Modified foot-and-mouth disease virus 3C proteases, compositions and methods thereof
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10385319-B2
Publication Date
2019-08-20
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
This invention relates 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 the FMDV P1 precursor protein and show a reduction in toxic or inhibitory effects associated with unmodified FMDV 3C protease on host cells used for recombinant production. Vectors carrying polynucleotides encoding these modified proteases induce production of FMDV virus-like particles (VLPs) in host cells when expressed, which can be used to produce immunogenic FMDV preparations capable of inducing immune responses.
The problem addressed by this invention arises from the toxicity of native FMDV 3C protease to host cells due to its promiscuous proteolytic activity, which limits recombinant production of FMDV antigens. Current FMD vaccines based on whole virus present shortcomings such as short vaccine shelf life, safety concerns from virus production facilities, and difficulties differentiating vaccinated from infected animals. Recombinant vaccine production is hindered as native 3C protease reduces host cell viability and expression yields. The inventors aim to develop modified FMDV 3C proteases that cleave the P1 precursor effectively but have reduced toxicity to host cells resulting in improved production of FMDV antigens and VLPs.
A key finding is that the 3C protease can be engineered by amino acid substitutions in surface regions distal from the active site and substrate binding cleft—specifically in residues 26-35, 125-134, or 138-150 of a wild-type 3C protease—to maintain processing ability while reducing toxicity. For example, introduction of mutations such as L127P within these regions significantly reduces host cell toxicity while retaining cleavage of the P1 precursor. This allows increased transgene expression, improved host cell viability, and enhanced yields of FMDV antigens and VLPs. The invention also describes vectors, host cells, and recombinant methods implementing these modified proteases for improved vaccine antigen production.
Claims Coverage
The patent includes multiple independent claims directed to modified FMDV 3C protease polynucleotides, proteins, vectors, and host cells expressing them, with specific amino acid substitutions affecting residues 26-35, 125-134, and 138-150, primarily focusing on the L127P substitution and others.
Modified polynucleotide encoding FMDV 3C protease with L127 substitution
A polynucleotide encoding a modified FMDV 3C protease comprising an amino acid substitution of leucine at position 127 (L127) of a wild-type FMDV 3C protease, optionally retaining residues H46, D84, C163, and maintaining high sequence identity to wild-type proteases from various serotypes.
Modified FMDV 3C protease with mutations in key surface regions
An FMDV 3C protease modified by one or more amino acid substitutions within residues 26-35, 125-134, or 138-150 of the wild-type protease, such as V28K, L127P, V141T, C142T, and variants thereof, which retain proteolytic activity on FMDV P1 precursor with reduced host cell toxicity.
Vectors comprising polynucleotides encoding modified 3C proteases
Vectors or polynucleotide constructs comprising sequences encoding the modified FMDV 3C protease mutants including L127P and optionally FMDV P1 precursor polypeptides or reporter genes such as GLuc or SGLuc linked via 2A or Δ1D2A translational interrupter sequences enabling independent protein expression.
Host cells transformed with vectors expressing modified 3C proteases and substrates
Host cells transformed or transfected with said vectors or polynucleotide constructs, expressing the modified FMDV 3C protease and optionally the FMDV P1 precursor polypeptide and reporter proteins, capable of processing P1 precursor and producing virus-like particles with improved viability and protein expression compared to wild-type 3C protease expression.
The claims mainly cover polynucleotides encoding FMDV 3C proteases modified at leucine 127 and other surface residues that reduce toxicity while preserving proteolytic function, the modified protease proteins themselves, vectors containing these sequences optionally linked to reporter genes or FMDV antigen genes, and host cells expressing such constructs with improved production and reduced cytotoxicity. These features collectively enable enhanced FMDV antigen production for vaccine applications.
Stated Advantages
Modified FMDV 3C proteases exhibit reduced cytotoxicity and inhibitory effects on host cells compared to wild-type proteases.
They maintain their ability to process the FMDV P1 precursor protein effectively, enabling proper cleavage into viral proteins.
Host cells expressing modified proteases show increased viability, growth rate, and recombinant expression yields of FMDV antigens and virus-like particles.
Vectors encoding modified proteases have higher transgene expression output and increased virus-like particle production, up to 20 times compared to vectors with wild-type protease.
Use of translational interrupter sequences (e.g., Δ1D2A) allows independent, equimolar expression of fused proteins and facilitates monitoring of expression via secreted luciferase reporter genes.
Modified proteases reduce proteolytic cleavage of host proteins such as eIF4AI, mitigating negative impacts on host cell protein synthesis machinery and overall cell function.
Documented Applications
Recombinant production of FMDV antigenic proteins and virus-like particles in various host cells including mammalian, insect, yeast, plant, and bacterial cells.
Use of modified FMDV 3C proteases in vectors and host cells for vaccine production, facilitating enhanced expression of FMDV P1 precursor and VLP assembly.
Development of immunogenic compositions and vaccines comprising FMDV virus-like particles produced using the modified proteases for inducing immune responses against FMDV in susceptible animals.
Methods for vaccinating subjects in need thereof with compositions containing VLPs, recombinant vectors encoding modified proteases and FMDV antigens, or cellular immunogens expressing these proteins in vivo.
Use of luciferase (GLuc/SGLuc) reporter chimeras with translational interrupters as markers for monitoring transgene expression in vitro and in vivo in vaccine research and development.
Expression and processing of diverse FMDV serotype P1 precursor proteins by modified proteases enabling broad-spectrum vaccine antigen production.
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