Classical swine fever virus replicon particles lacking the E$^{\rm rns}$ gene: a potential marker vaccine for intradermal application

Classical swine fever virus replicon particles (CSF-VRP) deficient for E$^{\rm rns}$ were evaluated as a non-transmissible marker vaccine. A cDNA clone of CSFV strain Alfort/187 was used to obtain a replication-competent mutant genome (replicon) lacking the sequence encoding the 227 amino acids of the glycoprotein E$^{\rm rns}$ (A187delE$^{\rm rns}$). For packaging of A187delE$^{\rm rns}$ into virus particles, porcine kidney cell lines constitutively expressing E$^{\rm rns}$ of CSFV were established. The rescued VRP were infectious in cell culture but did not yield infectious progeny virus. Single intradermal vaccination of two pigs with 10$^7$ TCID$_{50}$ of VRP A187delE$^{\rm rns}$ elicited neutralizing antibodies, anti-E2 antibodies, and cellular immune responses determined by an increase of IFN-$\gamma $ producing cells. No anti-E$^{\rm rns}$ antibodies were detected in the vaccinees confirming that this vaccine represents a negative marker vaccine allowing differentiation between infected and vaccinated animals. The two pigs were protected against lethal challenge with the highly virulent CSFV strain Eystrup. In contrast, oral immunization resulted in only partial protection, and neither CSFV-specific antibodies nor stimulated T-cells were found before challenge. These data represent a good basis for more extended vaccination/challenge trials including larger numbers of animals as well as more thorough analysis of virus shedding using sentinel animals to monitor horizontal spread of the challenge virus