Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana

Abstract

BackgroundHuman 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.ResultsWe 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.ConclusionOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0436-8) contains supplementary material, which is available to authorized users.

Production of vaccines for treatment of infectious diseases by transgenic plants

“…The expression of VP8*/VP6 was only successful when binary plant vector pEAQ-HT was used, but not when plasmids from pTRA family were used. The highly immunogenic epitope of VP8* was fused to either N-or C-terminal end of VP6 and successfully expressed in cytoplasm, whereas VP6/8*C was expressed faster and at higher levels than VP6/8*N. VP2 was coexpressed with all the proteins, expressed in cytoplasm, and was found to be expressed at the highest level when co-expressed with VP6 (Pera et al, 2015). The fusion proteins did not form VLPs but based on previous studies by other authors the team suspects they could still retain their high immunogenicity, which would have to be evaluated in animal experiments.…”

“…These experiments proved that rotavirus coat proteins can be successfully expressed in various plant tissues where they accumulate to relatively high levels, as high as 0.28 % TSP of VP6 in tobacco (Dong et al, 2005) and 1.5 % TSP of rotavirus VLPs in tobacco (Yang et al, 2011). Obtained proteins were also capable of inducing both mucosal and systemic immune response in orally immunized mice as well as provide passive immunity against rotavirus challenge in suckling mice born to immunized mother: 60 % of pups in the case of VP6-BSSV from C. amaranticolor (Zhou et al, 2010), 85 % of pups in case of bovine VP8* from transplantomic tobacco (Lentz et al, 2011) and bovine VP8* from tobacco (PerezFilgueira et al, 2004a (Pera et al, 2015). BergeronSandoval et al (2011) expressed codon-optimized full-length VP7, truncated VP4 and dimeric combinations of VP7, VP4 and fljB flagellin fused to 5'UTR of tobacco etch virus which works as a transcription enhancer, in leaves of N. benthamiana with relatively high yields: 5 µg/g for truncated VP4, 4 µg/g for flagellin, 31.97 µg/g for VP7-flagellin dimer and 12.3 µg/g for VP4-flagellin dimer.…”

“…As flagellin is known to have protected mice against chemical, bacterial, viral and radiation challenge without adverse side effects and induces many different cytoprotectant activities, Bergeron-Sandoval et al (2011) propose it as a promising candidate for vaccine adjuvant. Pera et al (2015) in term successfully expressed VP2 and VP6 in the form of VLPs in N. benthamiana by transient Agrobacterium-mediated transformation. Because the expression of VP7 and VP4 was not successful, the team tried to produce more appropriately immunogenic particles and created 3 fusion proteins, adding VP8* to the VP6 and co-expressing it with VP2.…”

“…For human rotavirus there are currently two live vaccines available, monovalent vaccine based on the most common serotype G1P[8] and pentavalentG1-G4 P[8] vaccine. Due to their expensiveness making them unavailable to the majority of the population in the developing world and the recent emergence of several novel serotypes there is however a big demand for developing new and better rotavirus vaccines (Pera et al, 2015), an aim for which also transgenic plants have been utilized a lot during the recent years. Rotavirus capsid proteins have been expressed by various research groups: VP6 has been expressed in tomato (Chung et al, 2000;Chung et al, 2001), alfalfa (Dong et al, 2005), potato (Yu and Langridge, 2003), VP7 has been expressed in potato (Wu et al, 2003;Choi et al, 2005), VP8* in transplastomic tobacco plants (Lentz et al, 2011), bovine VP8* in potato (Matsumura et al, 2002) and N. benthamiana in recombinant form (Perez-Filgueira et al, 2004a).…”

“…The capsid has 3 layers, named VP2, VP6 and VP7 (the outermost) with VP4 protein forming spikes on its surface. In the host intestine VP4 gets cleaved to VP5 and VP8* (Pera et al, 2015), especially the latter being highly immunogenic and playing a major role in rotavirus infectivity. The outer capsid layer, VP7, also contains several epitopes, important for viral activity.…”