Immunomodulation of cucumber mosaic virus infection by intrabodies selected in vitro from a stable single-framework phage display library.
Immunomodulation by the ectopic expression of intracellular antibodies ('intrabodies') has a great potential for interfering with physiological or pathological functions in vivo in a highly specific manner. One of the major obstacles of this technology is the inability of most antibodies to properly fold and function in the reducing environment of the cytoplasm, which prevents the formation of essential disulfide bonds. We wished to assess the intracellular performance of antibodies derived from a semi-synthetic single-chain variable fragment (scFv) phage display library ('F8 library') built on a thermodynamically stable single-framework scaffold. To this purpose, we chose to modulate the infection of a pandemic plant pathogen, the cucumber mosaic virus (CMV). After in vitro 'biopanning' on immobilized virions, two scFvs were biochemically characterized, showing high affinity toward the antigen. They were transiently expressed at high yields as soluble molecules in the cytoplasm of Nicotiana benthamiana plants. Subsequently, they were expressed in the cytoplasm of transgenic tomato plants. Challenge with high viral dose showed that both scFvs were able to elicit a phenotypic effect and led to the identification of a transgenic line fully resistant to infection. In these plants, the scFv binds the virus in the inoculated leaves preventing viral long distance movement. This work represents the first demonstration that the 'F8 library' can be directly screened in vitro to rapidly isolate antigen-specific scFvs that act as effective intrabodies in vivo. These antibodies represent powerful tools to interfere with several intracellular targets, modulating pathogen infectivity and/or cellular metabolism.
Villani
Maria Elena
ME
ENEA, Ente per le Nuove Tecnologie, l'Energia e l'Ambiente, UTS BIOTEC, Sezione Genetica e Genomica Vegetale, C.R. Casaccia, P.O. Box 2400, I-00100 Roma, Italy.
Roggero
Piero
P
Bitti
Orsola
O
Benvenuto
Eugenio
E
Franconi
Rosella
R
D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000918:Antibody Specificity; D017799:Cucumovirus / Q000235:genetics / Q000254:growth & development / Q000276:immunology*; D003593:Cytoplasm / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007113:Immunity, Innate / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D019151:Peptide Library; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics / Q000821:virology; D030821:Plants, Genetically Modified; D014026:Nicotiana / Q000235:genetics / Q000821:virology; D014162:Transfection
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library