DNA vaccines against foot-and-mouth disease virus
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10865389-B2
Publication Date
2020-12-15
Expiration Date
2036-09-08
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 modified foot-and-mouth disease virus (FMDV) 3C proteases that have been engineered by mutating polynucleotide sequences coding for the protease. These modified FMDV 3C proteases retain the ability to proteolytically cleave the FMDV P1 precursor polypeptide into viral proteins but exhibit a reduction in toxic or inhibitory effects on host cells used for recombinant protein production. This modification enables improved production of FMDV viral proteins and virus-like particles (VLPs) in host cells, including mammalian and bacterial cells.
The problem being addressed is the inherent toxicity of the native FMDV 3C protease to host cells, which reduces their viability and protein expression capacity. Conventional FMD vaccines use inactivated whole virus but have limitations including safety concerns, short shelf life, antigenic instability, and difficulty distinguishing vaccinated from infected animals. Recombinant production of FMDV antigens is hampered by the 3C protease's promiscuous proteolytic activity that damages host proteins, leading to low yields and cytotoxicity. Thus, there is a need for mutant FMDV 3C proteases that maintain proteolytic activity on viral precursors but reduce host cell toxicity to enable efficient recombinant vaccine production.
The invention demonstrates that modification of surface-exposed residues distal from the substrate binding cleft and active site, particularly within regions comprising residues 26-35, 125-134, or 138-150, such as the L127P mutation within the B2 beta-strand, reduces cytotoxicity while maintaining P1 processing activity. Polynucleotide constructs encoding these mutants, optionally combined with FMDV P1 precursor sequences and a translational interrupter sequence such as Δ1D2A, are used to express viral proteins and assemble VLPs efficiently in host cells. This approach enables improved protein yields and higher virus-like particle production compared to wild-type 3C protease expression.
Claims Coverage
The patent includes 15 claims centered on DNA vaccines and methods involving modified FMDV 3C proteases and FMDV P1 precursor polypeptides. The coverage focuses on inventive features related to the mutant 3C protease, vector constructs, vaccine formulations and administration methods.
Use of a modified FMDV 3C protease with L127 amino acid substitution
A DNA vaccine comprising a polynucleotide encoding a modified FMDV 3C protease having a leucine to proline substitution at position 127 (L127P) of the wild-type protease, along with a polynucleotide encoding a FMDV P1 precursor polypeptide.
Vector configuration for co-expression of modified 3C protease and P1 precursor
Vectors or polynucleotide constructs where the nucleotide sequences encoding the modified FMDV 3C protease and the FMDV P1 precursor polypeptide are contained in a single vector or two separate vectors, including arrangements as a fusion protein or a single open reading frame encoding both proteins.
Formulation and serotype coverage of the DNA vaccine
DNA vaccines formulated against multiple FMDV serotypes including 0, A, C, Asia 1, SAT1, SAT2, and SAT3, capable of multivalent use.
Additional substitutions in modified FMDV 3C protease
Inclusion of further amino acid substitutions such as at position C142 (e.g., C142T) in the modified FMDV 3C protease to enhance properties.
Delivery methods for the DNA vaccine
Administration of the vaccine by injection (subcutaneous, intradermal, intramuscular, jet), oral, intranasal, topical, electroporation, gene gun, transfection or liposome-mediated delivery.
Marker or DIVA vaccine aspects
The vaccine serves as a marker or Differentiating Infected from Vaccinated Animals (DIVA) vaccine by inducing specific immune responses distinct from natural infection.
Use of minicircle or viral vectors
The polynucleotides encoding the modified FMDV 3C protease and P1 precursor are carried in minicircle DNA vectors or viral vectors such as replication deficient adenovirus or vaccinia virus vectors.
Pharmaceutical composition comprising the modified 3C protease and P1 polypeptide
Pharmaceutical compositions comprising the polynucleotides encoding the FMDV P1 precursor polypeptide, the modified 3C protease (with L127 substitution), and a pharmaceutically acceptable carrier.
Method for inducing immune response using modified FMDV 3C protease and P1 precursor
Methods of vaccinating or inducing immune responses against FMDV by administering compositions comprising polynucleotides encoding the FMDV P1 precursor polypeptide and modified FMDV 3C protease (with L127 substitution), wherein the P1 precursor is expressed and processed by the co-expressed modified 3C protease.
Expanded substitutions and variants of the modified 3C protease
Use of additional substitutions such as the double mutant L127P/C142T to further enhance transgene output and processing.
The claims collectively cover DNA vaccine constructs and methods using polynucleotides encoding a modified FMDV 3C protease comprising at least an L127 amino acid substitution and a FMDV P1 precursor polypeptide, with various vector arrangements and formulations intended for multivalent vaccination, including delivery methods and compositions. The invention's core inventive features relate to the modified 3C protease mutations (especially L127P) that reduce toxicity while maintaining antigen processing, enabling effective DNA vaccines against FMDV.
Stated Advantages
The modified FMDV 3C proteases maintain proteolytic activity on FMDV P1 precursor protein while exhibiting reduced toxicity and inhibitory effects on host cells used for recombinant expression.
These modified proteases enable significantly increased transgene expression and virus-like particle production in host cells, sometimes up to 20-25 times higher than wild-type 3C protease.
The modifications allow safer and more efficient recombinant production of FMDV antigens and vaccines, avoiding limitations of current whole virus vaccines.
Use of mutant 3C proteases improves host cell viability, growth rates, and stability of expression during passages.
Incorporation of reporter genes such as secreted Gaussia luciferase chimeras facilitates monitoring of protein expression both in vitro and in vivo.
The DNA vaccine platform using these mutations provides rapid response capabilities, DIVA capability, and convenient administration routes with improved safety over live or whole virus vaccines.
Documented Applications
Vaccines against foot-and-mouth disease virus (FMDV) in susceptible animals such as cattle, pigs, sheep, goats, deer, and other cloven-hooved animals.
DNA vaccines comprising polynucleotides encoding modified FMDV 3C protease and P1 precursor for prophylactic or therapeutic immunization against multiple FMDV serotypes including 0, A, C, Asia 1, SAT1, SAT2, and SAT3.
Recombinant production of FMDV antigens and virus-like particles (VLPs) in a variety of host cells including mammalian, insect, yeast, plant, and bacterial cells for vaccine manufacture.
Use as marker or DIVA vaccines to differentiate infected from vaccinated animals using specific immune profiles.
Monitoring recombinant protein expression in vitro and in vivo via linked secreted luciferase reporters such as GLuc and SGLuc fused with translational interrupter sequences for vaccine development and assessment.
Interested in licensing this patent?