N-linked glycosylation alteration in E1 glycoprotein of classical swine fever virus and novel classical swine fever virus vaccine
Inventors
Borca, Manuel V. • Risatti, Guillermo R.
Assignees
US Department of Agriculture USDA
Publication Number
US-8426575-B2
Publication Date
2013-04-23
Expiration Date
2028-01-15
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Abstract
E1, along with Erns and E2 is one of the three envelope glycoproteins of Classical Swine Fever Virus (CSFV). Our previous studies indicated that glycosylation status of either E2 or Erns strongly influence viral virulence in swine. Here, we have investigated the role of E1 glycosylation of highly virulent CSFV strain Brescia during infection in the natural host. The three putative glycosylation sites in E1 were modified by site directed mutagenesis of a CSFV Brescia infectious clone (BICv). A panel of virus mutants was obtained and used to investigate whether the removal of putative glycosylation sites in the E1 glycoprotein would affect viral virulence/pathogenesis in swine. We observed that rescue of viable virus was completely impaired by removal of all three putative glycosylation sites in E1. Single mutations of each of the E1 glycosylation sites showed that CSFV amino acid N594 (E1.N3 virus), as well the combined mutation of N500 and N513 (E1.N1N2 virus) resulted in BICv attenuation. Infection of either E1.N1N2 or E1.N3 viruses were able to efficiently protected swine from challenge with virulent BICv at 3 and 28 days post-infection. These results, along with those demonstrating the role of glycosylation of Erns and E2, suggest that manipulation of the pattern of glycosylation could be a useful tool for development of CSF live-attenuated vaccines.
Core Innovation
The invention relates to the characterization and modification of glycosylation sites within the E1 glycoprotein of the highly virulent Classical Swine Fever Virus (CSFV) strain Brescia. By altering specific N-linked glycosylation sites in the E1 glycoprotein, the invention provides means to modify viral virulence and develop live-attenuated vaccines useful to protect swine from CSF infection.
The problem being solved is the incomplete understanding of the role that viral envelope protein glycosylation plays in virus replication, pathogenesis, and virulence in the natural host. While previous studies demonstrated that glycosylation of glycoproteins Erns and E2 influence CSFV virulence, the contribution of E1 glycosylation remained to be elucidated and exploited for vaccine development.
The invention uses site-directed mutagenesis to modify three putative glycosylation sites within the E1 glycoprotein of CSFV strain Brescia, generating virus mutants. Some mutants were attenuated or non-viable, indicating that glycosylation sites are critical for virus viability and virulence. In particular, mutation at amino acid N594 or combined mutations at N500 and N513 result in attenuation. Vaccination with these mutants elicited protective immunity in swine, supporting their use as live-attenuated vaccines.
Claims Coverage
The patent includes one independent claim encompassing isolated polynucleotide molecules encoding genetically modified CSFV mutants with specific mutations in E1 glycoprotein glycosylation sites, and their use in vaccines.
Isolated polynucleotide molecule encoding genetically modified CSFV mutant with altered E1 glycoprotein glycosylation
An isolated polynucleotide molecule comprising DNA encoding an infectious RNA molecule that encodes a genetically modified CSFV mutant of the highly pathogenic Brescia strain, having a modified E1 glycoprotein sequence with mutated non-glycosylated alanine residues replacing glycosylated asparagine residues at positions 500 and 513, yielding a distinct glycosylation pattern and attenuation of pathogenicity in swine.
Genetically modified CSFV mutant encoding modified E1 glycoprotein
A genetically modified CSFV mutant encoded by the isolated polynucleotide molecule, which does not produce classical swine fever disease in swine due to altered glycosylation sites on E1.
CSF vaccine comprising genetically modified CSFV mutant
A vaccine containing the genetically modified CSFV mutant that encodes the modified E1 glycoprotein with altered glycosylation pattern, which confers protection without causing disease.
Recombinant CSFV and plasmids for expression
Isolated recombinant CSFV viruses and plasmids capable of transfecting suitable host cells to express genetically modified CSFV with mutated E1 glycoprotein resulting in attenuation.
Methods of immunization and virus attenuation
Methods of immunizing animals against CSF by administering vaccines containing genetically modified CSFV and methods of producing attenuated recombinant CSFV by mutating E1 glycosylation sites to achieve attenuation.
The claims cover isolated nucleic acid molecules encoding genetically modified CSFV with specific E1 glycoprotein glycosylation mutations that confer attenuation, as well as vaccines comprising such viruses, recombinant expression constructs, and methods of use for immunization against CSF.
Stated Advantages
Modifying glycosylation in E1 glycoprotein attenuates the CSFV Brescia strain, allowing development of live-attenuated vaccines that protect swine from virulent challenge.
The genetically modified viruses elicit effective immunoprotective responses in swine, providing significant protection against classical swine fever disease without causing illness.
Mutations introduced provide a marker vaccine allowing serological distinction between vaccinated animals and those infected with wild-type CSFV.
Documented Applications
Use of genetically modified CSFV mutants with altered E1 glycoprotein glycosylation sites as live attenuated vaccines for protecting swine from classical swine fever.
Methods of immunizing swine by administering vaccines comprising genetically modified CSFV to confer protection against clinical CSF disease.
Production of recombinant CSFV and plasmids encoding genetically modified viruses for research, vaccine development, and immunization purposes.
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