Production and molecular characterization of monoclonal antibodies against rabies virus

Rabies continues to kill many thousands of people throughout the developing world every year. The murine monoclonal antibody (mAb) 62-71-3 was recently identified for its potential application in rabies post-exposure-prophylaxis (PEP). The purpose here was to establish a plant-based production system for a chimeric mouse-human version of mAb 62-71-3, to characterize the recombinant antibody and investigate at a molecular level its interaction with the rabies virus (RV) glycoprotein. The chimeric mAb 62-71-3 was successfully expressed in Nicotiana benthamiana and was shown to display plant-specific glycans in its Fc region. Functionality was analyzed by antigen binding ELISA and by neutralization of a panel of lyssaviruses. The antibody-antigen interaction was investigated using pseudotype virus expressing mutagenized RV glycoproteins. A critical role for antigenic site I of the glycoprotein, in particular for two specific amino acid residues, was identified. Because the selective pressure of a single mAb might give rise to resistant viruses (escape mutants), the long-term aim of this project is to develop a combination of neutralizing mAbs. The different rnAbs within a cocktail should complement each other in terms of functionality and specificity and should bind to distinct epitopes. MAb E559 is a well-characterized murine antibody which targets antigenic site 11 of the viral glycoprotein. We initially analysed the hybridoma-derived mAb E559 and subsequently investigated a plant-derived chimeric version of mAb E559, to be used in combination with the plant-derived mAb 62-71-3. In addition to chimeric mAbs, single chain antibodies and diabodies, we also investigated a plant-derived bispecific mAb (DVD-Ig) which incorporates the specificities of both mAb 62-71 -3 and mAb E559. Moreover, a small antibody fragment based on the 62-71-3 single chain was engineered for targeted delivery to neuronal cells. This antibody fragment will be explored in the future regarding its potential as novel prophylactic/therapeutic tool against rabies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo