1 SCIENTIFIC REPORTS | 6:30171 | DOI: 10.1038/srep30171 www.nature.com/scientificreports Fusion of the mouse IgG1 Fc domain to the VHH fragment (ARP1) enhances protection in a mouse model of rotavirus Gökçe Günaydın 1 , Shengze Yu 2 , Torbjörn Gräslund 2 , Lennart Hammarström 1 & Harold Marcotte 1 A variable fragment of a heavy chain antibody (VHH) directed against rotavirus, also referred to as anti-rotavirus protein 1 (ARP1), was shown to confer protection against rotavirus induced diarrhea in infant mouse model of rotavirus induced diarrhea. In this study, we have fused the mouse IgG1 Fc to ARP1 to improve the protective capacity of ARP1 by inducing an Fc-mediated effector function. We have shown that the Fc-ARP1 fusion protein confers significantly increased protection against rotavirus in a neonatal mouse model of rotavirus-induced diarrhea by reducing the prevalence, duration and severity of diarrhea and the viral load in the small intestines, suggesting that the Fc part of immunoglobulins may be engaged in Fc-mediated neutralization of rotavirus. Engineered conventional-like antibodies, by fusion of the Fc part of immunoglobulins to antigen-specific heavy-chain only VHH fragments, might be applied to novel antibody-based therapeutic approaches to enhance elimination of pathogens by activation of distinct effector signaling pathways. Rotavirus is a non-enveloped double stranded RNA virus that is associated with a severe dehydrating diar- rhea, infecting infants and children less than 5 years of age worldwide 1 . e rotavirus recognition involves the cell-surface Lewis b blood group antigen 2 and several intracellular receptors, and its replication is limited to mature enterocytes of the small intestinal villi 3 . Protection against rotavirus involves blocking of enterocyte infec- tion by neutralizing antibodies against outer capsid proteins VP4 and VP7 4,5 . However, the vast majority of anti- bodies is directed against the most abundant and highly conserved rotavirus inner capsid protein VP6, and has been shown to mediate intracellular neutralization 6,7 . IgG-based therapeutics have gained increasing importance for the treatment of a wide range of infectious diseases including rotavirus infection 8 . In addition to the receptor or ligand blocking capacity of antibodies, they can also trigger potent biological responses such as regulation of immune responses in cells through Fc/Fc recep- tor interactions. e receptors for IgG can be classified into the well-known Fc gamma receptor (FcγR) family, consisting of different proteins expressed on the surface of myeloid cells, and the neonatal Fc receptor (FcRn), expressed at various levels in different cell types 9 . FcRn is the only receptor known to be engaged in bidirectional transcytosis of IgG across the mucosal epithelium in individuals at any age 10,11 . It protects the captured antibody from lysosomal degradation and thus prolongs its half-life 12 . Another more recent and less-characterized receptor is the tripartite-motif containing protein 21 (TRIM21), a cytosolic Fc receptor found in all cells, but with high expression levels in immune and endothelial cells. TRIM21 is involved in intracellular antibody-mediated adeno- virus recognition and destruction of virus-antibody complexes using the proteasome degradation machinery 13 . According to the site and level of infection, either one or more Fc receptor(s) might be activated in concert to drive some well-defined effector functions, including virus degradation or cell phagocytosis 14 . Single domain variable fragments of camelid heavy chain-only antibodies (referred to as Nanobodies ® or VHHs) show high solubility and stability under different extreme conditions 15 and exhibit similar affini- ties as compared to full-sized antibodies 16,17 . e VHH molecules have been used in various prophylactic and 1 Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital, Huddinge, SE-14186 Stockholm, Sweden. 2 Division of Protein Technology, School of Biotechnology, KTH - Royal Institute of Technology, SE-106 91, Sweden. Correspondence and requests for materials should be addressed to H.M. (email: harold.marcotte@ki.se) Received: 25 February 2016 Accepted: 28 June 2016 Published: 21 July 2016 OPEN