Review article

 Influenza vaccine production in plants

Rigi Cheshmehali Garshasb1 – Mousavi Seyyed Amir2

1- PhD student in Biology – Molecular Genetics in National Institute of Genetic Engineering and Biotechnology (NIGEB)- Iran

2-Director, Department of Plant Biotechnology & International Relations Coordinate of National Institute of Genetic Engineering and Biotechnology (NIGEB)- Iran

 

A b s t r a c t

              Plants have been identified as promising expression systems for commercial production of vaccine antigens. Transgenic plants, including edible plant parts are uggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. As an important case the spectre of an influenza pandemic has led to a revision of national and global pandemic preparedness plans and has stressed the need for more efficient influenza vaccines and manufacturing practices. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens against influenza.

 

Keywords: transgenic plants, vaccine antigens, influenza, pandemic.

 

 Introduction

       Infectious diseases account for more than 45% of total deaths in developing countries (9). Vaccination is the most effective means to prevent infectious diseases. More than 30 million children in the world are not immunized against treatable or preventable diseases (www.care.org/campaigns/childrenpowerty/facts.asp) because the currently used approaches to vaccine production are technologically complex and expensive. Specialized requirements of packaging, cold chain and mode of delivery add to the cost.

      Currently used mammalian cell line based vaccine manufacturing requires large investment and expertise. These factors limit their scale up and thus, global availability. Advances in molecular biology techniques during the 1980s, helped in the development of new strategies for the production of subunit vaccines. These comprised of proteins derived from pathogenic viruses, bacteria or parasites. Although mammals, their tissue sand cell lines are currently utilized for commercial production of vaccines, these systems are expensive and their scale up is not easy (9). Toxins, infectious agents and other noxious compounds get carried in animal cell based processes and are often difficult to remove. Such production systems are prone to microbial contamination which sometimes escapes detection even in purified vaccines. Expression of recombinant antigen proteins in E. coli is often not feasible because of lack of a variety of post translational modifications and folding requirements.

          Some of the mammalian-type post translational processing and modifications in protein happen in yeast and insect cell lines. However, immunologically significant differences in the pattern of post translational modifications limit their deployment in the expression of vaccine antigens. As a major alternative, plants are emerging as a promising system to express and manufacture a wide range of functionally active proteins of high value to health industry. Various plant biotechnological techniques, such as, modern breeding methods, clonal propagation , somatic hybridization, protoplast/cell suspension culture, hairy root culture and genetic transformation can play a vital role in establishing the use of plants as “surrogate production organisms”. One or more immunoprotective antigens of pathogens can be produced in plants by the expression of gene(s) encoding the protein(s). In recent years, plant-based novel production systems aimed at developing “edible” or “oral” vaccines have also been discussed(9). Compared to traditional vaccines, edible vaccines offer simplicity of use, lower cost, convenient storage, economic delivery and mucosal immune response. Successful development of vaccine antigens against human and animal pathogen(s) in plants requires selection of one or more immunoprotective antigens and designing of genes and promoters that would express the antigen(s) at a high level in target plant tissue. Genetic transformation methods are then utilized for introducing the gene in the target plant species (figure 1).

This review focuses on applications of plants for vaccine production against Influenza.

 Introducing the Influenza and importance of it’s plant- based vaccine

      Influenza is a highly contagious and acute respiratory disease with a high degree of morbidity and mortality. It is estimated that influenza is responsible for 36,000 deaths and more than 200,000 hospitalizations in the United States annually. Globally, influenza epidemics result in 3–5 million hospitalizations and 300,000–500,000 deaths each year (4). New epidemic strains of influenza A arise due to point mutations within two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (figure 2). These changes in HA and NA enable emerging virus strains to evade the host’s immune system and therefore necessitates the annual revision of vaccine to include the new viruses (3, 4). Influenza type A virus is classified into several subtypes which vary in two of their transmembrane proteins, hemagglutinin (HA)1 and neuraminidase (NA) and possess similar nucleoprotein and matrix protein (3,4). A constant antigenic reassortment in surface-located HA and NA, which are, respectively, responsible for binding of the virus to its cellular receptor and promoting release of the mature virus from infected cells (4), makes vaccine development a complicated process. There are 16 known HA subtypes and nine known NA subtypes of influenza A viruses and many different combinations are possible. Three subtypes, (H1N1, H1N2, and H3N2), commonly circulate among humans and all known subtypes of influenza A viruses can be found in birds (4). Influenza A subtype H5N1 virus, known as avian influenza or bird flu, is endemic mainly in birds and does not usually infect people. However, because of its high virulence, H5N1 may be transmitted from infected birds to humans and cause fatal disease.

 Figure 1 . Steps in the production of plant-derived vaccine antigens (9)

       The recent swine H1N1 influenza pandemic (pH1N1) revealed the limitations of the current influenza vaccine manufacturing technologies. Regardless of the origin, mechanism of emergence or precise genetic makeup of the ‘next’ pandemic strain, recent experience with pH1N1demonstrate clearly that the current, egg-based manufacturing system would not be able to respond quickly enough in the face of a highly pathogenic influenza virus adapted for rapid human-tohuman pread.

     A plant-based manufacturing technology that can produce vaccine doses within one month of

the sequencing of a pandemic strain should be describe.

 Figure 2 . Schematic representation of the structural characteristics of viral particles and plant-made VLPs (virus-like particles) .  A. Cross-section showing internal differences. Hemagglutinin (HA) (green) andneuraminidase (NA) (orange) are the two major viral proteins protruding outside of the viral envelope (pink) , HA being the main antigenic determinant of the virus.  B. Transmission electron microscopy images of influenza viruses and plant-made VLPs (3).

 Influenza vaccines produced in plants

       Evidence of influenza antigen production in plants was only recently disclosed, in 2004, through an international patent application (1). In this application, it is concluded that influenza HA accumulates in calluses of NT1 tobacco cells transformed to express the complete coding sequence of influenza H5 from strain A⁄ turkey ⁄ Wisconsin ⁄ 1968 (H5N9). When extracted without detergent, the apoplastic fluid of the transgenic cells exhibited hemagglutination activity, indicating that plant-produced H5 was secreted and active. Rabbits immunized with a crude preparation of extracellular fluid from transgenic NT-1 cells producing H5 from strain A⁄ turkey ⁄ Wisconsin ⁄ 1968 (H5N9) showed a strong hemagglutination inhibiting (HI) antibody response 6 weeks after immunization in the presence of Freund’s adjuvant. 

      Expression of the HA ectodomain (the segment of the protein spanning outside of the viral envelope) fused to a KDEL peptide, to enhance accumulation through retention in the endoplasmic reticulum, and a poly-histidine purification tag has also been utilized as a mean for facilitating the production of recombinant influenza antigens in plants. Examples include the HA ectodomains from a human seasonal influenza strain (A ⁄ Wyoming ⁄ 03 ⁄ 03 (H3N2) (6) and highly pathogenic avian strains A⁄ Indonesia ⁄ 5 ⁄ 05 (7), A⁄ Bar-headed Goose ⁄ Qinghai ⁄ 1A ⁄ 05 and A⁄ Anhui ⁄ 1 ⁄ 05 (8). In immunogenicity studies in mice, HA ectodomains have been shown to induce significant HI responses when administered in conjunction with Quil A. However, although such composition induced a strong immune response with doses as low as 1 lg (A ⁄ Anhui ⁄ 1 ⁄ 05 (H5N1)) (8), two doses were required to obtain an HI antibody response. A ferret study further showed that three doses of 45 lg adjuvanted with Quil A were required to confer protection against a lethal challenge with the homologous strain of H5 ectodomain (7). Together, the above mentioned immunogenicity studies, performed with HA fragments in fusion with a carrier protein or with other peptides, provided strong indications that plant-made influenza antigens can be produced by agroinfiltration and that these HA fragments induce hemagglutination inhibition antibody response in model animals. However, the high dosage and multiple injections required to induce a protective immune response in ferrets from immunization with the ectodomain of H5 (A ⁄ Indonesia ⁄ 5 ⁄ 05 (H5N1)) suggests that a higher order of antigen organization is required for optimal stimulation of a protective immune response. More recently, attempts at producing the entire H5 protein (from strain A⁄ Vietnam ⁄ 1203 ⁄ 04 (H5N1)) or its HA1 domain by transient or stable transformation of N. benthamiana were reported unsuccessful as they led to only detectable accumulation of the mature H5 or HA1 domain (10). Fragments of 34 or 27 kDa from the HA1 domain, or fusions of the HA1 domain with 26 kDa fragment from a human or a mouse heavy chain constant region were reported to accumulate at higher levels. In an immunogenicity study in mice, two doses of 10 lg of the 34 kDa fragment from the antigenic region of H5 from influenza A⁄ Vietnam ⁄ 1203 ⁄ 04 (H5N1), administered in the presence of alum-CpG as adjuvant, induced high H5 specific antibody titers but failed to induce significant HI antibody titers (10), again highlighting the need for a higher degree of organization of multivalent antigens to stimulate a protective immune response.

       A similar strategy was recently proposed in the international patent application WO2007⁄ 011904 for the presentation of influenza M2e universal epitope onto CPMV particles. Cowpea plants rubbed with RNA1 and chimeric RNA2 encoding a CP-M2e fusion produced chimeric CPMV particles comprising CP-M2e (5). Similarly, expression of a chimeric cucumber mosaic virus (CMV) capsid protein fused to the M2e epitope in N. benthamiana using a potato virus X (PVX) expression vector also led to the production of chimeric CP-M2e. However, assembly of the capsid proteins into chimeric viral particles was not demonstrated in this study (4). Capsid proteins of the PVX expression vector have also been used for the display of H-2Db-restricted epitope from the influenza NP from strain A⁄ PR ⁄ 8 ⁄ 34 (2). In this study, the epitope coding sequence was fused to the CP gene of the PVX vector, creating chimeric PVX particles displaying the NP epitope. An immunogenicity study in mice, designed to evaluate the cellbased immune response, showed that when administered in the presence of incomplete Freund’s adjuvant, 50 lg of chimeric viral particles displaying the NP epitope activated ASNENMETM-specific CD8+ IFN-c secreting cells. Although chimeric viral particles antigen presentation platforms benefit from the display of selected antigenic epitopes in a multivalent fashion, this strategy also bear some intrinsic drawbacks. Only a limited number of antigenic epitopes of small size (less than 25 amino acids) can be displayed using this system. Therefore, it is easier for rapidly evolving viruses like influenza to evade the immune response induced by such vaccines by replacing a few amino acids in the selected immunogenic region. Conformational epitopes may also not fold properly, thereby inducing the production of antibodies that will not recognize the cognate native epitope. Finally, the use of chimeric plant viral particles as presentation devices will require regulatory acceptance in themselves in addition to the regulatory hurdles faced by new manufacturing platform.

 Conclusions and future prospects

 With the world's population at over 6.4 billion (http://esa.un.org) , majority of the poor need affordable technological solutions to health. Protection from viral infections is currently the most difficult area to address through drug development. Progress in plant genetic engineering has opened novel opportunities to use plants as bioreactors for safe and cost effective production of   Influenza vaccine antigens.

      As is clear from several examples cited in this review, the production of recombinant proteins in plant systems has a great potential. Recent developments in this area have significantly increased its utility and enabled various groups to explore the possibility of producing vaccine antigens from a variety of plants, which can be directly or indirectly used to develop commercial processes. Transgenic plants that can produce biologically active proteins or subunit oral vaccines and antibodies have been developed, though the applications of these technologies are at least a decade away.

      The need to establish safety, efficacy and functional equivalence of the vaccine antigens should guide future development and research in plant based preventive and therapeutic technologies.

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