Processing of a modified foot-and-mouth disease virus P1 polypeptide by an alternative protease
Inventors
Puckette, Michael • Rasmussen, Max
Assignees
US Department of Homeland Security
Publication Number
US-10308927-B2
Publication Date
2019-06-04
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 provides a platform for recombinant production of foot-and-mouth disease virus (FMDV) antigens and immunogens using an engineered FMDV P1 precursor polypeptide that contains Tobacco Etch Virus NIa (TEV) protease cleavage sites, and co-expression of a TEV protease. This system efficiently processes the P1 precursor into individual virus structural proteins without the need for the native, toxic FMDV 3C protease, thus avoiding cytotoxic effects on host cells commonly associated with 3C protease expression.
The problem being solved is that conventional recombinant production of FMDV antigens relies on the native FMDV 3C protease to cleave the P1 precursor polypeptide. However, the FMDV 3C protease exhibits promiscuous proteolytic activity leading to toxicity in host cells used for recombinant expression, which limits the yield and efficiency of antigen production. The invention addresses this by substituting or complementing the 3C protease with a potyvirus NIa protease, such as TEV protease, which recognizes engineered cleavage sites on the P1 precursor, allowing safer and more efficient antigen production.
The engineered P1 polypeptide contains NIa or TEV protease cleavage sites inserted at the junctions defining individual structural viral proteins, permitting their proteolytic separation by TEV protease. The system utilizes polynucleotides encoding these engineered P1 precursors and TEV protease, potentially along with translational interrupters such as 2A sequences. The invention also contemplates co-expression of modified or inactive 3C protease variants to explore their role in viral protein assembly. The platform can be used in various host cells including eukaryotic and prokaryotic systems, enabling production of structural proteins or virus-like particles (VLPs) for FMDV vaccines and diagnostics.
Claims Coverage
The patent includes a primary independent claim directed to a polynucleotide encoding an engineered FMDV P1 precursor polypeptide with TEV protease cleavage sites, along with optional encoding of TEV protease and modified 3C proteases. Other independent claims correspond to vectors comprising such polynucleotides, host cells transformed therewith, and methods of proteolytic processing and producing FMDV viral proteins or VLPs using these engineered components.
Engineered FMDV P1 precursor polypeptide with TEV protease cleavage sites
A polynucleotide encoding an engineered FMDV P1 precursor polypeptide comprising at least one TEV protease cleavage site selected from specific amino acid motifs (E1-D2-A3-Y4-T5-Q6-S7, E1-F2-L3-Y4-K5-Q6-G7, etc.) positioned in junctions between VP4 and VP2, VP2 and VP3, VP3 and VP1, or VP1 and 2A components.
Co-encoding of TEV protease and protease variants
Polynucleotides and vectors further comprising encoding of TEV protease (wild type or variants with at least 95% identity) capable of cleaving the engineered sites, and optionally encoding an engineered FMDV 3C protease with specific amino acid substitutions (e.g., L127P, C163A) that reduce toxicity or allow functional studies.
Vectors comprising engineered polynucleotides and translation interrupters
Vectors that carry the engineered polynucleotides encoding the modified FMDV P1 polypeptide with TEV protease cleavage sites, optionally combined with 2A, delta 1D2A, or other similar translational interrupters to facilitate expression.
Host cells expressing engineered polypeptides and proteases
Host cells transformed or transfected with the vectors encoding engineered FMDV P1 precursor polypeptide with TEV cleavage sites and TEV protease, optionally along with modified 3C protease, in a variety of expression systems including eukaryotic and prokaryotic cells.
Methods for proteolytic processing and production of viral proteins or VLPs
Methods comprising culturing host cells expressing the engineered polypeptides and proteases to proteolytically process the precursor polypeptides into individual viral proteins such as VP0, VP1, VP2, VP3, VP4, and assembling these proteins into virus-like particles (VLPs).
Diagnostic and antigenic compositions
Compositions containing the cleavage products of the engineered FMDV P1 precursor polypeptides produced by the methods, useful as antigenic or diagnostic products and kits.
The claims cover the core innovation of engineering FMDV P1 precursor polypeptides with TEV protease cleavage sites, co-expressing TEV protease and optionally engineered 3C protease variants, their incorporation into vectors and host cells, methods of proteolytic processing to produce viral proteins and VLPs, and their use in diagnostic or antigenic compositions. These inventive features collectively enable safe and efficient recombinant production of FMDV antigens without cytotoxicity associated with native 3C protease.
Stated Advantages
Avoidance of cytotoxic effects on host cells by eliminating the use of the toxic native FMDV 3C protease in processing FMDV P1 precursor polypeptide.
Efficient and high-yield recombinant expression and processing of FMDV structural proteins using TEV protease and engineered cleavage sites.
Improved safety and ease of production by avoiding the need to culture live, infectious FMDV.
Production of viral proteins capable of assembling into virus-like particles containing relevant epitopes for vaccines and diagnostics.
Capability to express all needed components from a single open reading frame using translational interrupters like 2A sequences, simplifying vector design and transformation.
Documented Applications
Production of FMDV antigens and immunogens for use in vaccines against foot-and-mouth disease.
Production of diagnostics and diagnostic products for foot-and-mouth disease based on cleavage products of engineered FMDV P1 precursor polypeptides.
Use in compositions comprising virus-like particles (VLPs) or individual viral proteins for therapeutic or diagnostic purposes.
Application in preventing or treating FMDV infection by administering compositions containing cleavage products or polynucleotides encoding engineered P1 precursors and proteases to susceptible animals, such as cloven-hoofed livestock.
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