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

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 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.

Core Innovation

This invention provides minicircle DNA vectors for vaccination against foot-and-mouth disease (FMD), comprising a transgene expression cassette. This cassette includes a eukaryotic translation initiation nucleotide sequence, a mutant nucleotide sequence encoding a foot-and-mouth disease virus (FMDV) capsid polyprotein precursor with at least one mutation removing 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 controlling protease expression. When transfected into mammalian host cells, these vectors enable intrinsic production of virus-like particles (VLPs) that stimulate adaptive immunity against FMD.

The problem addressed is the highly infectious nature of FMD, which affects cloven-hoofed animals and causes significant economic losses, necessitating effective vaccination and containment strategies. Existing vaccines have limitations including risk of virus escape, vaccine instability, short duration of immunity, and difficulties distinguishing infected from vaccinated animals. Additionally, formation of stable FMDV VLPs in host cells at sufficient concentrations is hindered by toxicity of the viral 3C protease and instability of empty capsids.

The disclosed minicircle DNA vectors circumvent these issues by encoding only the P1 and 3C viral proteins, lacking the complete FMDV genome to prevent accidental outbreaks during vaccine production or use. The 3C protease expression is downregulated via mutations or translational regulatory elements such as ribosomal frameshift sequences to reduce cytotoxicity while retaining cleavage activity for capsid formation. Silent mutations eliminate restriction sites in the capsid polyprotein precursor nucleotide sequence, facilitating cloning and sub-cloning without affecting protein sequence. The minicircle vectors lack bacterial sequences to enhance expression duration and reduce immune clearance in mammalian cells. This platform also enables differentiating infected animals from vaccinated ones through distinct antibody profiles, supporting DIVA (Differentiating Infected from Vaccinated Animals) strategies.

Claims Coverage

The patent includes two main independent claims covering methods of vaccinating a mammal with vectors comprising mutant nucleotide sequences encoding FMDV capsid polyprotein precursors with silent mutations removing restriction enzyme sites, and methods for determining vaccination or infection status via antibody detection.

The claims focus on mutant FMDV nucleotide sequences eliminating restriction sites, vectors inducing virus-like particle production in mammalian cells, modulation of viral protease expression to balance activity and toxicity, vaccination methods using minicircle DNA vectors, and serological methods to differentiate vaccinated from infected animals.

Stated Advantages

Documented Applications