virus Res. 1997;51: 35–41. doi: 10.1016/s0168-1702(97)00080-4 9381793

43. Shirato K, Azumano A, Nakao T, Hagihara D, Ishida M, Tamai K, et al. Middle East Respiratory Syndrome coronavirus infection not found in camels in Japan. Jpn J Infect Dis. 2015;68: 256–258. doi: 10.7883/yoken.JJID.2015.094 25993975

44. Fukushi S, Mizutani T, Saijo M, Kurane I, Taguchi F, Tashiro M, et al. Evaluation of a novel vesicular stomatitis virus pseudotype-based assay for detection of neutralizing antibody responses to SARS-CoV. J Med Virol. 2006;78: 1509–1512. doi: 10.1002/jmv.20732 17063504

45. Krämer B, Schildger H, Behrensdorf-Nicol HA, Hanschmann KM, Duchow K. The rapid fluorescent focus inhibition test is a suitable method for batch potency testing of inactivated rabies vaccines. Biologicals. 2009;37: 119–126. doi: 10.1016/j.biologicals.2009.01.001 19181541

46. Krämer B, Bruckner L, Daas A, Milne C. Collaborative study for validation of a serological potency assay for rabies vaccine (inactivated) for veterinary use. Pharmeur Bio Sci Notes. 2010;2010: 37–55. 21144488

47. Cho H, Excler J-L, Kim JH, Yoon I-K. Development of Middle East Respiratory Syndrome coronavirus vaccines–advances and challenges. Hum Vaccin Immunother. 2018;14: 304–313. doi: 10.1080/21645515.2017.1389362 29048984

48. Alharbi NK. Vaccines against Middle East Respiratory Syndrome coronavirus for humans and camels. Rev Med Virol. 2017;27: e1917.

49. Song F, Fux R, Provacia LB, Volz A, Eickmann M, Becker S, et al. Middle East Respiratory Syndrome coronavirus spike protein delivered by Modified Vaccinia Virus Ankara efficiently induces virus-neutralizing antibodies. J Virol. 2013;87: 11950–11954. doi: 10.1128/JVI.01672-13 23986586

50. Malczyk AH, Kupke A, Prüfer S, Scheuplein VA, Hutzler S, Kreuz D, et al. A highly immunogenic and protective Middle East Respiratory Syndrome coronavirus vaccine based on a recombinant measles virus vaccine platform. J Virol. 2015;89: 11654–11667. doi: 10.1128/JVI.01815-15 26355094

51. Guo X, Deng Y, Chen H, Lan J, Wang W, Zou X, et al. Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East Respiratory Syndrome coronavirus. Immunology. 2015;145: 476–484. doi: 10.1111/imm.12462 25762305

52. Kim E, Okada K, Kenniston T, Raj VS, AlHajri MM, Farag EABA, et al. Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice. Vaccine. 2014;32: 5975–5982. doi: 10.1016/j.vaccine.2014.08.058 25192975

53. Blaney JE, Marzi A, Willet M, Papaneri AB, Wirblich C, Feldmann F, et al. Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine. PLoS Pathog. 2013;9: e1003389. doi: 10.1371/journal.ppat.1003389 23737747

54. Siler CA, McGettigan JP, Dietzschold B, Herrine SK, Dubuisson J, Pomerantz RJ, et al. Live and killed rhabdovirus-based vectors as potential hepatitis C vaccines. Virology. 2002;292: 24–34. doi: 10.1006/viro.2001.1212 11878905

55. Li Y, Wan Y, Liu P, Zhao J, Lu G, Qi J, et al. A humanized neutralizing antibody against MERS-CoV targeting the receptor-binding domain of the spike protein. Cell Res. 2015;25: 1237–1249. doi: 10.1038/cr.2015.113 26391698

56. Uyeki TM, Erlandson KJ, Korch G, O’Hara M, Wathen M, Hu-Primmer J, et al. Development of medical countermeasures to Middle East Respiratory Syndrome coronavirus. Emerg Infect Dis. 2016;22.

57. World Health Organization. Rabies vaccines: WHO position paper–April 2018. Wkly Epidemiol Rec. 2018;16: 201–220.

58. Papaneri AB, Wirblich C, Cann JA, Cooper K, Jahrling PB, Schnell MJ, et al. A replication-deficient rabies virus vaccine expressing Ebola virus glycoprotein is highly attenuated for neurovirulence. Virology. 2012;434: 18–26. doi: 10.1016/j.virol.2012.07.020 22889613

59. Blaney JE, Wirblich C, Papaneri AB, Johnson RF, Myers CJ, Juelich TL, et al. Inactivated or live-attenuated bivalent vaccines that confer protection against rabies and Ebola viruses. J Virol. 2011;85: 10605–10616. doi: 10.1128/JVI.00558-11 21849459

60. Yu GM, Zu SL, Zhou WW, Wang XJ, Shuai L, Wang XL, et al. Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels. J Vet Sci. 2017;18: 351–359. doi: 10.4142/jvs.2017.18.S1.351 27515260

61. Al-Amri SS, Abbas AT, Siddiq LA, Alghamdi A, Sanki MA, Al-Muhanna MK, et al. Immunogenicity of candidate MERS-CoV DNA vaccines based on the spike protein. Sci Rep. 2017;7: 44875. doi: 10.1038/srep44875 28332568

62. Chi H, Zheng X, Wang X, Wang C, Wang H, Gai W, et al. DNA vaccine encoding Middle East Respiratory Syndrome coronavirus S1 protein induces protective immune responses in mice. Vaccine. 2017;35: 2069–2075. doi: 10.1016/j.vaccine.2017.02.063 28314561

63. Kim E, Okada K, Kenniston T, Raj VS, AlHajri MM, Farag EABA, et al. Immunogenicity of an adenoviral-based Middle East Respiratory Syndrome coronavirus vaccine in BALB/c mice. Vaccine. 2014;32: 5975–5982. doi: 10.1016/j.vaccine.2014.08.058 25192975

64. Iwata-Yoshikawa N, Uda A, Suzuki T, Tsunetsugu-Yokota Y, Sato Y, Morikawa S, et al. Effects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccine. J Virol. 2014;88: 8597–8614. doi: 10.1128/JVI.00983-14 24850731

65. Reusken CBEM, Messadi L, Feyisa A, Ularamu H, Godeke G-J, Danmarwa A, et al. Geographic distribution of MERS coronavirus among dromedary camels, Africa. Emerg Infect Dis. 2014;20: 1370–1374. doi: 10.3201/eid2008.140590 25062254

66. Reusken CB, Haagmans BL, Müller MA, Gutierrez C, Godeke G-J, Meyer B, et al. Middle East Respiratory Syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect Dis. 2013;13: 859–866. doi: 10.1016/S1473-3099(13)70164-6 23933067

Článek vyšel v časopise


2019 Číslo 10

Nejčtenější v tomto čísle

Tomuto tématu se dále věnují…


Zvyšte si kvalifikaci online z pohodlí domova

Léčba bolesti v ordinaci praktického lékaře
nový kurz
Autoři: MUDr. PhDr. Zdeňka Nováková, Ph.D.

Revmatoidní artritida: včas a k cíli
Autoři: MUDr. Heřman Mann

Jistoty a nástrahy antikoagulační léčby aneb kardiolog - neurolog - farmakolog - nefrolog - právník diskutují
Autoři: doc. MUDr. Štěpán Havránek, Ph.D., prof. MUDr. Roman Herzig, Ph.D., doc. MUDr. Karel Urbánek, Ph.D., prim. MUDr. Jan Vachek, MUDr. et Mgr. Jolana Těšínová, Ph.D.

Léčba akutní pooperační bolesti
Autoři: doc. MUDr. Jiří Málek, CSc.

Nové antipsychotikum kariprazin v léčbě schizofrenie
Autoři: prof. MUDr. Cyril Höschl, DrSc., FRCPsych.

Všechny kurzy
Kurzy Doporučená témata Časopisy
Zapomenuté heslo

Nemáte účet?  Registrujte se

Zapomenuté heslo

Zadejte e-mailovou adresu se kterou jste vytvářel(a) účet, budou Vám na ni zaslány informace k nastavení nového hesla.


Nemáte účet?  Registrujte se