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

Date Published: May 2, 2013

Publisher: Public Library of Science

Author(s): Celina G. Vega, Marina Bok, Anastasia N. Vlasova, Kuldeep S. Chattha, Silvia Gómez-Sebastián, Carmen Nuñez, Carmen Alvarado, Rodrigo Lasa, José M. Escribano, Lorena L. Garaicoechea, Fernando Fernandez, Karin Bok, Andrés Wigdorovitz, Linda J. Saif, Viviana Parreño, Barbara Sherry.


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.

Partial Text

Diarrhea is the second most common cause of childhood mortality worldwide, causing 1.3 million deaths among children younger than 5 years of age [1]. Group A rotavirus (RVA) is the leading cause of severe diarrhea in children worldwide and is responsible for approximately 29% of all diarrheal deaths, causing 453,000 deaths per year [2]–[5]. Human rotaviruses (Group A, B and C) have also been implicated as causative agents of diarrheal outbreaks occurring in nursing homes [6], among travelers [7], in day-care centers [8], and in patients suffering from a variety of immunodeficiency conditions [9], [10].

The VP6 constitutes the inner capsid of rotavirus and it is the most abundant and immunodominant viral protein. The Abs directed to VP6 are highly cross-reactive among RVA so VP6 immunization could potentially provide heterotypic protection [62]. The main objective of the present study was to determine the efficacy of the oral administration of G6P[1] VP6-specific VHH nanoAbs against Wa G1P[8]I1 human RVA-induced diarrhea in a Gn pig model of infection and disease. The 3B2 VHH clone protected all treated animals from RVA-induced diarrhea when administered daily as milk supplement at a final Wa RVA ELISA Ab titer of 4096 (VN titer of 256) during nine consecutive days. The protection observed was comparable to that in pigs treated with porcine IgG Abs to Wa RVA at the same ELISA and VN titers. This latter group of pigs was considered the positive control group, simulating the protection conferred by homologous Abs [63], [64]. The presence of diarrhea in 2 out of 4 animals from the IgG group suggests that this passive treatment with homologous Abs is not completely capable of preventing RVA-induced diarrhea against the challenge dose administered. However, the homologous treatment still conferred a great degree of protection, highlighting the importance of passive maternal Abs in newborns. These results reinforce the importance of the use of RVA vaccines (to induce active immunity) but also state the precedent that additional RVA-specific strategies might be useful to control and prevent RVA diarrhea in human infants.




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