Recombinant Monovalent Llama-Derived Antibody Fragments (VHH) to Rotavirus VP6 Protect Neonatal Gnotobiotic Piglets against Human Rotavirus-Induced Diarrhea

Abstract

Group A Rotavirus (RVA) is the leading cause of severe diarrhea in children. The aims of the present study were to determine the neutralizing activity of VP6-specific llama-derived single domain nanoantibodies (VHH nanoAbs) against different RVA strains in vitro and to evaluate the ability of G6P[1] VP6-specific llama-derived single domain nanoantibodies (VHH) to protect against human rotavirus in gnotobiotic (Gn) piglets experimentally inoculated with virulent Wa G1P[8] rotavirus. Supplementation of the daily milk diet with 3B2 VHH clone produced using a baculovirus vector expression system (final ELISA antibody -Ab- titer of 4096; virus neutralization -VN- titer of 256) for 9 days conferred full protection against rotavirus associated diarrhea and significantly reduced virus shedding. The administration of comparable levels of porcine IgG Abs only protected 4 out of 6 of the animals from human RVA diarrhea but significantly reduced virus shedding. In contrast, G6P[1]-VP6 rotavirus-specific IgY Abs purified from eggs of hyperimmunized hens failed to protect piglets against human RVA-induced diarrhea or virus shedding when administering similar quantities of Abs. The oral administration of VHH nanoAb neither interfered with the host's isotype profiles of the Ab secreting cell responses to rotavirus, nor induced detectable host Ab responses to the treatment in serum or intestinal contents. This study shows that the oral administration of rotavirus VP6-VHH nanoAb is a broadly reactive and effective treatment against rotavirus-induced diarrhea in neonatal pigs. Our findings highlight the potential value of a broad neutralizing VP6-specific VHH nanoAb as a treatment that can complement or be used as an alternative to the current strain-specific RVA vaccines. Nanobodies could also be scaled-up to develop pediatric medication or functional food like infant milk formulas that might help treat RVA diarrhea.

Rotavirus VP6 preparations as a non-replicating vaccine candidates

“…To support these results, generated VHH molecules directed VP6 in insect cells indicate the same functionality as those expressed in E. coli inducing a broad neutralization activity in vitro and protection against diarrhea and RV shedding in a neonatal mouse model [114]. It has also been shown that the VHH nanoAb can block the infection independently of the RV strain genotypes in vitro and observed a high protection against RV Wa strain in gnotobiotic pigs in vivo [115] as well as VP6-specific IgY Abs from egg yolk [116]. However, DLP2/6 was found to induce neither neutralizing Ab titer nor protecting neonatal Gnotobiotic Piglets against RV challenge [117].…”

Rotavirus VP6 as a potential vaccine candidate

“…Safe therapeutic approach and passive protection have also been reported following p.o. administration of RV VP6‐VHH nano‐antibodies in neonatal gnotobiotic piglets and mice . These findings demonstrate the potential application of VHH against VP6 to prevent RV‐induced diarrhea and suggest that VHHs should be further investigated as a suitable treatment for gastroenteritis.…”

“…The smaller size of the VHH molecule than conventional antibodies seems to play an important role in this scenario . Furthermore, virus neutralization activity in vitro and in vivo has been reported by insect cells and larvae derived VHH nano‐antibodies to VP6 . Safe therapeutic approach and passive protection have also been reported following p.o.…”

Immunogenicity and antigenicity of a recombinant chimeric protein containing epitopes of poliovirus type 1

“…4h). VP6 possess broad neutralizing activity in vitro and confer protection against diarrhea in mice (Garaicoechea et al, 2008) and neonatal gnotobiotic piglets (Vega et al, 2013).…”