A strain-specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant-based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high-molecular-weight structures. H5-containing structures were purified and examination by transmission electron microscopy confirmed virus-like particle (VLP) assembly. High-performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP-producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 microg of purified influenza H5-VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 microg of H5-VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.
"Medicago, Inc., has also developed their own CPMV vector-based technology which produces virus-like particles (VLPs) carrying influenza virus antigens . These VLPs express a lipid-anchored recombinant HA and are fully protected against lethal viral challenge in both mice and ferrets. "
[Show abstract][Hide abstract] ABSTRACT: Plant made biologics have elicited much attention over recent years for their potential in assisting those in developing countries who have poor access to modern medicine. Additional applications such as the stockpiling of vaccines against pandemic infectious diseases or potential biological warfare agents are also under investigation. Plant virus expression vectors represent a technology that enables high levels of pharmaceutical proteins to be produced in a very short period of time. Recent advances in research and development have brought about the generation of superior virus expression systems which can be readily delivered to the host plant in a manner that is both efficient and cost effective. This review presents recent innovations in plant virus expression systems and their uses for producing biologics from plants.
"However, biosafety issues and difficulties with growth and yield of certain isolates may cause constraints during production. Besides whole virus vaccines, subunit vaccines comprising hemagglutinin (HA) and virus-like particles (VLPs) are considered as alternative strategies and have been shown to successfully induce neutralizing immune response          . "
"Protein assays are frequently described in the literature for the quantification of purified VLPs as illustrated in Table 1. D’Aoust et al. demonstrated 80% purity of plant derived VLP produced purified by size exclusion chromatography . Additionally, protein assays such as the bicinchoninic acid (BCA) assay  or the Quant-IT assay kit with bovine serum albumin (BSA)  were used to quantify the total protein content in purified samples. "
[Show abstract][Hide abstract] ABSTRACT: Influenza virus-like particle vaccines are one of the most promising ways to respond to the threat of future influenza pandemics. VLPs are composed of viral antigens but lack nucleic acids making them non-infectious which limit the risk of recombination with wild-type strains. By taking advantage of the advancements in cell culture technologies, the process from strain identification to manufacturing has the potential to be completed rapidly and easily at large scales. After closely reviewing the current research done on influenza VLPs, it is evident that the development of quantification methods has been consistently overlooked. VLP quantification at all stages of the production process has been left to rely on current influenza quantification methods (i.e. -- Hemagglutination assay (HA), Single Radial Immunodiffusion assay (SRID), NA enzymatic activity assays, Western blot, Electron Microscopy). These are analytical methods developed decades ago for influenza virions and final bulk influenza vaccines. Although these methods are time-consuming and cumbersome they have been sufficient for the characterization of final purified material. Nevertheless, these analytical methods are impractical for in-line process monitoring because VLP concentration in crude samples generally falls out of the range of detection for these methods. This consequently impedes the development of robust influenza-VLP production and purification processes. Thus, development of functional process analytical techniques, applicable at every stage during production, that are compatible with different production platforms is in great need to assess, optimize and exploit the full potential of novel manufacturing platforms.
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