Development of a marker foot and mouth disease virus vaccine candidate that is attenuated in the natural host
Inventors
Rieder, Aida E. • Rodriguez, Luis L. • Hollister, Jason R. • Uddowla, Sabena
Assignees
US Department of Agriculture USDA
Publication Number
US-9180179-B1
Publication Date
2015-11-10
Expiration Date
2031-06-09
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Abstract
We have generated novel molecularly marked FMDV A24LL3DYR and A24LL3BPVKV3DYR vaccine candidates. The mutant viruses contain a deletion of the leader coding region (LL) rendering the virus attenuated in vivo and negative antigenic markers introduced in one or both of the viral non-structural 3Dpol and 3B proteins. The vaccine platform includes unique restriction endonuclease sites for easy swapping of capsid proteins for different FMDV subtypes and serotypes. The mutant viruses produced no signs of FMD and no shedding of virulent virus in cattle. No clinical signs of disease or fever were observed and no transmission to in-contact animals was detected in pigs inoculated with live A24LL3DYR. Cattle immunized with chemically inactivated vaccine candidates showed an efficacy comparable to a polyvalent commercial FMDV vaccine. These vaccine candidates used in conjunction with a cELISA provide a suitable target for DIVA companion tests.
Core Innovation
We have generated novel molecularly marked Foot and Mouth Disease Virus (FMDV) vaccine candidates, A24LL3DYR and A24LL3BPVKV3DYR, that are genetically modified to contain a deletion of the leader proteinase (Lpro) coding region rendering the virus attenuated in vivo. These mutants additionally contain negative antigenic markers introduced in the non-structural proteins 3Dpol and 3B, eliminating two antigenic epitopes recognized by specific antibodies. This design provides targets for DIVA (Differentiation of naturally Infected from Vaccinated Animals) serological tests.
The vaccine platform is engineered with unique restriction enzyme sites flanking the capsid-coding region to facilitate easy swapping of capsid proteins for different FMDV subtypes and serotypes, enabling the creation of custom vaccines. Live mutant viruses produced no signs of foot and mouth disease and no shedding or transmission of virulent virus was detected in cattle and pigs, demonstrating attenuation in the natural host. Chemically inactivated vaccines derived from these mutants showed efficacy comparable to commercial polyvalent FMD vaccines.
The problem addressed is the need for safe and effective FMD vaccines that allow differentiation between naturally infected and vaccinated animals. Conventional chemically inactivated vaccines are slow acting, do not always permit serological differentiation between infected and vaccinated animals, and involve the production of large volumes of virulent virus in high containment facilities with risk of escape. Existing empirical attenuation methods have failed or been risky due to potential reversion to virulence. Therefore, there is an urgent need for genetically engineered marker vaccines that are attenuated, safe for production, and compatible with companion diagnostic tests for effective FMD control and eradication.
Claims Coverage
The patent contains multiple independent claims focusing on methods to distinguish infected animals from vaccinated animals using specific molecular markers in FMDV and related diagnostic assays.
Marker vaccine with specific amino acid substitutions for differentiation
A method of distinguishing animals exposed to wild-type FMDV versus those vaccinated with a marker FMDV vaccine, wherein the marker involves replacement of amino acids His27 and Asn31 in FMDV 3Dpol with Tyr and Arg from bovine rhinovirus type 2 (BRV2), using competitive ELISA with monoclonal antibody F32-44 to detect inhibition patterns specific to infection or vaccination.
Double marker vaccine enabling differentiation with two epitopes
A method of differentiating animals exposed to wild-type FMDV from those vaccinated with a double marker vaccine where the markers are the 3Dpol substitutions His27/Asn31 to Tyr/Arg and the 3B substitution of amino acids RQKP to PVKV from BRV2. The method uses competitive ELISA with monoclonal antibodies F8B and F32-44 to detect inhibition of binding to 3B and 3Dpol antigens respectively, distinguishing infected from vaccinated animals.
Use of specific 3B peptide antigen in diagnostic assays
The use of the 3B peptide GPYAGPLETQKPLK (SEQ ID NO:40) as the 3B antigen in competitive immunoassays to detect antibodies distinguishing infected from vaccinated animals with the double marker vaccine.
Application of serological tests to animals vaccinated with specific genetically modified FMDVs
Use of serum from animals vaccinated with genetically modified FMDVs encoded by the DNA sequences SEQ ID NO:1 for the single marker vaccine and SEQ ID NO:3 for the double marker vaccine, respectively, in the competitive ELISA methods for differentiation.
Application to chimeric marker vaccines with swapped capsid regions
Use of serum from animals vaccinated with chimeric marker FMDV vaccines where the capsid region has been replaced with that of different FMDV strains, enabling differentiation by the described competitive ELISA testing methods relying on the negative antigenic markers.
The claims cover methods for serologically distinguishing animals infected with wild-type FMDV from those vaccinated with monopartite or bipartite marker FMDV vaccines through specific amino acid substitutions in viral proteins 3Dpol and 3B, and companion competitive ELISA assays employing monoclonal antibodies and specific peptide antigens. These claims also include applications to vaccines encoded by specified polynucleotide sequences and chimeric vaccines with capsid exchanges.
Stated Advantages
The vaccines are attenuated in the natural host, reducing the risk of disease and transmission.
The platform allows for easy swapping of capsid proteins enabling custom vaccines for different FMDV serotypes and subtypes.
The negative antigenic markers enable serological differentiation (DIVA) between vaccinated and naturally infected animals.
Chemically inactivated vaccines derived from the mutants show protection efficacy comparable to commercial vaccines.
Reduced risk during vaccine production due to genetic attenuation reducing need for high containment facilities.
Documented Applications
Use of the genetically modified FMDV vaccine candidates to protect cattle and swine against clinical foot and mouth disease.
Vaccination with chemically inactivated mutant viruses inducing protective immune responses.
Use of companion competitive ELISA assays for serological differentiation of vaccinated animals from naturally infected animals as part of FMD control and eradication programs.
Production of custom vaccines by swapping capsid coding regions from different FMDV subtypes and serotypes utilizing unique restriction sites engineered in the vaccine platform.
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