Authors: N. Pant (1), S. Bezemer (2), P. Hermans (2), A. Einerhand (3), J. Gray (4), K. Bellamy (5), S. Sarker (6), L. Hammarstrom (1), M. v.d. Vaart (7), and L. Frenken (7) ([email protected])
(1) Division of Clinical Immunology, Karolinska University Hospital, Stockholm, Sweden,
(2) BAC BV, Naarden, The Netherlands,
(3) Croklaan, Wormerveer, The Netherlands,
(4) Enteric Virus Unit, Health Protection Agency, London, UK,
(5) Unilever Research and Development Colworth House Sharnbrook, UK,
(6) ICDDR,B: Centre for Health and Population Research, GPO Box 128, Dhaka 1000, Bangladesh,
(7) Unilever Foods and Health Research Institute, Vlaardingen, The Netherlands
Objective:
The study was conducted to determine the feasibility of producing llama-derived antibody fragments (VHH) by Saccharomyces cerevisiae, which can neutralize rotavirus in an in-vivo mouse model and have a broad specificity towards common human rotavirus strains.
Methodology:
A llama was immunized with a G3 rotavirus strain (RRV). Using the modern biotechnological techniques, cDNA fragments encoding for binding domains of heavy chain only antibodies (VHH fragments) were obtained and expressed in Escherichia coli and S. cerevisiae. ELISA tests were used for identifying the VHH fragments which recognized RRV (G3,P[3]) and CK5 (G3). Rotavirus neutralization activity of rotavirus-specific VHH fragments was tested in vitro via plaque neutralization assays and infection inhibition using an indirect immunofluorescence assay (IFA). A mouse pup model of rotavirus-associated diarrhoea was used for testing the same in vivo. Immunoelectron microscopy (IEM) was used for determining the specificity towards human rotavirus isolates from faecal samples.
Results: From ELISA tests, 23 unique VHH fragments were identified which were able to bind to RRV and CK5. Nine of these fragments were able to neutralize CK5 in a plaque assay. After transferring the DNA fragments to the baker's yeast S. cerevisiae, the VHH fragments produced were re-tested in the plaque neutralization assay and in an IFA. Four fragments were selected for further in-vitro and in-vivo experiments. From the in-vivo model it was found that one fragment in particular was able to reduce the occurrence (30 % vs 100% in untreated group) and the duration (0.33 days vs 2.87 days in untreated group) of diarrhoea in the mouse pups. This fragment, VHH1, also showed broad specificity and in-vitro neutralization capacity towards very common circulating rotavirus stereotypes worldwide.
Conclusion: Monovalent antibody fragments directed against a common G3 rotavirus strain can be effectively produced by the food grade yeast S. cerevisiae and can reduce morbidity of rotavirus-induced diarrhoea in mice.
Acknowledgements: Part of this work was supported by an EU framework grant.
