Minicircle DNA vector vaccine platform for foot-and-mouth disease and methods thereof
Inventors
Puckette, Michael • Rasmussen, Max • Neilan, John
Assignees
US Department of Homeland Security
Publication Number
US-10513542-B2
Publication Date
2019-12-24
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
This invention relates to minicircle DNA vectors for vaccination against foot-and-mouth disease (FMD). The minicircle DNA vector's transgene expression cassette comprises a eukaryotic translation initiation nucleotide sequence, a mutant nucleotide sequence encoding a FMD virus (FMDV) capsid polyprotein precursor containing at least one mutation to eliminate a restriction enzyme recognition site, a nucleotide sequence encoding a protease that cleaves the capsid polyprotein precursor into multiple FMDV capsid proteins, and a translational regulatory element to regulate protease expression. When transfected into mammalian host cells, these minicircle vectors intrinsically produce virus-like particles (VLPs) that stimulate adaptive immunity to FMD.
The problem addressed is the difficulty in producing stable and immunogenic FMD vaccines. Whole-virus vaccines possess risks such as virus escape, instability, and inability to differentiate vaccinated from infected animals via antibody profiles. Existing recombinant VLP production is hindered by viral 3C protease cytotoxicity and instability issues of empty capsids. Additionally, preparing vaccines using intermediate host cell systems is costly and labor-intensive. The inability to rapidly generate vaccines addressing antigenic diversity and mutations affects outbreak response.
The invention solves these problems by engineering mutant FMDV nucleotide sequences with silent mutations that remove restriction enzyme sites, facilitating cloning and swapping of capsid sequences for various serotypes. The minicircle DNA vector enables direct transfection into mammalian cells, bypassing intermediate host systems and reducing costs. Use of translational regulatory elements or protease mutations downregulates 3C protease expression to balance effective capsid processing and host cell viability. This approach yields stable VLPs, with the vaccine encoding only part of the FMDV genome (P1 and 3C), preventing risks of accidental outbreaks and allowing differentiation of infected from vaccinated animals via antibody profiles.
Claims Coverage
The patent discloses six main inventive features covering mutant nucleotide sequences, vectors, host cells, virus-like particles, and methods related to dent removal and expression regulation for FMDV vaccination.
Mutant nucleotide sequence encoding FMDV capsid polyprotein with silent mutations removing restriction sites
A mutant nucleotide sequence encoding a FMDV capsid polyprotein precursor, comprising one or more silent mutations that remove all occurrences of specific restriction enzyme recognition sites from the wild-type nucleotide sequence, facilitating cloning without altering the amino acid sequence.
Vector comprising mutant nucleotide sequence with expression control elements
A vector containing the mutant nucleotide sequence encoding the FMDV capsid polyprotein precursor together with a eukaryotic translation initiation sequence 5′ to the mutant sequence, a nucleotide sequence encoding a protease, and a translational regulatory element positioned between them to reduce protease expression, where the vector may be a minicircle vector and expresses protease functional to cleave the polyprotein into FMDV capsid proteins (VP1, VP2, VP3, VP4). The translational regulatory element may comprise a ribosomal frameshift sequence reducing protease translation to no more than 20%, preferably 5-10%, of full translation.
Transformed mammalian host cell producing FMDV virus-like particles
A transformed mammalian host cell comprising the mutant nucleotide sequence encoding the FMDV capsid polyprotein precursor that removes restriction enzyme sites, which is functional to produce FMDV virus-like particles.
Virus-like particles produced from expression of mutant nucleotide sequence vector
Virus-like particles comprising polypeptides produced from expression of vectors containing the mutant nucleotide sequence encoding FMDV capsid polyprotein precursor with silent mutations removing restriction enzyme recognition sites, optionally including expression control elements and protease sequences.
Method of vaccinating mammals using vector encoding mutant nucleotide sequence producing VLPs
A method of vaccinating a mammal against FMDV by administering a vector comprising the mutant nucleotide sequence encoding the capsid polyprotein precursor with silent mutations removing restriction enzyme sites, which induces production of FMD virus-like particles by host cells; the method may include co-administration of adjuvants.
Method of differentiating vaccinated from infected mammals via antibody detection
A method of determining FMDV vaccination or infection status in a mammal by immunoassay detecting presence of antibodies specific to vaccination with the mutant nucleotide sequence vector (producing VLPs) and absence of antibodies against FMDV non-structural proteins, indicating vaccination rather than infection.
The claims cover the compositions of mutant nucleotide sequences and vectors with expression regulation for FMDV capsid protein production, transformed host cells and VLPs produced therefrom, and methods of vaccination and differential diagnosis using these vectors, focusing on silent mutations eliminating restriction sites and controlled protease expression within minicircle DNA vectors.
Stated Advantages
Minicircle vectors lack prokaryotic sequences, resulting in less foreign DNA perception and extended transgene expression duration in mammalian cells.
Silent mutations removing restriction enzyme recognition sites facilitate cloning and rapid swapping of capsid sequences for different FMDV serotypes.
Downregulation or mutation of 3C protease expression reduces host cell cytotoxicity while maintaining capsid precursor processing, enabling stable VLP formation.
Direct transfection into mammalian cells using minicircle vectors reduces costs and labor by eliminating the need for intermediate host cell production systems.
The vaccine encodes only P1 and 3C proteins, reducing the risk of accidental FMD outbreaks during production or vaccination and allowing differentiation between infected and vaccinated animals through distinct antibody profiles.
Minicircle DNA vaccines can be rapidly modified to tailor vaccines to specific outbreak strains or serotypes, enabling a rapid response to FMD outbreaks.
FMD DNA vaccines do not require cold chain delivery and are amenable to lyophilization, improving vaccine stability and ease of distribution.
Documented Applications
Vaccination of mammals, including cloven-hoofed animals, against foot-and-mouth disease virus serotypes O, A, C, Asia 1, SAT 1, SAT 2, and SAT 3 using minicircle DNA vectors encoding mutant FMDV capsid polyprotein precursor producing virus-like particles for immunization.
Use of minicircle DNA vectors to produce FMDV virus-like particles in host mammalian cells to stimulate adaptive immunity.
Diagnostic methods to differentiate vaccinated from naturally infected animals by detecting antibodies specific to vaccination and absence of antibodies against FMDV non-structural proteins (DIVA strategy).
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