title: Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.
abstract: BACKGROUND
  Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties.
  
  
  RESULTS
  We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2.
  
  
  CONCLUSIONS
  Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher overall efficacy for the existing vaccines. The production of rotavirus proteins in plants would probably result in lower manufacturing costs, making it more affordable for developing countries. Further investigation is required to evaluate the immunogenic potential of the VLPs and fusion proteins created in this study.
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. DFGFRA001@myuct.ac.za.
authors.forename: Francisco F P G
authors.initials: FF
authors.lastname: Pêra
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. dl.mutepfa@gmail.com.
authors.forename: David L R
authors.initials: DL
authors.lastname: Mutepfa
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. eyeshamo8@gmail.com.
authors.forename: Ayesha M
authors.initials: AM
authors.lastname: Khan
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. Hendrik.Els@alumni.uct.ac.za.
authors.forename: Johann H
authors.initials: JH
authors.lastname: Els
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. MBWSAN001@myuct.ac.za.
authors.forename: Sandiswa
authors.initials: S
authors.lastname: Mbewana
authors.affiliation: Biochemistry Division, North-West University, Potchefstroom, South Africa. Albie.VanDijk@nwu.ac.za.
authors.forename: Alberdina A A
authors.initials: AA
authors.lastname: van Dijk
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. Ed.rybicki@uct.ac.za.|Institute of Infectious Disease and Molecular Medicine, Faculty of Health Science, University of Cape Town, Cape Town, South Africa. Ed.rybicki@uct.ac.za.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Rybicki
authors.affiliation: Biopharming Research Unit, Department of Molecular and Cell Biology, University of Cape Town, Rondebosch, Cape Town, South Africa. inga.hitzeroth@uct.ac.za.
authors.forename: Inga I
authors.initials: II
authors.lastname: Hitzeroth
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology; D005759:Gastroenteritis / Q000517:prevention & control / Q000821:virology; D005838:Genotype; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012401:Rotavirus / Q000145:classification / Q000235:genetics / Q000276:immunology*; D012400:Rotavirus Infections / Q000517:prevention & control* / Q000821:virology; D022243:Rotavirus Vaccines / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000235:genetics; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D058425:Vaccines, Virus-Like Particle / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification
chemical_list: D000956:Antigens, Viral; D036022:Capsid Proteins; D011993:Recombinant Fusion Proteins; D022243:Rotavirus Vaccines; C442545:VP2 protein, Rotavirus; C061332:VP6 protein, Rotavirus; D014614:Vaccines, Synthetic; D058425:Vaccines, Virus-Like Particle