Microbiology Independent Research Journal (MIR Journal)

Расширенный поиск

Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line

Полный текст:


The biological basis for the restricted immunogenicity of some live attenuated influenza vaccine strains generated on the backbone of the cold adapted (ca) A/Singapore/1/1957/ca (H2N2) influenza A virus master strain and produced in the Vero cells was investigated. According to our previous results the vaccine candidate made from A/Hong Kong/1035/1998 (H1N1) Vero-derived virus did not provoke a measurable antibody titers following the intranasal immunization of humans. We report here that the hemagglutinin (HA) of A/Hong Kong/1035/1998 virus contained the mutation 10Ile→Val in the HA2 subunit, that increased the pH threshold of HA conformational change (pH of activation) by 0.3 pH units and therefore might be responsible for the lack of immune response in humans. Similar effect was shown for the reassortant made from the Vero-derived A/Switzerland/5389/1995 (H1N1) (5389wt) virus which had the HA2 mutation 3Phe→Leu leading to the lack of immune response in mice. Another factor compromising the immunogenicity of a vaccine candidate is the incompatibility of epidemic virus HA with the M gene of the master strain. In mice the 6/2 A/Switzerland/5389/1995 reassortant induced antibodies that were directed predominantly to the HA2 subunit and were detectable by ELISA but not by a hemagglutination inhibition (HAI) test. In contrast, the 5/3 reassortant, bearing the HA, neuraminidase (NA), and M genes from the epidemic virus induced an equivalent amount of antibodies against the HA1 and HA2 subunits detected by HAI and ELISA. By comparing the sensitivity of the viruses to amantadine, we showed that the M2 ion channel of the master strain had lower activity than that of the A/Switzerland/5389/1995. These data suggest that M2 of the master strain was not sufficiently active to keep the pH of the transGolgi network high enough to prevent the conformational change of the acid sensitive HA to the low pH form.
Overall, the adaptation mutations in the HA of the vaccine candidate that increase the pH of HA activation as well as the incompatibility of HA and M genes must be taken into consideration when constructing the reassortant strains for the live attenuated vaccine.

Об авторах

A. Wolkerstorfer
Department of Biotechnology, University for Natural Resources and Life Sciences


D. Katinger
Polymun Scientific Immunbiologische Forschung GmbH


Ju. Romanova
Department of Biotechnology, University for Natural Resources and Life Sciences

Julia Romanova 


Список литературы

1. Kendal AP. Cold-adapted live attenuated influenza vaccines developed in Russia: can they contribute to meeting the needs for influenza control in other countries? Eur J Epidemiol. 1997;13(5), 591-609.

2. Rudenko L, Desheva J, Korovkin S, Mironov A, Rekstin A, Grigorieva E, Donina S, Gambaryan A, Katlinsky A. Safety and immunogenicity of live attenuated influenza reassortant H5 vaccine (phase I-II clinical trials). Influenza Other Respi Viruses 2008; 2(6), 203-9.

3. Rudenko LG, Arden NH, Grigorieva E, Naychin A, Rekstin A, Klimov AI, Donina S, Desheva J, Holman RC, DeGuzman A, Cox NJ, Katz JM. Immunogenicity and efficacy of Russian live attenuated and US inactivated influenza vaccines used alone and in combination in nursing home residents. Vaccine 2000; 19(2-3), 308-18.

4. Rudenko LG, Lonskaya NI, Klimov AI, Vasilieva RI, Ramirez A. Clinical and epidemiological evaluation of a live, cold-adapted influenza vaccine for 3-14-yearolds. Bull World Health Organ. 1996; 74(1), 77-84.

5. Rudenko L, Isakova-Sivak I, Naykhin A, Kiseleva I, Stukova M, Erofeeva M, Korenkov D, Matyushenko V, Sparrow E, Kieny MP. H7N9 live attenuated influenza vaccine in healthy adults: a randomised, doubleblind, placebo-controlled, phase 1 trial. Lancet Infect Dis. 2016; 16(3), 303-10. doi: 10.1016/S1473-3099(15)00378-3.

6. Maassab HF, Heilman CA, Herlocher ML. Coldadapted influenza viruses for use as live vaccines for man. Adv Biotechnol Processes. 1990; 14, 203-42.

7. Ambrose CS, Luke C, Coelingh K. Current status of live attenuated influenza vaccine in the United States for seasonal and pandemic influenza. Influenza Other Respir Viruses. 2008; 2(6), 193-202. doi: 10.1111/j.1750-2659.2008.00056.x.

8. Robertson JS. An overview of host cell selection. Dev Biol Stand. 1999; 98, 7-11; discussion 73-4.

9. Robertson JS, Cook P, Attwell AM, Williams SP. Replicative advantage in tissue culture of eggadapted influenza virus over tissue-culture derived virus: implications for vaccine manufacture. Vaccine. 1995; 13(16), 1583-8.

10. Hardy CT, Young SA, Webster RG, Naeve CW, Owens RJ. Egg fluids and cells of the chorioallantoic membrane of embryonated chicken eggs can select different variants of influenza A (H3N2) viruses. Virology. 1995; 211(1), 302-6. doi: 10.1006/viro.1995.1405.

11. Liu J, Shi X, Schwartz R, Kemble G. Use of MDCK cells for production of live attenuated influenza vaccine. Vaccine. 2009; 27(46), 6460-3. doi: 10.1016/j.vaccine.2009.06.024.

12. Ghendon YZ, Markushin SG, Akopova II, Koptiaeva IB, Nechaeva EA, Mazurkova LA, Radaeva IF, Kolokoltseva TD. Development of cell culture (MDCK) live cold-adapted (CA) attenuated influenza vaccine. Vaccine. 2005; 23(38), 4678-84. doi: 10.1016/j.vaccine.2005.04.039.

13. Barrett PN, Portsmouth D, Ehrlich HJ. Developing cell culture-derived pandemic vaccines. Curr Opin Mol Ther. 2010; 12(1), 21-30.

14. Barrett PN, Portsmouth D, Ehrlich HJ. Vero cell culturederived pandemic influenza vaccines: preclinical and clinical development. Expert Rev Vaccines. 2013; 12(4), 395-413. doi: 10.1586/erv.13.21.

15. Murakami S, Horimoto T, Ito M, Takano R, Katsura H, Shimojima M, Kawaoka Y. Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion. J Virol. 2012; 86(3), 1405-10. doi: 10.1128/JVI.06009-11.

16. Romanova J, Katinger D, Ferko B, Vcelar B, Sereinig S, Kuznetsov O, Stukova M, Erofeeva M, Kiselev O, Katinger H, Egorov A. Live cold-adapted influenza A vaccine produced in Vero cell line. Virus Res. 2004; 103(1-2), 187-93. doi: 10.1016/j.virusres.2004.02.032.

17. Noble GR, Kaye HS, Yarbrough WB, Fiedler BK, Reed CJ, Felker MB, Kendal AP, Dowdle WR. Measurement of hemagglutination-inhibiting antibody to influenza virus in the 1976 influenza vaccine program: methods and test reproducibility. J Infect Dis. 1977; 136 Suppl: S429-34.

18. Matrosovich MN, Gambaryan AS, Tuzikov AB, Byramova NE, Mochalova LV, Golbraikh AA, Shenderovich MD, Finne J, Bovin NV. Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology. 1993; 196(1), 111- 21. doi: 10.1006/viro.1993.1459.

19. Nakowitsch S, Waltenberger AM, Wressnigg N, Ferstl N, Triendl A, Kiefmann B, Montomoli E, Lapini G, Sergeeva M, Muster T, Romanova JR. Egg- or cell culture-derived hemagglutinin mutations impair virus stability and antigen content of inactivated influenza vaccines. Biotechnol J. 2014; 9(3), 405-14. doi: 10.1002/biot.201300225.

20. Nakowitsch S, Wolschek M, Morokutti A, Ruthsatz T, Krenn BM, Ferko B, Ferstl N, Triendl A, Muster T, Egorov A, Romanova J. Mutations affecting the stability of the haemagglutinin molecule impair the immunogenicity of live attenuated H3N2 intranasal influenza vaccine candidates lacking NS1. Vaccine. 2011; 29(19), 3517-24. doi: 10.1016/j.vaccine.2011.02.100.

21. Krenn BM, Egorov A, Romanovskaya-Romanko E, Wolschek M, Nakowitsch S, Ruthsatz T, Kiefmann B, Morokutti A, Humer J, Geiler J, Cinatl J, Michaelis M, Wressnigg N, Sturlan S, Ferko B, Batishchev OV, Indenbom AV, Zhu R, Kastner M, Hinterdorfer P, Kiselev O, Muster T, Romanova J. Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1. PLoS ONE. 2011; 6(4), e18577. doi: 10.1371/journal.pone.0018577.

22. Cross KJ, Wharton SA, Skehel JJ, Wiley DC, Steinhauer DA. Studies on influenza haemagglutinin fusion peptide mutants generated by reverse genetics. EMBO J. 2001; 20(16), 4432-42. doi: 10.1093/emboj/20.16.4432.

23. Clements DA, Langdon L, Bland C, Walter E. Influenza A vaccine decreases the incidence of otitis media in 6- to 30-month-old children in day care. Arch Pediatr Adolesc Med. 1995; 149(10), 1113-7.

24. Klimov AI, Sokolov NI, Orlova NG, Ginzburg VP. Correlation of amino acid residues in the M1 and M2 proteins of influenza virus with high yielding properties. Virus Res. 1991; 19(1), 105-14.

25. Florent G. Gene constellation of live influenza A vaccines. Arch Virol. 1980; 64(2), 171-3.

26. Snyder MH, Buckler-White AJ, London WT, Tierney EL, Murphy BR. The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys. J Virol. 1987; 61(9), 2857-63.

27. Subbarao EK, Perkins M, Treanor JJ, Murphy BR. The attenuation phenotype conferred by the M gene of the influenza A/Ann Arbor/6/60 cold-adapted virus (H2N2) on the A/Korea/82 (H3N2) reassortant virus results from a gene constellation effect. Virus Res. 1992; 25(1-2), 37-50.

28. Yasuda J, Bucher DJ, Ishihama A. Growth control of influenza A virus by M1 protein: analysis of transfectant viruses carrying the chimeric M gene. J Virol. 1994; 68(12), 8141-6.

29. Grambas S, Hay AJ. Maturation of influenza A virus hemagglutinin--estimates of the pH encountered during transport and its regulation by the M2 protein. Virology. 1992; 190(1), 11-8.

30. Liu T, Muller J, Ye Z. Association of influenza virus matrix protein with ribonucleoproteins may control viral growth and morphology. Virology. 2002; 304(1), 89-96.

31. Scholtissek C, Stech J, Krauss S, Webster RG. Cooperation between the hemagglutinin of avian viruses and the matrix protein of human influenza A viruses. J Virol. 2002; 76(4), 1781-6.

32. Ciampor F, Thompson CA, Grambas S, Hay AJ. Regulation of pH by the M2 protein of influenza A viruses. Virus Res. 1992; 22(3), 247-58.

33. Pinto LH, Holsinger LJ, Lamb RA. Influenza virus M2 protein has ion channel activity. Cell. 1992; 69(3), 517-28.

34. Wang C, Lamb RA, Pinto LH. Direct measurement of the influenza A virus M2 protein ion channel activity in mammalian cells. Virology. 1994; 205(1), 133-40. doi: 10.1006/viro.1994.1628.

35. Wang C, Lamb RA, Pinto LH. Activation of the M2 ion channel of influenza virus: a role for the transmembrane domain histidine residue. Biophys J. 1995; 69(4), 1363-71. doi: 10.1016/S0006-3495(95)80003-2.

36. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera- -a visualization system for exploratory research and analysis. J Comput Chem. 2004; 25(13), 1605-12. doi: 10.1002/jcc.20084.

37. Katz JM, Naeve CW, Webster RG. Host cell-mediated variation in H3N2 influenza viruses. Virology. 1987; 156(2), 386-95.

38. Katz JM, Webster RG. Efficacy of inactivated influenza A virus (H3N2) vaccines grown in mammalian cells or embryonated eggs. J Infect Dis. 1989; 160(2), 191-8.

39. Meyer WJ, Wood JM, Major D, Robertson JS, Webster RG, Katz JM. Influence of host cell-mediated variation on the international surveillance of influenza A (H3N2) viruses. Virology. 1993; 196(1), 130-7. doi: 10.1006/viro.1993.1461.

40. Romanova J, Katinger D, Ferko B, Voglauer R, Mochalova L, Bovin N, Lim W, Katinger H, Egorov A. Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern. Virology. 2003; 307(1), 90-7.

41. Lin YP, Wharton SA, Martin J, Skehel JJ, Wiley DC, Steinhauer DA. Adaptation of egg-grown and transfectant influenza viruses for growth in mammalian cells: selection of hemagglutinin mutants with elevated pH of membrane fusion. Virology. 1997; 233(2), 402-10.

42. Washington N, Steele RJ, Jackson SJ, Bush D, Mason J, Gill DA, Pitt K, Rawlins DA. Determination of baseline human nasal pH and the effect of intranasally administered buffers. Int J Pharm. 2000; 198(2), 139- 46.

43. Vareille M, Kieninger E, Edwards MR, Regamey N. The airway epithelium: soldier in the fight against respiratory viruses. Clin Microbiol Rev. 2011; 24(1), 210-29. doi: 10.1128/CMR.00014-10.

44. England RJ, Homer JJ, Knight LC, Ell SR. Nasal pH measurement: a reliable and repeatable parameter. Clin Otolaryngol Allied Sci. 1999; 24(1), 67-8.

45. Arora P, Sharma S, Garg S. Permeability issues in nasal drug delivery. Drug Discov Today. 2002; 7(18), 967-75.

46. Hehar SS, Mason JD, Stephen AB, Washington N, Jones NS, Jackson SJ, Bush D. Twenty-four hour ambulatory nasal pH monitoring. Clin Otolaryngol Allied Sci. 1999; 24(1), 24-5.

47. McShane D, Davies JC, Davies MG, Bush A, Geddes DM, Alton EW. Airway surface pH in subjects with cystic fibrosis. Eur Respir J. 2003; 21(1), 37-42.

48. Fischer H, Widdicombe JH. Mechanisms of acid and base secretion by the airway epithelium. J Membr Biol. 2006; 211(3), 139-50.

49. Takeuchi K, Lamb RA. Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport. J Virol. 1994; 68(2), 911-9.

50. Garten W, Braden C, Arendt A, Peitsch C, Baron J, Lu Y, Pawletko K, Hardes K, Steinmetzer T, BottcherFriebertshauser E. Influenza virus activating host proteases: Identification, localization and inhibitors as potential therapeutics. Eur J Cell Biol. 2015; 94(7- 9), 375-83. doi: 10.1016/j.ejcb.2015.05.013.

51. Alvarado-Facundo E, Gao Y, Ribas-Aparicio RM, Jimenez-Alberto A, Weiss CD, Wang W. Influenza virus M2 protein ion channel activity helps to maintain pandemic 2009 H1N1 virus hemagglutinin fusion competence during transport to the cell surface. J Virol. 2015; 89(4), 1975-85. doi: 10.1128/JVI.03253-14.

52. Holsinger LJ, Nichani D, Pinto LH, Lamb RA. Influenza A virus M2 ion channel protein: a structure-function analysis. J Virol. 1994; 68(3), 1551-63.

53. Balannik V, Carnevale V, Fiorin G, Levine BG, Lamb RA, Klein ML, Degrado WF, Pinto LH. Functional studies and modeling of pore-lining residue mutants of the influenza a virus M2 ion channel. Biochemistry. 2010; 49(4), 696-708. doi: 10.1021/bi901799k.

54. Schnell JR, Chou JJ. Structure and mechanism of the M2 proton channel of influenza A virus. Nature. 2008; 451(7178). 591-5. doi: 10.1038/nature06531.

55. Wang J, Qiu JX, Soto C, DeGrado WF. Structural and dynamic mechanisms for the function and inhibition of the M2 proton channel from influenza A virus. Curr Opin Struct Biol. 2011; 21(1), 68-80. doi: 10.1016/

56. Pielak RM, Chou JJ. Influenza M2 proton channels. Biochim Biophys Acta. 2011; 1808(2), 522-9. doi: 10.1016/j.bbamem.2010.04.015.

57. Betakova T, Ciampor F, Hay AJ. Influence of residue 44 on the activity of the M2 proton channel of influenza A virus. J Gen Virol. 2005; 86(Pt 1), 181-4. doi: 10.1099/vir.0.80358-0.

58. O’Donnell CD, Vogel L, Matsuoka Y, Jin H, Subbarao K. The matrix gene segment destabilizes the acid and thermal stability of the hemagglutinin of pandemic live attenuated influenza virus vaccines. J Virol. 2014; 88(21), 12374-84. doi: 10.1128/JVI.01107-14.

59. Kim JH, Lingwood CA, Williams DB, Furuya W, Manolson MF, Grinstein S. Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor. J Cell Biol. 1996; 134(6), 1387-99.

60. Watanabe T, Watanabe S, Ito H, Kida H, Kawaoka Y. Influenza A virus can undergo multiple cycles of replication without M2 ion channel activity. J Virol. 2001; 75(12), 5656-62. doi: 10.1128/JVI.75.12.5656-5662.2001.

Для цитирования:

Wolkerstorfer A., Katinger D., Romanova J. Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line. Microbiology Independent Research Journal (MIR Journal). 2016;3(1):13-24.

For citation:

Wolkerstorfer A., Katinger D., Romanova J. Factors affecting the immunogenicity of the live attenuated influenza vaccine produced in continuous cell line. Microbiology Independent Research Journal (MIR Journal). 2016;3(1):13-24.

Просмотров: 47

Creative Commons License
Контент доступен под лицензией Creative Commons Attribution-NonCommercial-ShareAlike 4.0.

ISSN 2500-2236 (Online)