1. Field of the Invention
This invention relates to a rationally designed engineered attenuated antigenic marker vaccine-virus production platform comprising a deletion of the Lpro coding sequence resulting in complete attenuation and mutations (negative markers) introduced in two non-structural viral proteins resulting in the elimination of two antigenic epitopes recognized by specific antibodies, one located in 3B and the other in 3D, thus providing a target for DIVA (Differentiation of naturally Infected from Vaccinated Animals) serological tests. The attenuated marker vaccine production virus also comprises unique restriction endonuclease sites flanking the capsid-coding region to facilitate the replacement of the capsid region making possible the exchange of cassettes representing relevant capsid coding regions of different serotypes and subtypes of FMDV field isolates for the design of custom vaccines.
2. Description of the Relevant Art
Foot and mouth disease (FMD) is an extremely contagious viral disease of cloven-hoofed ungulates which include domestic animals (cattle, pigs, sheep, goats, and others) and a variety of wild animals. The most prominent disease symptoms in FMDV-infected cattle include vesicular lesions of the epithelium of the mouth, tongue, teats and feet. Although some countries, among them United States, Canada, Mexico, Australia and most of Europe, are considered to be free of FMD, the disease is distributed worldwide and has a great economic impact on the export industry. Indeed, several economically devastating outbreaks have occurred over the past decade on almost every continent.
Control methods to eradicate FMD depend upon the prevalence of the disease in particular geographical regions/states and often include mass annual prophylactic vaccination campaigns and the application of stringent zooprophylactic measures following outbreaks. A chemically inactivated whole virus vaccine has been used to contain the disease, but it is slow acting and does not always permit distinction between infected and vaccinated animals. Indeed, in recent years the differentiation of infected animals from those that have been vaccinated is of paramount importance as a protective activity following emergency vaccination. Historically, the use of non-structural viral protein as serological indicators of viral replication has been widely applied. Among these proteins, the highly conserved FMDV 3D polymerase (3Dpol) of 52-KDa has been identified as the main determinant of infection and has been called the FMD-Virus Infection-Associated Antigen (FMD-VIAA; Berger et al. 1990. Vaccine 8:213-216; Bergmann et al. 1993. Am. J. Vet. Res. 54:825-831; Cowan and Graves. 1966. Virology 30:528-540; McVicar and Sutmoller. 1970. Am. J. Epidemiol. 92:273-278; Sorensen et al. 1998. Arch. Virol. 143:1461-1476). Studies by Newman and Brown (1997. J. Virol. 71: 7657-7662; Newman et al. 1994. Proc. Natl. Acad. Sci. USA 91:733-737) suggested that purified 140S FMDV preparations contain small quantities of 3Dpol and therefore, could account for seroconversion to 3Dpol in animals that have received inactivated FMDV vaccines.
Previous strategies to select highly attenuated vaccines for FMDV have relied on the selection of less-pathogenic variants produced by serial passages of the virus in non-natural hosts such as embryonated chicken eggs and rabbits (Giraudo et al. 1990. Virology 177:780-783; Xin et al. 2009. Vet. Microbiol.). Those empirical strategies failed when tested in susceptible species due to reversion to virulence by the mutant viruses harboring point-mutations and therefore, were not pursued for being too risky (Sutmoller, P. 2001. Rev. Sci. Tech. 20:715-722; Sutmoller et al. 2003. Virus Res. 91:101-144). Modern approaches to produce genetically engineered FMDV with altered virulence have relied on the deletion of the cell-receptor binding site (Mason et al. 1994. Proc. Natl. Acad. Sci. USA 91:1932-1936; McKenna et al. 1995. J. Virol. 69:5787-5790; Rieder et al. 1996. Proc. Natl. Acad. Sci. USA 93:10428-33), the viral leader coding sequence (Lpro, Piccone et al. 1995. J. Virol. 69:5376-82) or elements within the non-translated region (NTR) (Rodriguez et al. 2009. J. Virol. 83:3475-3485). The FMDV Lpro together with the 3Cpro and 2A proteinases play an important role in processing of the viral polyprotein. In addition to cleaving itself from the nascent polyprotein, Lpro cleaves the eukaryotic initiation factor 4G (eIF4G) causing inhibition of the cellular translation machinery. Lpro is also known to relocate to the nucleus in the FMDV-infected cells and to induce degradation of nuclear factor kappa B (NF-κB) with the consequent inhibition of the innate immune response (de Los Santos et al. 2006. J. Virol. 80:1906-1914). FMDVs of serotype A lacking Lpro have been shown to be infectious, to grow more slowly in BHK-21 cells (A12-LLV2, (Piccone et al., supra) and be attenuated for pigs (Brown et al. 1996. J. Virol. 70:5638-5641; Chinsangaram et al. 1998. Vaccine 16:1516-1522; Mason et al. 1997. Virology 227:96-102).
Currently killed-antigen FMDV vaccines are produced in expensive biological containment facilities, by growing large volumes (thousands of liters) of virulent FMDV that has been adapted to grow in cells, which can be sometimes difficult. This process has resulted in escape of virulent virus from the manufacturing facility causing costly outbreaks in livestock (see Cottam et al. 2008. PLoS Pathogen 4:1-8). After growth, virus is then inactivated using chemicals and antigen concentrates are prepared, followed by purification steps required to remove contaminant proteins, making it difficult to differentiate infected from vaccinated animals (DIVA) through serological diagnostic tests. There is little to no cross protection across serotypes and subtypes requiring the appropriate matching between vaccine and circulating field strains to achieve protection. Despite these shortcomings of the vaccines, billions of doses are manufactured every year around the world. Their use has been the basis for eradicating FMDV from Europe and for controlling the disease in many parts of the world through mass vaccination campaigns. Thus, there is an urgent need for the development of effective marker FMDV vaccine candidates with DIVA capabilities.