VHH directed to rotavirus VP6 with neutralizing activity in vitro independent of viral genotypes was expressed in Baculovirus Larva System

GARAICOECHEA, LORENA; GOMEZ SEBASTIAN, SILVIA; AVARADO, CARMEN; OLICHON, AURELIEN; SURREY, THOMAS; SAIF, LINDA J.; ESCRIBANO, JOSE ANGEL; WIGDOROVICH, ANDRES; PARREÑO, VIVIANA

Hamilton Island, Australia

Simposio; X International Symposium on Double-Stranded RNA Viruses; 2009

Double-Stranded RNA Viruses commetee

Group A rotavirus RV. is one of the main causes of severe diarrhoea worldwide. The outer capsid proteins, VP4 and VP7, are highly variable and represent the major neutralizing antigens classifying RV into serotypes. The inner capsid protein VP6 is conserved among group A RV, is highly immunogenic and is the target antigen of most immuno-diagnosis tests. Conventional antibodies directed to VP6 have not shown neutralizing activity to RV infection in vitro. Llama derived single chain antibody fragments VHH. are the smallest molecules with antigen binding capacity. A library containing the VHH genes of a llama immunized with recombinant inner capsid protein VP6 was generated and four binders directed to VP6 were selected, characterized and expressed in E coli. The selected VHH recognized all human and animal RV strains tested by ELISA and three of them showed neutralizing activity to RV infection in vitro, independently of  the strains serotypes. Aditionally, two of the VHH induced partial protection against RV diarrhoea in a mouse model. With the future aims to develop a diagnosis test using the recombinant molecules and to test the protective capacity of one of the VHH in a gnotobiotic pig model, two VHH were successfully engineered and expressed in a non fermentative system based on baculovirus and insect larva.with good yields about 1 mg/larva.. Furthermore, the binders engineered and expressed in larva recognized all the RV strains tested and kept the neutralizing activity in vitro . The low cost production of VHH directed to VP6, in baculovirus and insect  larva, open a possibility of the development of a new strategy against rotavirus diarrhoea based on VHH antibodies, especially in developing countries where disease cause more than 600 thousand children deaths every year.