Recombinant rinderpest vaccines expressing membrane-anchored proteins as genetic markers: Evidence of exclusion of marker protein from the virus envelope

Rinderpest virus (RPV) causes a severe disease of cattle resulting in serio us economic losses in parts of the developing world. Effective control and elimination of this disease require a genetically marked rinderpest vaccine that allows serological differentiation between animals that have been vac cinated against rinderpest and those which have recovered from natural infe ction. We have constructed two modified cDNA clones of the vaccine strain R NA genome of the virus, with the coding sequence of either a receptor site mutant form of the influenza virus hemagglutinin (HA) gene or a membrane-an chored form of the green fluorescent protein (GFP) gene (ANC-GFP), inserted as a potential genetic marker. Infectious recombinant virus was rescued in cell culture from both constructs. The RPVINS-HA and RPVANC-GFP viruses we re designed to express either the HA or ANC-GFP protein on the surface of v irus-infected cells with the aim of stimulating a strong humoral antibody r esponse to the marker protein. In vitro studies showed that the marker prot eins were expressed on the surface of virus-infected cells, although to dif ferent extents, but neither was incorporated into the envelope of the virus particles. RPVINS-HA- or RPVANC-GFP-vaccinated cattle produced normal leve ls of humoral anti-RPV antibodies and significant Levels of anti-HA or anti -GFP antibodies, respectively. Both viruses were effective in stimulating p rotective immunity against RPV and antibody responses to the marker protein in all animals when tested in a cattle vaccination trial.