Puumala virus nucleocapsid protein expressed in transgenic plants is not immunogenic after oral administration.

Transgenic plants expressing foreign genes are suitable systems for the production of relevant immunogens in high amounts that can be used to develop a new generation of vaccines against a variety of infectious diseases. Transgenic tobacco and potato plants expressing the nucleocapsid protein of Hantavirus serotype Puumala were generated and established. Puumala virus is a human pathogen causing hemorrhagic fever with renal syndrome. To investigate oral immunogenicity of the nucleocapsid protein expressed in plants, mice were fed with tubers of transgenic potato and tobacco leaf powder. The resulting antibodies were compared among groups. No significant difference could be found between the control group and the groups of animals, which had been fed with the recombinant plants expressing Puumala nucleocapsid protein. Hence, the effect of different enzymes, present in the gastro-intestinal tract, on the plant-derived antigen was investigated. It was found that the recombinant viral protein was completely degraded by trypsin and/or pepsin. In conclusion, the enzymes present in the intestine can degrade major antigenic domains of antigens, expressed in transgenic plants, thus preventing the induction of antibodies against the ingested viral antigen.

Characterization of expression of Puumala virus nucleocapsid protein in transgenic plants.

Transgenic plants expressing a foreign gene are a suitable system for the production of relevant immunogens in high amounts that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study, the expression of the nucleocapsid (N) protein of hantavirus serotype Puumala in tobacco and potato plants was investigated. Transgenic tobacco and potato plants were generated and established. These transgenic plants expressed the N protein of Puumala virus strain CG-1820. No major differences were observed when the phenotype and growth rates of transgenic plants were compared to those of normal plants. However, it was found that the leaves of transgenic tobacco plants were more slender and the tubers of transgenic potato plants were smaller than those in normal plants. In order to investigate the distribution of the expression of the foreign gene in transgenic plants, the proteins of leaves and roots of the individual transgenic tobacco and potato plants were examined by Western blot analyses. It was found that all transgenic tobacco and potato plants expressed the N protein in the leaves, whereas transgenic potato plants are able to significantly express the viral proteins also in the tubers and roots. The antigens were expressed at a level of 1 ng of protein/5 microg of dried leaves. The hantaviral recombinant N proteins obtained from transgenic tobacco and potato plants were able to elicit specific humoral and mucosal immune responses when administered intraperitoneally or orally to rabbits and mice. The expression of viral proteins in plants has two major advantages compared to other expression systems: firstly, there is no risk of contamination with mammalian viruses or other pathogens, and secondly, the production of high amounts of antigens is cheap and therefore of great economic interest.