A plant-based system for rapid production of influenza vaccine antigens

Fraunhofer USA Center for Molecular Biotechnology, Newark, DE 19711, USA.
Influenza and Other Respiratory Viruses (Impact Factor: 1.9). 10/2011; 6(3):204-10. DOI: 10.1111/j.1750-2659.2011.00295.x
Source: PubMed

ABSTRACT Influenza virus is a globally important respiratory pathogen that causes a high degree of annual morbidity and mortality. Significant antigenic drift results in emergence of new, potentially pandemic, virus variants. The best prophylactic option for controlling emerging virus strains is to manufacture and administer pandemic vaccines in sufficient quantities and to do so in a timely manner without impacting the regular seasonal influenza vaccine capacity. Current, egg-based, influenza vaccine production is well established and provides an effective product, but has limited capacity and speed.
To satisfy the additional global demand for emerging influenza vaccines, high-performance cost-effective technologies need to be developed. Plants have a potential as an economic and efficient large-scale production platform for vaccine antigens.
In this study, a plant virus-based transient expression system was used to produce hemagglutinin (HA) proteins from the three vaccine strains used during the 2008-2009 influenza season, A/Brisbane/59/07 (H1N1), A/Brisbane/10/07 (H3N2), and B/Florida/4/06, as well as from the recently emerged novel H1N1 influenza A virus, A/California/04/09.
The recombinant plant-based HA proteins were engineered and produced in Nicotiana benthamiana plants within 2 months of obtaining the genetic sequences specific to each virus strain. These antigens expressed at the rate of 400-1300 mg/kg of fresh leaf tissue, with >70% solubility. Immunization of mice with these HA antigens induced serum anti-HA IgG and hemagglutination inhibition antibody responses at the levels considered protective against these virus infections.
These results demonstrate the feasibility of our transient plant expression system for the rapid production of influenza vaccine antigens.

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Available from: Brian J Green, Jan 21, 2014
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    • "Similar experiments were done with HAs from the strains comprising the 2008–2009 seasonal vaccine (A/Brisbane/10/07, A/Brisbane/59/07 and B/Flori- da/04/06) and California/04/09 (H1N1) (Shoji et al., 2012 "
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    ABSTRACT: Many examples of a successful application of plant-based expression systems for production of biologically active recombinant proteins exist in the literature. These systems can function as inexpensive platforms for the large scale production of recombinant pharmaceuticals or subunit vaccines. Hemagglutinin (HA) is a major surface antigen of the influenza virus, thus it is in the centre of interests of various subunit vaccine engineering programs. Large scale production of recombinant HA in traditional expression systems, such as mammalian or insect cells, besides other limitations, is expensive and time-consuming. These difficulties stimulate an ever-increasing interest in plant-based production of this recombinant protein. Over the last few years many successful cases of HA production in plants, using both transient and stable expression systems have been reported. Various forms of recombinant HA, including monomers, trimers, virus like particles (VLPs) or chimeric proteins containing its fusion with other polypeptides were obtained and shown to maintain a proper antigenicity. Immunizations of animals (mice, ferrets, rabbits or chickens) with some of these plant-derived hemagglutinin variants were performed, and their effectiveness in induction of immunological response and protection against lethal challenge with influenza virus demonstrated. Plant-produced recombinant subunit vaccines and plant-made VLPs were successfully tested in clinical trials (Phase I and II) that confirmed their tolerance and immunogenicity.
    Acta biochimica Polonica 09/2014; · 1.39 Impact Factor
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    • "It has been found to be extremely versatile for genetic manipulation and tissue culture research (Ganapathi et al., 2004). Several recombinant proteins have been expressed in transgenic tobacco as bioreactors for the production of commercially important pharmaceutical molecules (Shoji et al., 2012). Many immunogenic proteins have been synthesised in tobacco and several plant-based vaccine candidates have demonstrated efficacy against a large number of human and animal pathogens after oral administration (Daniell et al., 2001). "
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    ABSTRACT: Plants engineered with genes encoding for the antigenic proteins of various microorgan-isms have shown to correctly express the pro-teins that elicit the production of antibodies in mammalian hosts. In livestock, plant-based vaccines could represent an innovative strate-gy for oral vaccination, especially to prevent infection by enteric pathogens. The aim of this study was to evaluate tobacco plants as a seed-specific expression system for the production of the flgK flagellar hook-associated protein from a wild type Salmonella typhimurium strain, as a model of an edible vaccine. The flgK gene is the principal component of bacte-rial flagella and is recognised as virulence fac-tor by the innate immune system. It was isolat-ed from the Salmonella typhimurium strain by PCR. The encoding sequence of flgK was trans-ferred into a pBI binary vector, under control of soybean basic 7S globulin promoter for the seed-specific. Plant transformation was car-ried out using recombinant EHA 105 Agrobacterium tumefaciens. A transgenic popu-lation was obtained made up of independently kanamycin-resistant transgenic plants, which had a similar morphological appearance to the wild-type plants. Molecular analyses of seeds confirmed the integration of the gene and the average expression level of flgK was estimated to be about 0.6 mg per gram of seeds, corre-sponding to 0.33% of the total amount of solu-ble protein in tobacco seeds. This study showed that the foreign flgK gene could be sta-bly incorporated into the tobacco plant genome by transcription through the nuclear apparatus of the plant, and that these genes are inherited by the next generation.
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    ABSTRACT: Antibodies directed against the influenza hemagglutinin (HA) protein largely mediate virus neutralization and confer protection against infection. Consequently, many studies and assays of influenza vaccines are focused on HA-specific immune responses. Recombinant HA (rHA) proteins can be produced in a number of protein expression and cell culture systems. These range from baculovirus infection of insect cell cultures, to transient transfection of plants, to stably transfected human cell lines. Furthermore, the rHA proteins may contain genetic modifications, such as histidine tags or trimerization domains, intended to ease purification or enhance protein stability. However, no systematic study of these different forms of the HA protein have been conducted. It is not clear which, if any, of these different protein expression systems or structural modifications improve or diminish the biological behavior of the proteins as immunogens or antigens in immune assays. Therefore we set out to perform systematic evaluation of rHA produced in different proteins expression systems and with varied modifications. Five rHA proteins based on recent strains of seasonal influenza A and five based on influenza B HA were kindly provided by the Biodefense and Emerging Infections Reagent Repository (BEIR). These proteins were evaluated in a combination of biochemical and structural assays, in vitro humoral and cellular immune assays, and in an animal vaccination model. Marked differences in the behavior of the individual proteins was evident suggesting that they are not equal when being used to detect an immune response. They were, nevertheless, similar at eliciting neutralizing antibody responses.
    Vaccine 05/2012; 30(31):4606-16. DOI:10.1016/j.vaccine.2012.05.005 · 3.49 Impact Factor
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