Vaccines and pharmaceutical compositions against foot-and-mouth disease virus
Inventors
Puckette, Michael • Rasmussen, Max V.
Assignees
US Department of Homeland Security
Publication Number
US-10858634-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 genetically modified by mutating polynucleotide sequences encoding the FMDV 3C protease to reduce their cytotoxicity when expressed in host cells. These modified FMDV proteases retain or enhance their ability to proteolytically process the FMDV P1 precursor polypeptide, cleaving it into individual viral proteins such as VP0, VP1, VP2, VP3 and VP4, and support the assembly of these into virus-like particles (VLPs).
The problem addressed arises from the native FMDV 3C protease being highly toxic to host cells used for recombinant expression of FMDV antigens, due to the protease's promiscuous cleavage of host proteins and interference with host cellular processes, which substantially reduces the yield of viral proteins and VLPs needed for vaccine production. Current vaccines based on whole inactivated virus have limitations including difficulty in distinguishing vaccinated animals from infected ones, immunogenic instability, short shelf-life, high production cost, and safety risks of virus escape from manufacturing facilities.
To overcome these issues, the inventors engineered mutant FMDV 3C proteases with amino acid substitutions particularly within residues 26-35, 125-134 or 138-150, which are surface regions distant from catalytic and substrate binding sites. Notably, an L127P mutation in the B2 β-strand region, among others, resulted in a protease that maintains proteolytic activity to process P1 polypeptide effectively while markedly reducing cytotoxicity to host cells. This allows for improved transgene expression, increased production of FMDV viral proteins and VLPs, and creates safer and more efficient platforms for producing immunogenic FMDV vaccine components, including for multiple serotypes.
Claims Coverage
The independent claims focus on vaccines, pharmaceutical compositions and methods for inducing immune responses against FMDV, emphasizing the use of modified FMDV 3C proteases with specific amino acid substitutions.
Use of modified FMDV 3C protease with L127 substitution in vaccine production
A vaccine comprising at least one FMDV viral protein or VLP produced by culturing a host cell expressing a FMDV P1 precursor polypeptide and a modified FMDV 3C protease containing an L127 amino acid substitution of the wild-type protease, followed by recovery of the viral protein.
Virus-like particle formation with specific viral protein compositions
The vaccine includes FMDV viral proteins in the form of VLPs, comprising at least VP0, VP1 and VP3, or VP1, VP2, VP3 and VP4.
Formulation against multiple FMDV serotypes and polyvalency
The vaccine is formulated against at least one of the major FMDV serotypes O, A, C, Asia 1, SAT1, SAT2, or SAT3, and may be polyvalent or multivalent to protect against multiple strains.
Additional mutation inclusion in modified 3C protease
The modified FMDV 3C protease may include a further C142 substitution, e.g., the L127P/C142T double mutation.
Pharmaceutical composition comprising recovered FMDV proteins with carriers
A pharmaceutical composition including recovered FMDV viral proteins or VLPs together with a pharmaceutically acceptable carrier.
Methods of inducing immune response or vaccination using said compositions
Methods for inducing immune response, vaccinating against FMDV or reducing severity by administering an effective amount of recovered FMDV viral proteins or VLPs produced using host cells expressing P1 precursor and modified 3C protease with L127 substitution.
Routes and schedules of administration
The compositions may be administered by multiple routes such as injection (subcutaneous, intradermal, intramuscular, jet), oral, intranasal, topical, electroporation, gene gun, or liposome-mediated delivery, using single, two-dose, or multiple-dose schedules.
Host range applicability and subject species
The methods and compositions are applicable to various susceptible mammals including livestock such as cows, pigs, sheep, goats, buffalo, and wildlife that are at risk or infected.
The independent claims primarily cover vaccines and pharmaceutical compositions comprising FMDV proteins or VLPs produced using modified FMDV 3C proteases with specific amino acid substitutions, notably L127P and optionally C142T, host cell-based production methods, and their use in immunizing or treating subjects against various FMDV serotypes, allowing improved transgene expression, VLP formation, and safety compared to wild-type 3C protease usage.
Stated Advantages
Modified FMDV 3C proteases retain the ability to process viral P1 precursor proteins into structural proteins while exhibiting reduced toxicity or inhibitory effects on host cells.
Host cells expressing modified proteases show increased growth rates, viability, passage stability, and significantly enhanced production yields of viral proteins and virus-like particles compared to cells expressing unmodified proteases.
The modifications allow safer and more efficient recombinant production of FMDV antigens, reducing risks associated with whole virus vaccines.
Composition and vaccine platforms using modified proteases enable improved immunogenic stability and stronger immune responses against FMDV.
Inclusion of translational interrupter sequences such as Δ1D2A with luciferase reporters permits effective monitoring and quantification of transgene expression both in vitro and in vivo.
Documented Applications
Production of vaccines against foot-and-mouth disease virus comprising viral proteins or virus-like particles.
Recombinant production of FMDV P1 precursor proteins and processed viral proteins or VLPs in host cells transformed with vectors encoding modified FMDV 3C proteases.
Use in methods for vaccinating animals susceptible to FMDV infection such as cattle, pigs, sheep, goats, and wildlife species.
Administration of DNA vaccines or immunogenic compositions to induce protective immunity against multiple FMDV serotypes, including O, A, C, Asia 1, SAT1, SAT2, and SAT3.
Use of luciferase fusion constructs to monitor and quantify expression of recombinant FMDV proteins in host cells and potentially in vivo.
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