Method for producing foot-and-mouth disease virus (FMDV) viral proteins utilizing a modified FMDV 3C protease
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10858633-B2
Publication Date
2020-12-08
Expiration Date
2036-09-08
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Abstract
This application is directed generally to foot-and-mouth disease virus (FMDV) 3C proteases that have been modified by mutating a polynucleotide sequence coding for the FMDV 3C protease. The modified FMDV proteases exhibit proteolytic activity on FMDV P1 precursor protein and exhibit a reduction in one or more toxic or inhibitory properties associated with an unmodified FMDV 3C protease on a host cell used to recombinantly produce it. Vectors carrying polynucleotides encoding modified FMDV 3C protease sequences can induce production of FMDV virus-like particles in a host cell when expressed in the host cell. The modified FMDV 3C proteases can generally be used to produce immunogenic FMDV preparations capable of inducing an immune response against FMDV.
Core Innovation
The invention relates to foot-and-mouth disease virus (FMDV) 3C proteases that have been modified by mutating a polynucleotide sequence coding for the FMDV 3C protease. The modified FMDV proteases exhibit proteolytic activity on the FMDV P1 precursor protein while exhibiting a reduction in one or more toxic or inhibitory properties associated with unmodified FMDV 3C proteases on host cells used to recombinantly produce these proteins.
The problem addressed by the invention is the cytotoxicity of native FMDV 3C protease to host cells during recombinant production of FMDV viral proteins, which limits yields of immunogenic FMDV antigens. The native 3C protease, while necessary for processing P1 precursor protein into structural viral proteins, also cleaves host proteins and inhibits host cell functions, leading to reduced recombinant expression and cell viability.
The inventors discovered that by modifying surface regions of the FMDV 3C protease distal from the active site and substrate binding cleft, specifically by introducing amino acid substitutions within residues 26-35, 125-134, or 138-150 of the protease, they were able to retain proteolytic processing activity on the P1 precursor but significantly reduce toxicity to host cells. For example, substituting the leucine residue at position 127 with proline (L127P) maintains processing ability while enhancing host cell viability and transgene expression.
Claims Coverage
The patent contains multiple claims focusing on methods for producing FMDV viral proteins using host cells expressing a modified FMDV 3C protease, especially featuring the L127P substitution. The claims cover constructs, polynucleotides, host cells, and methods involving these modified proteases and their application in producing FMDV virus-like particles (VLPs).
Use of modified FMDV 3C protease with L127P substitution for protein production
Culturing host cells expressing (i) a polynucleotide encoding a FMDV P1 precursor polypeptide and (ii) a polynucleotide encoding a modified FMDV 3C protease containing a leucine to proline substitution at position 127, enabling production of FMDV viral proteins and VLPs.
Construction of vectors encoding modified 3C protease and P1 precursor polypeptide
Vectors or polynucleotide constructs carrying the modified 3C protease gene and/or the P1 precursor gene for expression in host cells, including fusion proteins with translation interrupter sequences such as 2A or Δ1D2A to enable individual expression of components.
Expression in various host cells and enhanced protein recovery
Host cells, including prokaryotic and eukaryotic cells (mammalian, insect, plant, yeast), transformed with vectors encoding the modified 3C protease and P1 precursor, enabling higher yields of processed viral proteins (VP0, VP1, VP2, VP3, VP4) and VLPs, with reduced toxicity and enhanced viability.
Inclusion of luciferase reporters in constructs for monitoring expression
Polynucleotide constructs further encoding secreted luciferases (Gaussia luciferase or super-luminescent variants) fused or linked via translational interrupter sequences to monitor real-time expression and processing efficiency by measuring luminescence in culture media.
Production of immunogenic FMDV viral proteins and VLPs for vaccine use
Methods and compositions involving modified 3C proteases enable production of immunogenic proteins or VLPs suitable for vaccines or immunogens comprising one or more processed FMDV viral capsid proteins or assembled VLPs.
The claims collectively cover polynucleotides, vectors, host cells, and methods for producing FMDV viral proteins and VLPs using modified FMDV 3C proteases, particularly those with the L127P substitution, enabling reduced host cell toxicity and enhanced recombinant yield. Additionally, constructs incorporating reporter luciferases facilitate monitoring of protein expression.
Stated Advantages
Modified FMDV 3C proteases retain proteolytic activity on the P1 precursor while exhibiting reduced cytotoxicity and inhibitory effects on host cells, improving cell viability.
Expression of modified 3C proteases in host cells results in significantly enhanced transgene output and recombinant protein yields, up to 25 times higher than with wild-type 3C.
The modifications allow production of fully processed FMDV capsid proteins and formation of virus-like particles capable of inducing immune responses.
Use of modified proteases reduces proteolytic degradation of host proteins such as eIF4A1, supporting normal cell function and further enhancing protein expression.
Inclusion of secreted luciferase reporters linked by translational interrupter sequences allows real-time monitoring of protein expression without lysing cells, facilitating efficient experimentation and vaccine development.
Documented Applications
Production of recombinant FMDV P1 precursor polypeptides and processed of viral proteins VP0, VP1, VP2, VP3, VP4, and assembly of FMDV virus-like particles in host cells for use as immunogenic preparations or vaccines.
Creation of DNA vaccine compositions expressing modified FMDV 3C protease and P1 precursor to induce protective immune responses against FMDV in susceptible animal hosts.
Use of secreted luciferase reporters fused to constructs for monitoring recombinant protein expression in vitro and in vivo.
Production of FMDV viral proteins or VLPs in various expression systems including mammalian cells, insect cells, yeast, plant cells, or prokaryotic cells such as E. coli.
Application in veterinary vaccines to immunize cloven-hooved animals susceptible to FMDV infection including cattle, pigs, sheep, goats, and wildlife.
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