Virulence determinant within the E2 structural glycoprotein of classical swine fever virus
Inventors
Borca, Manuel V. • Risatti, Guillermo R.
Assignees
US Department of Agriculture USDA
Publication Number
US-8846055-B2
Publication Date
2014-09-30
Expiration Date
2026-05-30
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Abstract
Classical Swine Fever Virus (CSFV) E2 glycoprotein is a major inducer of neutralizing antibodies and protective immunity in swine. E2 mediates virus adsorption to the target cell, and harbors genetic determinants associated with virus virulence. CSFV E2 also contains between residues 829 and 837 a discrete epitope (TAVSPTTLR) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related Pestiviruses Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV). In this report, a CSFV infectious clone of the virulent Brescia isolate (BICv) was used to progressively mutate the mAb WH303 epitope of CSFV E2 to the homologous amino acid sequence of BVDV strain NADL E2 (TSFNMDTLA). While the resulting virus mutants T1v (TSFSPTTLR), T2v (TSFNPTTLR), T3v (TSFNMTTLR) demonstrated in vitro growth characteristics similar to those of parental BICv, mutants T4v (TSFNMDTLR) and T5v (TSFNMDTLA) exhibited a 10-fold decrease in virus yield and a significant decrease in plaque size relative to parental BICv. Immunohistochemical reactivity with WH303 was lost only in T3v, T4v and T5v. Interestingly, progressive mutation of the WH303 epitope had an additive effect on attenuation for the virus in swine, with mutants T1v, T2v or T3v inducing progressively milder but invariably lethal CSF, T4v inducing only mild and transient clinical disease, and T5v inducing no disease. Swine infected with either T4v or T5v showed decreased virus replication in tonsils, draining lymph node, spleen and kidney and a significant reduction in virus shedding. Finally, T5v-infected animals were protected from clinical disease when challenged with virulent Brescia virus at 3 or 21 days post T5v inoculation. These results indicate that amino acid residues 830 to 834 of E2 are critical for virulence of CSFV in swine and that engineering at this locus may provide basis for a rationally designed live attenuated CSF vaccine.
Core Innovation
The invention identifies a novel virulence determinant within the E2 structural glycoprotein of Classical Swine Fever Virus (CSFV). It involves progressive mutation of the amino acid sequence of the WH303 epitope of the virulent Brescia CSFV strain to correspond more closely to the homologous amino acid sequence from the Bovine Viral Diarrhea Virus (BVDV) strain NADL. This modification results in attenuation of the virus, with increasing mutations producing progressively milder disease in swine, culminating in complete attenuation after six amino acid changes.
The CSFV E2 glycoprotein is critical for virus replication and mediates virus adsorption to target cells, harboring genetic determinants associated with virulence. By mutating residues 829 to 837 of the E2 glycoprotein and altering the WH303 epitope, the invention shows an additive effect in reducing viral virulence, reduced virus replication in tissues, decreased virus shedding, and loss of reactivity with monoclonal antibody WH303. These changes provide the basis for rational design of live attenuated CSF vaccines that lessens disease severity and confers protection.
The problem addressed by the invention stems from the prior uncertainty regarding the genetic basis of CSFV virulence and the need for improved, rationally designed live attenuated vaccines that provide rapid and effective protection against Classical Swine Fever in swine, while allowing serological distinction between vaccinated and infected animals.
Claims Coverage
The patent includes seven claims covering inventive features of recombinant CSFV with mutated E2 glycoprotein sequences and methods for vaccination and virus attenuation.
Recombinant CSFV with E2 epitope substitution to TSFNMDTLR resulting in attenuation
A recombinant classical swine fever virus comprising DNA encoding a mutated E2 glycoprotein where amino acids 829-837 are substituted with the sequence TSFNMDTLR, producing attenuation of CSFV.
Recombinant CSFV with E2 epitope substitution to TSFNMDTLA resulting in attenuation
A recombinant classical swine fever virus comprising DNA encoding a mutated E2 glycoprotein of Brescia strain with amino acids 829-837 substituted by TSFNMDTLA, where this modification results in attenuation.
Classical swine fever vaccine using recombinant live attenuated CSFV with specific E2 mutations
A classical swine fever vaccine comprising a recombinant rationally designed live attenuated CSFV according to either of the above mutations in the E2 glycoprotein.
Strategy for producing attenuated recombinant CSFV by progressive mutation of virulence determinant
A strategy for attenuation comprising identifying a virulence determinant in pathogenic strain Brescia, identifying a homologous determinant in a related non-pathogenic virus, progressively mutating the DNA encoding this determinant to resemble the non-pathogenic form, thereby achieving attenuation.
Methods of immunizing and protecting animals against CSF using the recombinant attenuated viruses
Methods comprising administering to animals vaccines containing the recombinant CSFV with mutated E2 glycoprotein sequences effective to immunize or protect from clinical CSF disease.
The claims cover recombinant CSFV viruses with specific amino acid substitutions in the E2 glycoprotein epitope that result in attenuation, vaccines comprising these viruses, a method to produce such attenuated viruses via progressive mutation, and methods for immunization and protection of animals against Classical Swine Fever.
Stated Advantages
Provides a rationally designed live attenuated CSF vaccine that lessens the severity of classical swine fever.
Induces rapid and complete protection against lethal CSFV challenge as early as three days post-inoculation.
Allows serological differentiation between vaccinated animals and infected animals through loss of the WH303 epitope reactivity.
Results in significantly decreased virus replication in tonsils and target organs and reduced virus shedding, enhancing safety.
Documented Applications
Use as a live attenuated vaccine for protecting swine against clinical disease caused by classical swine fever virus.
Development of marker vaccines enabling serological distinction between vaccinated and infected swine.
Use of recombinant CSFV mutants for rapid induction of immunity in swine during CSF outbreaks.
Interested in licensing this patent?