Processing engineered FMDV P1 polypeptide using an alternative TEV protease
Inventors
Puckette, Michael • Rasmussen, Max
Assignees
US Department of Homeland Security
Publication Number
US-11008564-B2
Publication Date
2021-05-18
Expiration Date
2037-01-17
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Abstract
Polynucleotide constructs that express an engineered foot-and-mouth disease (FMDV) P1 precursor protein and a non-FMDV TEV protease and methods for safe and efficient recombinant production of FMDV antigens and immunogens. Recombinant production of FMDV antigens avoids the need to culture highly-infectious FMDV, while conventional culture methods for producing FMDV antigens rely on the native FMDV 3C protease which exerts toxic effects on host cells. The inventors have developed a new system that efficiently and safely processes FMDV P1 precursor without the FMDV 3C protease, thus avoiding the toxic effects associated with use of the 3C protease. The invention is also directed to the FMDV antigens and virus-like particles produced by this system as well as to FMDV vaccines, diagnostics and other biologics.
Core Innovation
The invention relates to a protein engineering and expression platform that uses a protease to process a precursor polypeptide into individual subunit proteins while minimizing protease-associated damage to host cells used for recombinant expression. Specifically, it involves an engineered foot-and-mouth disease virus (FMDV) P1 precursor polypeptide modified to contain Tobacco Etch Virus (TEV) protease cleavage sites at junctions between viral structural proteins, and a TEV protease that recognizes these sites. This approach provides an alternative to the native FMDV 3C protease conventionally used for processing FMDV antigens.
The problem addressed is that the native FMDV 3C protease is toxic to many host cells used for recombinant production of FMDV proteins, severely limiting expression yields and complicating vaccine production. Conventional methods relying on the 3C protease also involve culturing highly-infectious FMDV, posing significant safety risks. Additionally, whole-virus vaccines have drawbacks such as difficulty distinguishing infected from vaccinated animals, immunogenic instability, short shelf life, and high production costs.
The invention overcomes these issues by developing a new TEV protease-based system that efficiently and safely processes FMDV P1 precursor without the 3C protease, thereby eliminating the cytotoxic effects on host cells. This system produces high yields of virus structural proteins capable of assembling into virus-like particles (VLPs). The platform may be extended to other precursor polypeptides engineered with TEV or other potyvirus NIa protease cleavage sites and can be combined with 2A or 2A-like translational interrupter sequences or modified 3C proteases with reduced toxicity. This enables safer, more efficient recombinant production of FMDV antigens suitable for vaccines, diagnostics, and other biologics.
Claims Coverage
The patent includes multiple independent claims covering pharmaceutical compositions, methods of processing FMDV P1 precursor polypeptides, methods for treating FMDV infection, and host cells expressing engineered polypeptides and proteases. The main inventive features involve engineered FMDV P1 polypeptides containing TEV protease cleavage sites, TEV protease use instead of FMDV 3C protease, and compositions comprising virus-like particles produced by such processing.
Engineered FMDV P1 precursor polypeptides with TEV protease cleavage sites
Polynucleotides encoding at least one engineered FMDV P1 precursor polypeptide containing at least one TEV protease cleavage site positioned to allow processing into separate viral proteins VP0, VP1, VP2, VP3, and VP4.
Use of TEV protease for processing engineered FMDV P1 polypeptides
Expression of TEV protease that recognizes and cleaves the engineered cleavage sites in the FMDV P1 polypeptide to produce individual viral proteins without the use of toxic FMDV 3C protease.
Pharmaceutical compositions comprising virus-like particles
Pharmaceutical compositions containing one or more FMDV structural proteins (VP0, VP1, VP2, VP3, VP4) assembled into virus-like particles produced by proteolytic processing of engineered FMDV P1 precursor polypeptides containing TEV protease cleavage sites, formulated with pharmaceutically acceptable buffers and optionally adjuvants or other active agents.
Methods for preventing or treating FMDV infection
Administering subjects with pharmaceutical compositions of virus-like particles produced from engineered FMDV P1 precursor polypeptides processed by TEV protease, or administering host cells expressing engineered FMDV P1 polypeptides with TEV cleavage sites and TEV protease to induce protective immunity.
Host cells expressing engineered polypeptides and proteases
Host cells comprising vectors encoding engineered FMDV P1 precursor polypeptides with TEV protease cleavage sites and TEV protease, optionally co-expressing modified FMDV 3C protease variants with reduced toxicity, enabling safe and efficient proteolytic processing and production of viral proteins or virus-like particles.
The claims collectively cover engineered FMDV P1 polypeptides containing TEV protease cleavage sites, the use of TEV protease to process these polypeptides safely and efficiently, compositions of virus-like particles produced therefrom, methods of treating FMDV infection using such compositions or cells, and host cells engineered to express these components, providing comprehensive protection for the novel expression platform and its applications.
Stated Advantages
Avoids cytotoxic effects on host cells caused by native FMDV 3C protease, enabling more efficient recombinant protein production.
Eliminates the need to culture live, highly infectious FMDV, improving safety of vaccine production.
Produces high yields of structurally processed viral proteins capable of assembling into virus-like particles with relevant epitopes for immunogenicity.
Provides a flexible platform applicable to other polypeptides engineered with NIa or TEV protease cleavage sites, allowing modular protein processing.
Documented Applications
Recombinant production of FMDV antigens and immunogens for use in vaccines to protect against foot-and-mouth disease.
Production of diagnostic products, antigenic compositions, and diagnostic kits utilizing processed viral proteins or virus-like particles obtained by TEV protease-mediated cleavage of engineered FMDV P1 polypeptides.
Methods for preventing or treating FMDV infection in susceptible animals by administering compositions comprising virus-like particles produced by the inventive system or host cells expressing engineered proteins and TEV protease.
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