Formation of self-assembled triple-layered rotavirus-like particles (tlRLPs) by constitutive co-expression of VP2, VP6, and VP7 in stably transfected high-five insect cell lines

Shoja, Zabihollah; Tagliamonte, Maria; Jalilvand, Somayeh; Mollaei-Kandelous, Yaghoub; Stradis, A. De; Tornesello, Maria Lina; Buonaguro, Franco M.

doi:10.1002/jmv.23973

Cited by 16 publications

(5 citation statements)

References 65 publications

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“…These dysregulations in Ca 2+ concentrations could be responsible for the cytotoxicity and cell death responses observed when expressing VP7 [ 41 ]. Despite some studies having reported expression of VP7 in transgenic potatoes and insect cells, it is important to note that these used simian rotavirus VP7 [ 42 ] and human group A G1 VP7 [ 43 – 45 ], while our work focused on human rotavirus G9 VP7. Amino acid differences between the proteins in specific domains could be responsible for this effect in N. benthamiana .…”

Section: Resultsmentioning

confidence: 99%

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

Pera

Mutepfa

Khan

et al. 2015

Virol J

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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.

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Section: Resultsmentioning

confidence: 99%

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

Pera

Mutepfa

Khan

et al. 2015

Virol J

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“…The High Five cell line is conventionally accepted as a higher protein producer host compared to Sf9 cells (14,46). Therefore, several works have focused on using this cell line for VLP production (15,(47)(48)(49). However, quality assessment of VLPs and the associated contaminants has not been addressed in detail.…”

Section: Discussionmentioning

confidence: 99%

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

et al. 2020

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Advancements in the field of characterization techniques have broadened the opportunities to deepen into nanoparticle production bioprocesses. Gag‐based virus‐like particles (VLPs) have shown their potential as candidates for recombinant vaccine development. However, comprehensive characterization of the production process is still a requirement to meet the desired critical quality attributes. In this work, the production process of Gag VLPs by baculovirus (BV) infection in the reference High Five and Sf9 insect cell lines is characterized in detail. To this end, the Gag polyprotein was fused in frame to the enhanced green fluorescent protein (eGFP) to favor process evaluation with multiple analytical tools. Tracking of the infection process using confocal microscopy and flow cytometry revealed a pronounced increase in the complexity of High Five over Sf9 cells. Cryogenic transmission electron microscopy (cryo‐TEM) characterization determined that changes in cell complexity could be attributed to the presence of occlusion‐derived BV in High Five cells, whereas Sf9 cells evidenced a larger proportion of the budded virus phenotype (23‐fold). Initial evaluation of the VLP production process using spectrofluorometry showed that higher levels of the Gag‐eGFP polyprotein were obtained in High Five cells (3.6‐fold). However, comparative analysis based on nanoparticle quantification by flow virometry and nanoparticle tracking analysis (NTA) proved that Sf9 cells were 1.7‐ and 1.5‐fold more productive in terms of assembled VLPs, respectively. Finally, analytical ultracentrifugation coupled to flow virometry evidenced a larger sedimentation coefficient of High Five‐derived VLPs, indicating a possible interaction with other cellular compounds. Taken together, these results highlight the combined use of microscopy and flow cytometry techniques to improve vaccine development processes using the insect cell/BV expression vector system. © 2020 International Society for Advancement of Cytometry

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“…It has been shown by several studies that the structural proteins (VP2, VP6, VP7, and VP4) of RV can be expressed in different prokaryotic and eukaryotic expression systems and form SLPs (VP2), DLPs (VP2 and VP6), and TLPs (VP2, VP6, and VP7 or VP2, VP6, VP7, and VP4) [132][133][134][135][136][137][138][139][140][141][142][143][144][145][146][147][148]. Furthermore, expressing VP6 alone in vitro [14,29] can form spherical, tubular, and trimer structures similar to VP6 purified from the RVs [31].…”

Section: The Different Structures Of Vp6 (Trimers Tubules and Sphermentioning

confidence: 99%

Rotavirus VP6 preparations as a non-replicating vaccine candidates

Jalilvand

Marashi

Shoja

2015

Vaccine

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Formation of self-assembled triple-layered rotavirus-like particles (tlRLPs) by constitutive co-expression of VP2, VP6, and VP7 in stably transfected high-five insect cell lines

Cited by 16 publications

References 65 publications

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

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

Coupling Microscopy and Flow Cytometry for a Comprehensive Characterization of Nanoparticle Production in Insect Cells

Rotavirus VP6 preparations as a non-replicating vaccine candidates

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