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 MEW capsid polyprotein precursor into 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.

Polynucleotide constructs that express an engineered foot-and-mouth disease (FMDV) P1 precursor protein and a non-FMDV TEV protease and methods for safe and efficient recombinant production of FMDV antigens and immunogens. Recombinant production of FMDV antigens avoids the need to culture highly-infectious FMDV, while conventional culture methods for producing FMDV antigens rely on the native FMDV 3C protease which exerts toxic effects on host cells. The inventors have developed a new system that efficiently and safely processes FMDV P1 precursor without the FMDV 3C protease, thus avoiding the toxic effects associated with use of the 3C protease. The invention is also directed to the FMDV antigens and virus-like particles produced by this system as well as to FMDV vaccines, diagnostics and other biologics.

The present invention provides a recombinant foot and mouth disease virus (FMDV) capsid precursor protein comprising a modified VP1 protein and optionally further comprising a modified VP4 protein. The invention further relates to an isolated nucleic acid molecule and an expression vector comprising the nucleic acid molecule for recombinant expression of the modified capsid precursor protein. In further aspects, the invention relates to a virus-like particle (VLP) obtained from the modified capsid precursor protein and a vaccine for use in the protection of a subject against an infection with FMDV produced from the VLP.

The invention concerns a method of producing a foot and mouth disease virus (FMDV) virus-like particle (VLP) in a baculovirus expression system, the method comprising the steps of (i) infecting an insect cell with a baculovirus expression vector, (ii) culturing the insect cell in cell culture medium for 5 days or more post infection and (iii) harvesting the FMDV VLP from the cell culture medium. The invention further relates to a vaccine for use in the protection of a subject against an infection with FMDV, the vaccine being obtainable by the method of the invention.

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.

The invention concerns a method of producing a foot and mouth disease virus (FMDV) virus-like particle (VLP) in a baculovirus expression system, the method comprising the steps of (i) infecting an insect cell with a baculovirus expression vector, (ii) culturing the insect cell in cell culture medium for 4 days or more post infection, (iii) separating the insect cells from the cell culture to obtain cell-free cell culture medium, and (iv) harvesting the FMDV VLP from the cell-free cell culture medium. The invention further relates to a vaccine for use in the protection of a subject against an infection with FMDV, the vaccine being obtainable by the method of the invention.

The invention concerns a modified recombinant foot and mouth disease virus (FMDV) VP2 protein and further concerns an FMDV capsid precursor protein P1 comprising the modified VP2 protein. In a specific aspect, the present invention concerns a VP2 protein or a capsid precursor protein P1 comprising the VP2 protein, wherein the amino acid sequence of the VP2 protein is modified to improve the stability of FMDV capsids. The invention further relates to an isolated nucleic acid molecule and an expression vector comprising the nucleic acid molecule for recombinant expression of the modified VP2 protein or a capsid precursor protein P1 comprising the VP2 protein. In further aspects, the invention relates to a virus-like particle (VLP) obtained from the modified capsid precursor protein P1 and a vaccine for use in the protection of a subject against an infection with FMDV produced from the VLP.

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.

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.

A type O foot-and-mouth disease virus-like particle antigen is provided, wherein the type O foot-and-mouth disease virus-like particle antigen is type O CATHAY topotype foot-and-mouth disease virus-like particle antigen, and the type O CATHAY topotype foot-and-mouth disease virus-like particle antigen is assembled by VP0, VP3 and VP1 antigen proteins of type O CATHAY topotype foot-and-mouth disease virus. The type O foot-and-mouth disease virus-like particle antigen has good immunogenicity. The prepared vaccine can produce complete protection against the O-type foot-and-mouth disease virus on the 14th day after immunization. The antibody titer produced is higher than that of the commercial inactivated vaccine, and the duration of immune protection can be maintained for at least 133 days. The prepared vaccine composition, preparation method and use thereof are also provided.