Microbiology Independent Research Journal (MIR Journal)

Advanced search

H5N1 influenza vaccine quality is affected by hemagglutinin conformational stability


Since 1997, highly pathogenic H5N1 avian influenza viruses have circulated in wild and domestic birds and sporadically have infected humans. Conventional inactivated vaccines made from these viruses were shown to have decreased HA content and immunogenicity compared to seasonal preparations. We assumed that the high pH threshold (5.6-6.0) known for the HA conformational change (pH of fusion or activation) of avian highly pathogenic influenza viruses resulted in the low stability of native HA conformation and affected the vaccine quality. The 58Lys→Ile mutation introduced into the HA2 subunit of the HA of A/chicken/Kurgan/5/05 (H5N1) virus decreased the pH threshold of the HA activation. The mutant virus demonstrated increased HA stability toward acidic pH and elevated temperature, decreased binding efficiency to the monoclonal antibody IIF4 that recognizes the HA low pH form, and increased HA resistance to trypsin digestion. Virus with modified HA was less susceptible to freezing stress and showed an increased content of immunocompetent HA in inactivated vaccine preparation compared to the analogous virus with original HA. Therefore, we have shown a way to increase the quality of inactivated vaccines made from highly pathogenic avian influenza viruses.

About the Authors

M. Sergeeva
Research Institute of Influenza
Russian Federation
Russian Federation 197376, Saint Petersburg, Prof. Popova str., 15/17

A. Krokhin
Green Hills Biotechnology AG
Vienna, Austria

M. Matrosovich
Institute of Virology, Philipps-University of Marburg
Marburg, Germany

T. Matrosovich
Institute of Virology, Philipps-University of Marburg
Marburg, Germany

M. Wolschek
Green Hills Biotechnology AG
Vienna, Austria

O. Kiselev
Research Institute of Influenza
Russian Federation
Saint Petersburg, Russian Federation

J. Romanova
Research Institute of Influenza; Green Hills Biotechnology AG
Russian Federation
Saint Petersburg, Russian Federation; Vienna, Austria


1. Bresson JL, Perronne C, Launay O, Gerdil C, Saville M, Wood J, Hoschler K, Zambon MC. Safety and immunogenicity of an inactivated split-virion influenza A/Vietnam/1194/2004 (H5N1) vaccine: phase I randomised trial. Lancet 2006; 367,1657-64.

2. Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety and immunogenicity of an inactivated subvirion influenza A (H5N1) vaccine. N Engl J Med 2006; 354,1343-51.

3. Ehrlich HJ, Muller M, Oh HM, Tambyah PA, Joukhadar C, Montomoli E, Fisher D, Berezuk G, Fritsch S, Low-Baselli A, Vartian N, Bobrovsky R, Pavlova BG, Pollabauer EM, Kistner O, Barrett PN. A clinical trial of a whole-virus H5N1 vaccine derived from cell culture. N Engl J Med 2008; 358, 2573-84.

4. 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.

5. Maeda T, Ohnishi S. Activation of influenza virus by acidic media causes hemolysis and fusion of erythrocytes. FEBS Lett 1980; 122, 283-7.

6. Scholtissek C. Stability of infectious influenza A viruses at low pH and at elevated temperature. Vaccine 1985; 3, 215-8.

7. Gray TE, Guzman K, Davis CW, Abdullah LH, Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 1996; 14, 104-12.

8. Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD. Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A 2004; 101, 4620-4.

9. Romanova J, Krenn BM, Wolschek M, Ferko B, Romanovskaja-Romanko E, Morokutti A, Shurygina AP, Nakowitsch S, Ruthsatz T, Kiefmann B, Konig U, Bergmann M, Sachet M, Balasingam S, Mann A, Oxford J, Slais M, Kiselev O, Muster T, Egorov A. Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine. PLoS One 2009; 4, e5984.

10. Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 2000; 97, 6108-13.

11. Horimoto T, Takada A, Fujii K, Goto H, Hatta M, Watanabe S, Iwatsuki-Horimoto K, Ito M, Tagawa-Sakai Y, Yamada S, Ito H, Ito T, Imai M, Itamura S, Odagiri T, Tashiro M, Lim W, Guan Y, Peiris M, Kawaoka Y. The development and characterization of H5 influenza virus vaccines derived from a 2003 human isolate. Vaccine 2006; 24, 3669-76.

12. Reed LJ, Muench H. A simple method of estimating fifty percent endpoints. The American Journal of Hygiene 1938; 27, 493–7.

13. Vareckova E, Wharton SA, Mucha V, Gocnik M, Kostolansky F. A monoclonal antibody specific to the HA2 glycoprotein of influenza A virus hemagglutinin that inhibits its fusion activity reduces replication of the virus. Acta virologica 2003; 47, 229-36.

14. Wood JM, Schild GC, Newman RW, Seagroatt V. An improved single-radial-immunodiffusion technique for the assay of influenza haemagglutinin antigen: application for potency determinations of inactivated whole virus and subunit vaccines. Journal of biological standardization 1977; 5, 237-47.

15. 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, e18577.

16. Reed ML, Bridges OA, Seiler P, Kim JK, Yen HL, Salomon R, Govorkova EA, Webster RG, Russell CJ. The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus pathogenicity and transmissibility in ducks. J Virol 2009; 84, 1527-35.

17. Smeenk CA, Wright KE, Burns BF, Thaker AJ, Brown EG. Mutations in the hemagglutinin and matrix genes of a virulent influenza virus variant, A/FM/1/47-MA, control different stages in pathogenesis. Virus Res 1996; 44, 79-95.

18. Daniels RS, Downie JC, Hay AJ, Knossow M, Skehel JJ, Wang ML, Wiley DC. Fusion mutants of the influenza virus hemagglutinin glycoprotein. Cell 1985; 40, 431-9.

19. 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, 1605-12.

20. Skehel JJ, Bayley PM, Brown EB, Martin SR, Waterfield MD, White JM, Wilson IA, Wiley DC. Changes in the conformation of influenza virus hemagglutinin at the pH optimum of virus-mediated membrane fusion. Proc Natl Acad Sci U S A 1982; 79, 968-72.

21. Russell RJ, Kerry PS, Stevens DJ, Steinhauer DA, Martin SR, Gamblin SJ, Skehel JJ. Structure of influenza hemagglutinin in complex with an inhibitor of membrane fusion. Proc Natl Acad Sci U S A 2008; 105, 17736-41.

22. 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, 405-14.

23. Amorij JP, Meulenaar J, Hinrichs WL, Stegmann T, Huckriede A, Coenen F, Frijlink HW. Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze-drying. Vaccine 2007; 25, 6447-57.

24. Eichelberger SL, Sultana I, Gao J, Getie-Kebtie M, Alterman M, Eichelberger MC. Potency under pressure: the impact of hydrostatic pressure on antigenic properties of influenza virus hemagglutinin. Influenza Other Respir Viruses 2013; 7, 961-8.

25. Harvey R, Nicolson C, Johnson RE, Guilfoyle KA, Major DL, Robertson JS, Engelhardt OG. Improved haemagglutinin antigen content in H5N1 candidate vaccine viruses with chimeric haemagglutinin molecules. Vaccine 2010; 28, 8008-14.

26. Carr CM, Chaudhry C, Kim PS. Influenza hemagglutinin is spring-loaded by a metastable native conformation. Proc Natl Acad Sci USA 1997; 94, 14306–13.

27. Khurana S, Chearwae W, Castellino F, Manischewitz J, King LR, Honorkiewicz A, Rock MT, Edwards KM, Del Giudice G, Rappuoli R, Golding H. Vaccines with MF59 adjuvant expand the antibody repertoire to target protective sites of pandemic avian H5N1 influenza virus. Sci Transl Med 2010; 2, 15ra5.

28. Budimir N, Huckriede A, Meijerhof T, Boon L, Gostick E, Price DA, Wilschut J, de Haan A. Induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity. PLoS ONE 2012; 7, e30898.

29. Wei CJ, Xu L, Kong WP, Shi W, Canis K, Stevens J, Yang ZY, Dell A, Haslam SM, Wilson IA, Nabel GJ. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J Virol 2008; 82, 6200-8.

30. Sato SB, Kawasaki K, Ohnishi S. Hemolytic activity of influenza virus hemagglutinin glycoproteins activated in mildly acidic environments. Proc Natl Acad Sci U S A 1983; 80, 3153-7.

31. Babiuk S, Skowronski DM, De Serres G, HayGlass K, Brunham RC, Babiuk L. Aggregate content influences the Th1/ Th2 immune response to influenza vaccine: evidence from a mouse model. J Med Virol 2004; 72, 138-42.

32. 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, 3517-24.

33. Imai M, Watanabe T, Hatta M, Das SC, Ozawa M, Shinya K, Zhong G, Hanson A, Katsura H, Watanabe S, Li C, Kawakami E, Yamada S, Kiso M, Suzuki Y, Maher EA, Neumann G, Kawaoka Y. Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets. Nature 2012; 486, 420-8.


For citations:

Sergeeva M., Krokhin A., Matrosovich M., Matrosovich T., Wolschek M., Kiselev O., Romanova J. H5N1 influenza vaccine quality is affected by hemagglutinin conformational stability. Microbiology Independent Research Journal (MIR Journal). 2014;1(1):1-11. (In Russ.)

Views: 144

ISSN 2500-2236 (Online)