Summary
The genus Flavivirus includes a number of important human pathogens that impose major health problems in large regions of the world. The emergence of flaviviruses in new geographic regions (e.g., West Nile virus in North America) and rapid socioeconomic changed in many developing countries where flaviviruses such as dengue virus and Japanese encephalitis virus and endemic demand the development of new vaccines against these diseases. Using tick-borne encephalitis virus as a model we have established a new method to generate attenuated flavivirus strains that may be useful for generating cost-effective and safe live vaccines. This method relies on the specific introduction of deletions into one of the structural proteins, the capsid protein C. These deletions remove parts or all of an internal stretch of hydrophobic amino acid residues that probably is involved in virion assembly. We observed that remarkably long deletions were tolerated, yielding viable viral mutants that were highly attenuated in the mouse model but efficiently induced protective immunity. Biochemical analyses suggested that attenuation was caused by an assembly defect of infectious virions but the mutants produced ample amounts of non-infections subviral particles. The generation of viable mutants with deletions longer that 16 amino acid residues depended on additional, spontaneously emerging mutations within protein C that increased the hydrophobicity of the mutant protein. Although the second-site mutations increased infectivity, they did not restore neuroinvasiveness. Mouse experiments demonstrated excellent safety and immunogenicity profiles for these mutants.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsPreview
Unable to display preview. Download preview PDF.
References
Allison SL, Stadler K, Mandl CW, Kunz C, Heinz FX (1995) Synthesis and secretion of recombinant tick-borne encephalitis virus protein E in soluble and particulate form. J Virol 69:5816–5820
Arroyo J, Guirakhoo F, Fenner S, Zhang ZX, Monath TP, Chambers TJ (2001) Molecular basis for attenuation of neurovirulence of a yellow fever virus/Japanese encephalitis virus chimera vaccine (ChimeriVax-JE). J Virol 75:934–942
Braun P, von Heijne G (1999) The aromatic residues Trp and Phe have different effects on the positioning of a transmembrane helix in the microsomal membrane. Biochem 38:9778–9782
Burke DS, Monath TP (2001) Flaviviruses, pp 1043–1125. In: Knipe DM, Howley PM et al. (eds), Fields virology, 4th edn. Lippincot Williams & Wilkins, Philadelphia, Pa
Ferlenghi I, Clarke M, Ruttan T, Allison SL, Schalich J, Heinz FX, Harrison SC, Rey FA, Fuller SD (2001) Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Mol Cell 7:593–602
Heinz FX, Allison SL, Stiasny K, Schalich J, Holzmann H, Mandl CW, Kunz C (1995) Recombinant and virion-derived soluble and particulate immunogens for vaccination against tick-borne encephalitis. Vaccine 13:1636–1642
Heinz FX, Tuma W, Guirakhoo F, Kunz C (1986) A model study of the use of monoclonal antibodies in capture enzyme immunoassays for antigen quantification exploiting the epitope map of tick-borne encephalitis virus. J Biol Stand 14:133–141
Holzmann H, Stiasny K, Ecker M, Kunz C, Heinz FX (1997) Characterization of monoclonal antibody-escape mutants of tick-borne encephalitis virus with reduced neuroinvasiveness in mice. J Gen Virol 78:31–37
Kofler RM, Heinz FX, Mandl CW (2002) Capsid protein C of tick-borne encephalitis virus tolerates large internal deletions and is a favorable target for attenuation of virulence. J Virol 76:3534–3543
Kofler RM, Leitner A, O’Riordain G, Heinz FX, Mandl CW (2003) Spontaneous mutations restore the viability of tick-borne encephalitis virus mutants with large deletions in protein C. J Virol 77:443–451
Lindenbach BD, Rice CM (2001) Flaviviridae: The viruses and their replication. In:Knipe DM, Howley PM et al. (eds), Fields Virology. 4th edn. Lippincott Williams & Wilkins, Philadelphia, pp 991–1041
Mandl CW, Allison SL, Holzmann H, Meixner T, Heinz FX (2000) Attenuation of tick-borne encephalitis virus by structure-based site-specific mutagenesis of a putative flavivirus receptor binding site. J Virol 74:9601–9609
Mandl CW, Ecker M, Holzmann H, Kunz C, Heinz FX (1997) Infectious cDNA clones of tick-borne encephalitis virus European subtype prototypic strain Neudoerfl and high virulence strain Hypr. J Gen Virol 78:1049–1057
Mandl CW, Heinz FX, Kunz C (1988) Sequence of the structural proteins of tick-borne encephalitis virus (Western subtype) and comparative analysis with other flaviviruses. Virology 166:197–205
Mandl CW, Heinz FX, Stockl E, Kunz C (1989) Genome sequence of tick-borne encephalitis virus (Western subtype) and comparative analysis of non structural proteins with other flaviviruses. Virology 173:291–301
Mandl CW, Holzmann H, Meixner T, Rauscher S, Stadler PF, Allison SL, Heinz FX (1998) Spontaneous and engineered deletions in the 3′ noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus. J Virol 72:2132–2140
Mandl CW, Kroschewski H, Allison SL, Kofler R, Holzmann H, Meixner T, Heinz FX (2001) Adaptation of tick-borne encephalitis virus to BHK-21 cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo. J Virol 75:5627–5637
Markoff L, Falgout B, Chang A (1997) A conserved internal hydrophobic domain mediates the stable membrane integration of the dengue virus capsid protein. Virology 233:105–117
Mason PW, Pincus S, Fournier MJ, Mason TL, Shope RE, Paoletti E (1991) Japanese encephalitis virus-vaccinia recombinants produce particulate forms of the structural membrane proteins and induce high levels of protection against lethal JEV infection. Virology 180:294–305
McMinn PC (1997) The molecular basis of virulence of the encephalitogenic flaviviruses. J Gen Virol 78:2711–2722
Ruggli N, Rice CM (1999) Functional cDNA clones of the flaviviridae: strategies and applications. Adv Virus Res 53:183–207
Schalich J, Allison SL, Stiasny K, Mandl CW, Kunz C, Heinz FX (1996) Recombinant subviral particles from tick-borne encephalitis virus are fusogenic and provide a model system for studying flavivirus envelope glycoprotein functions. J Virol 70:4549–4557
van Regenmortel MHV, Faquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, McGeoch DJ, Pringle CR, Wickner RB (eds) (2000) Virus taxonomy: classification and nomenclature of viruses. Academic Press, London, pp 859–878
Wallner G, Mandl CW, Ecker M, Holzmann H, Stiasny K, Kunz C, Heinz FX (1996) Characterization and complete genome sequences of high-and low-virulence variants of tick-borne encephalitis virus. J Gen Virol 77:1035–1042
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Springer-Verlag Wien
About this chapter
Cite this chapter
Kofler, R.M., Heinz, F.X., Mandl, C.W. (2004). A novel principle of attenuation for the development of new generation live flavivirus vaccines. In: Calisher, C.H., Griffin, D.E. (eds) Emergence and Control of Zoonotic Viral Encephalitides. Archives of Virology. Supplementa, vol 18. Springer, Vienna. https://doi.org/10.1007/978-3-7091-0572-6_17
Download citation
DOI: https://doi.org/10.1007/978-3-7091-0572-6_17
Publisher Name: Springer, Vienna
Print ISBN: 978-3-211-20454-2
Online ISBN: 978-3-7091-0572-6
eBook Packages: Springer Book Archive