Development of a genetically marked recombinant rinderpest vaccine expressing green fluorescent protein

In order to effectively control and eliminate rinderpest, a method is required to allow serological differentiation between animals that have been vaccinated and those which have recovered from natural infection. One way of doing this would be to engineer the normal vaccine to produce a genetically marked rinderpest virus (RPV) vaccine. We constructed two modified cDNA clones of the RPV RBOK vaccine strain with the coding sequence of the green fluorescent protein (GFP) gene inserted as a potential genetic marker. RPVINS-GFP virus was designed to produce independent and high level expression of GFP inside infected cells, whilst the GFP expressed by RPVSIG-GFP virus was designed to be efficiently secreted. Infectious recombinant virus was rescued in cell culture from both constructs. The effectiveness of these viruses in stimulating protective immunity and antibody responses to the marker protein was tested by vaccination of cattle and goats. All of the vaccinated animals were completely protected when challenged with virulent virus: RPV in cattle or peste-des-petits ruminants virus in the goats. ELISA showed that all of the animals produced good levels of anti-RPV antibodies. Three of the four cattle and the two goats vaccinated with RPVSIG-GFP produced detectable levels of anti-GFP antibodies. In contrast, no anti-GFP antibodies were produced in the four cattle and two goats vaccinated with RPVINS-GFP. Therefore, secretion of the GFP marker protein was absolutely required to elicit an effective humoral antibody response to the marker protein.