Genetically engineered foot and mouth disease virus and related proteins, polynucleotides, compositions, methods and systems
Inventors
Rieder, Aida E. • De Los Santos, Teresa B. • Rodriguez, Luis L. • RAI, Devendra • DIAZ-SAN SEGUNDO, Fayna C. • Hoeprich, Paul D.
Assignees
University of Connecticut • Lawrence Livermore National Security LLC • US Department of Agriculture USDA
Publication Number
US-11813320-B2
Publication Date
2023-11-14
Expiration Date
2037-05-09
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Abstract
Genetically engineered Foot and Mouth Disease Virus (FMDV) and related engineered proteins and polynucleotides, nanolipoprotein particles, compositions, methods and systems are described. The genetically engineered FMDV is modified by the strategic insertion of a protein tag into select regions of the FMDV genome which encode viral proteins that are exposed on the surface of the FMDV viral capsid. The inserted protein tag is displayed as a decoration or attachment on the viral capsid surface.
Core Innovation
The invention describes genetically engineered Foot and Mouth Disease Virus (FMDV) modified by the strategic insertion of a protein tag into select regions of the FMDV genome encoding viral proteins exposed on the surface of the viral capsid. These inserted protein tags are displayed as decorations on the viral capsid surface. The genetically modified FMD viruses comprise a genetically modified VP1 protein engineered either to fuse at the C-terminus a 2A protein with an inserted protein tag or to include a protein tag inserted within the VP1 protein at specified insertion positions relative to the 2A protein or VP1 C-terminus. These modifications allow the protein tag to be presented on an external surface of the viral capsid.
The problem solved is the challenge in controlling and preventing FMD due to the lack of cross-protection offered by strains of different serotypes and subtypes, and the antigenic mismatch reducing vaccine efficacy. Existing vaccines are limited in protection across geographical viral variants, and there is a need for improved vaccines and methods enabling better diagnostics, purification, and vaccine formulations. The genetically engineered FMDV with surface-displayed protein tags provides novel tools for vaccine development, diagnostics, and purification specifically tailored to address these challenges.
The summaries detail different aspects including genetically modified FMD viruses having VP1 modified with inserted protein tags at positions selected to maintain viral viability and capsid integrity, polynucleotides encoding such viruses or VP1 proteins, FMDV functionalized nanolipoprotein particles (NLPs) presenting tag substrates capable of binding to the protein tags, and compositions including vaccines comprising the genetically modified viruses and NLPs with suitable vehicles. Methods and systems to produce tagged FMDV and detect antibodies against tagged viruses are also described. The functional tagged FMDV can be purified via affinity to metal ion tags, detected by antibodies, formulated with adjuvanted NLPs to enhance immunogenicity, and enable differentiation of vaccinated from infected animals.
Claims Coverage
The independent claims primarily cover a vaccine comprising genetically modified FMDV with protein tags presented on the capsid surface and methods to treat or prevent FMD using these vaccines.
FMDV exhibiting capsid protein tags on external surface
A vaccine containing one or more genetically modified FMDV having a viral capsid with a protein tag up to 100 amino acids inserted in an engineered VP1 protein and displayed on the capsid external surface.
Specific tag types and insertion positions in FMDV VP1 protein
The protein tag is from 1 to 20 amino acids and selected from known peptide tags including polyhistidine-tag (His-tag). Insertion point is selected from positions −1 to −4 relative to the VP1 C-terminus with specific sequences HHHHHHKQ or HHHHHHIIAPAK as examples.
FMDV encoded by nucleotide sequence with designed tag insertions
The genetically modified FMDV is encoded by a polynucleotide, exemplified by SEQ ID NO:3, encoding VP1 with the inserted tag.
Functionalized nanolipoprotein particles binding tagged FMDV
Nanolipoprotein particles comprise a scaffold protein and functionalized membrane forming lipid presenting a tag substrate capable of binding the protein tag on FMDV capsid, enabling composition and vaccine formulations.
Inclusion of adjuvants in nanolipoprotein particles
Functionalized nanolipoprotein particles further comprise one or more adjuvants attached to the tag substrate on the NLP surface, including MPLA and other immunostimulatory molecules, to enhance vaccine efficacy.
Chemically inactivated vaccine and treatment method
The vaccine can comprise chemically inactivated genetically engineered FMDV. Methods of treating or preventing FMD comprise administering an effective amount of such vaccine, providing protection or delaying onset or severity of disease.
The claims cover genetically modified FMD viruses with externally displayed protein tags on VP1, compositions including functionalized nanolipoprotein particles bound to tagged virus, vaccine formulations optionally chemically inactivated and adjuvanted, and methods for preventing or treating FMD by administration of said vaccines. The inventive features enable efficient purification, detection, and improved immunogenicity.
Stated Advantages
Provides genetically modified FMDV that are viable and display protein tags on the capsid surface for affinity purification and detection.
Enables one-step purification of FMDV viruses using divalent cation affinity chromatography through polyhistidine tags.
Facilitates formulation of vaccines with nanolipoprotein particles presenting corresponding tag substrates and adjuvants to enhance immunogenicity.
Allows differentiation (DIVA) between vaccinated and infected animals by serological recognition of the inserted protein tag.
Supports generation of tagged FMD viruses for broad vaccine development by cassette swapping of capsid regions via unique restriction sites.
Documented Applications
Use of genetically modified FMDV as chemically-inactivated marker vaccines to protect cloven-hoofed animals against foot-and-mouth disease.
Application in diagnostic arrays for detection of antibodies against different FMDV strains by capturing tagged viruses on substrates.
Affinity purification of genetically modified FMDV by metal ion chelate substrates.
Formulation of vaccines combining genetically modified FMDV with adjuvanted nanolipoprotein particles presenting tag substrates for enhanced immune response.
Use of fluorescently labeled molecules binding to protein tags for live imaging and tracking of virus in infected cells.
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