Co-expression of anti-rotavirus proteins (llama VHH antibody fragments) in Lactobacillus: development and functionality of vectors containing two expression cassettes in tandem.

Gökçe Günaydın,Lennart Hammarström,Harold Marcotte,Yin Lin,Beatriz Álvarez,Prosper N Boyaka

doi:10.1371/journal.pone.0096409

Gökçe Günaydın, Lennart Hammarström + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0096409

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Journal: PloS one	Publication Date: Apr 29, 2014
Citations: 42	License type: CC BY 4.0

Affiliation: Karolinska University Hospital, Karolinska Institutet

Abstract

Rotavirus is an important pediatric pathogen, causing severe diarrhea and being associated with a high mortality rate causing approximately 500 000 deaths annually worldwide. Even though some vaccines are currently available, their efficacy is lower in the developing world, as compared to developed countries. Therefore, alternative or complementary treatment options are needed in the developing countries where the disease burden is the largest. The effect of Lactobacillus in promoting health and its use as a vehicle for delivery of protein and antibody fragments was previously shown. In this study, we have developed co-expression vectors enabling Lactobacillus paracasei BL23 to produce two VHH fragments against rotavirus (referred to as anti-rotavirus proteins 1 and 3, ARP1 and ARP3) as secreted and/or surface displayed products. ARP1 and ARP3 fragments were successfully co-expressed as shown by Western blot and flow cytometry. In addition, engineered Lactobacillus produced VHH antibody fragments were shown to bind to a broad range of rotavirus serotypes (including the human rotavirus strains 69M, Va70, F45, DS1, Wa and ST3 and simian rotavirus strains including RRV and SA11), by flow cytometry and ELISA. Hereby, we have demonstrated for the first time that when RRV was captured by one VHH displayed on the surface of co-expressor Lactobacillus, targeting other epitope was possible with another VHH secreted from the same bacterium. Therefore, Lactobacillus producing two VHH antibody fragments may potentially serve as treatment against rotavirus with a reduced risk of development of escape mutants. This co-expression and delivery platform can also be used for delivery of VHH fragments against a variety of mucosal pathogens or production of other therapeutic molecules.

Highlights

Rotavirus, one of the most crucial pediatric pathogens, is transmitted by the fecal-oral route and infects the enterocytes of the upper- and mid- section of the small intestine [1]
Modified lactobacilli producing VHH antibody fragments offer a novel approach to the prevention and treatment of rotavirusinduced diarrhea and could complement current vaccine-based forms of prophylaxis
We have developed vectors consisting two expression cassettes cloned in tandem to allow the co-expression of the two functional ARP antibody fragments, targeting different epitopes simultaneously

Summary

Introduction

One of the most crucial pediatric pathogens, is transmitted by the fecal-oral route and infects the enterocytes of the upper- and mid- section of the small intestine [1]. According to a surveillance report by WHO, during 2010, the common rotavirus G types (G1–G4 and G9) represented approximately 70% of all rotavirus infections [8]. The frequency of G–P combinations vary geographically, for example G1 P[8], the main component of rotavirus vaccines, was identified in more than 80% of all reported rotavirus infections in America, Europe and the West pacific region, but less than 40 and 20% in Africa and Asia, respectively [9]. Uncommon rotavirus G/P combinations (G12 P[8], G12 P[6], G2 P[6], G3 P[6], and G1 P[6] viruses), with an emerging G12 type [10], were found in 30 and 50% of all rotavirus infections in Africa and Asia, respectively [9]. A successful rotavirus therapy should serve as a good heterotypic protection against human rotaviruses with high diversity and capacity of acquiring mutations

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