Methods of making and using vaccines utilizing minicircle DNA expression vectors for production of foot-and-mouth-disease virus proteins and virus-like particles
Inventors
Puckette, Michael • Rasmussen, Max • Neilan, John
Assignees
US Department of Homeland Security
Publication Number
US-9975926-B2
Publication Date
2018-05-22
Expiration Date
2035-12-08
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Abstract
This application is directed generally to minicircle DNA vectors for the vaccination of foot-and-mouth disease (FMD). The transgene expression cassette in the minicircle DNA vector includes: a eukaryotic translation initiation nucleotide sequence, a mutant nucleotide sequence that encodes a foot-and-mouth disease virus (FMDV) capsid polyprotein precursor that contains at least one mutation to eliminate a restriction enzyme recognition site, a nucleotide sequence that encodes a protease that cleaves the FMDV capsid polyprotein precursor into a plurality of FMDV capsid proteins and a translational regulatory element to regulate the expression of the protease. The minicircle DNA vectors can be transfected directly into the cell of a mammalian host. When transfected into the mammalian host cell, virus-like particles can be produced intrinsically to stimulate the mammalian host's immune system to develop adaptive immunity toward foot-and-mouth disease.
Core Innovation
The invention provides mutant nucleotide sequences encoding foot-and-mouth disease virus (FMDV) capsid polyprotein precursors, wherein the sequences include mutations that remove restriction enzyme recognition sites to facilitate cloning without altering the encoded protein. These mutant sequences can be incorporated into transgene expression cassettes that also include a eukaryotic translation initiation sequence, a nucleotide sequence encoding a protease to cleave the polyprotein into capsid proteins, and a translational regulatory element to control protease expression.
The minicircle DNA vectors containing these transgene expression cassettes can be directly transfected into mammalian host cells, enabling intrinsic production of FMDV virus-like particles (VLPs) that stimulate adaptive immunity against foot-and-mouth disease. The vectors may utilize translational regulatory elements such as ribosomal frameshift sequences to reduce protease expression and limit cytotoxicity while preserving cleavage activity necessary for capsid formation.
The problem addressed is the need for effective, stable, and safe vaccines against FMD, a highly contagious disease affecting cloven-hoofed animals and causing substantial economic losses. Traditional vaccine methods have limitations including potential virus escape, vaccine instability, short immunity duration, and difficulty differentiating vaccinated from infected animals. Additionally, production of stable empty capsids or VLPs is hindered by protease toxicity, and previous methods require complicated intermediate host systems for production.
Claims Coverage
The patent contains seven independent claims that focus on mutant nucleotide sequences, vectors carrying such sequences with additional functional elements, and regulatory features controlling protease expression.
Mutant nucleotide sequence encoding FMDV capsid polyprotein precursor
A nucleotide sequence encoding the FMDV capsid polyprotein precursor that includes mutations removing restriction enzyme recognition sites, specifically comprising SEQ ID NO: 1.
Vector with mutant nucleotide sequence encoding FMDV capsid polyprotein precursor
A vector comprising the mutant nucleotide sequence encoding the capsid polyprotein precursor removing a restriction enzyme site, including SEQ ID NO: 1.
Vector comprising mutant nucleotide sequence with specific nucleotide sequence
A vector comprising the mutant nucleotide sequence encoding the FMDV capsid polyprotein precursor with sequences comprising SEQ ID NO: 2.
Vector comprising mutant nucleotide sequence and translational regulatory elements
A vector comprising the mutant nucleotide sequence, a eukaryotic translation initiation sequence 5' to the mutant sequence, a nucleotide sequence encoding a protease, and a translational regulatory element 3' to the mutant sequence and 5' to the protease sequence, with the vector comprising SEQ ID NO: 3.
Vector comprising ribosomal frameshift regulatory element
A vector as above where the translational regulatory element comprises a DNA or RNA sequence responsible for a ribosomal frameshift, including the nucleotide sequence of SEQ ID NO: 6.
Vector comprising nucleotide sequence encoding protease
A vector as above where the nucleotide sequence encoding the protease is SEQ ID NO: 7.
Vector comprising mutated protease nucleotide sequence
A vector as above where the nucleotide sequence encoding the protease is SEQ ID NO: 9, representing a mutated 3C protease.
The claims collectively cover mutant nucleotide sequences that remove restriction enzyme sites, vectors carrying these sequences with additional elements like translation initiation sequences, protease encoding sequences, and regulatory elements such as ribosomal frameshift sequences, enabling expression of FMDV capsid precursors and controlled protease activity in host cells.
Stated Advantages
Enables stable and efficient vaccine production against FMD without the risk of accidental FMD outbreak since the complete viral genome is not encoded.
Allows direct transfection into mammalian host cells without requiring intermediate host systems, reducing costs and complexity.
Mutation of restriction enzyme sites facilitates cloning and sub-cloning of different FMDV serotypes to rapidly respond to outbreaks.
Regulation of 3C protease expression reduces host cell toxicity while maintaining necessary cleavage activity for capsid formation.
Minicircle DNA vectors, lacking bacterial sequences, improve transfection efficiency and prolong transgene expression.
Allows differentiation between vaccinated and naturally infected animals by producing distinct antibody profiles.
Documented Applications
Vaccination of mammals against foot-and-mouth disease virus by administering vectors expressing mutant FMDV capsid polyprotein precursor producing virus-like particles.
Diagnostic method to determine whether a mammal is vaccinated or infected by detecting presence or absence of specific antibodies associated with vaccination or natural infection using immunoassays such as ELISA.
Production of virus-like particles (VLPs) in mammalian cells through transfection with minicircle DNA vectors encoding mutant FMDV capsid polyprotein precursor and protease.
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