Llama VHH directed to rotavirus VP 6 protein possess broad neutralizing 2 activity in vitro and confer protection against diarrhoea in mice 3 4 Running title 5 Anti-VP 6 VHH Neutralize Rotaviruses 6

6 Group A rotavirus is one of the most common causes of severe diarrhoea in 7 human infants and newborn animals. Rotavirus virions are triple layered particles. The 8 outer capsid proteins VP4 and VP7 are highly variable and represent the major 9 neutralizing antigens. The inner capsid protein VP6 is conserved among group A 10 rotavirus, is highly immunogenic and is the target antigen of most immuno-diagnosis 11 tests. Llama derived single chain antibody fragments (VHH) are the smallest molecules 12 with antigen binding capacity and can therefore be expected to have properties different 13 from conventional antibodies. In this study a library containing the VHH genes of a llama 14 immunised with recombinant inner capsid protein VP6 was generated. Binders directed to 15 VP6, in its native conformation within the viral particle, were selected and characterized. 16 Four selected VHH directed to conformational epitopes of VP6 recognized all human and 17 animal rotavirus strains tested and could be engineered for their use in immunodiagnostic 18 tests for Group A rotavirus detection. Furthermore this result was confirmed by in vivo 19 partial protection against rotavirus challenge in a neonatal mouse model. This study 20 demonstrates for the first time a broad neutralization activity of VP6 specific VHH in 21 vitro and in vivo. Neutralizing VHH directed to VP6 promise to become an essential tool 22 for prevention and treatment of rotavirus diarrhoea. 23