Plant-derived pharmaceuticals--the road forward

Plant-derived pharmaceuticals are poised to become the next major commercial development in biotechnology. The advantages they offer in terms of production scale and economy, product safety, ease of storage and distribution cannot be matched by any current commercial system; they also provide the most promising opportunity to supply low-cost drugs and vaccines to the developing world. However, despite the promised benefits, the commercialization of plant-derived pharmaceutical products is overshadowed by the uncertain regulatory terrain, particularly with regard to the adaptation of good manufacturing practice regulations to field-grown plants. The success of such products also depends on careful negotiation of the intellectual property landscape, particularly the achievement of freedom-to-operate licenses for use in developing countries.     

Vaccine manufacturing: challenges and solutions

The recent influenza vaccine shortages have provided a timely reminder of the tenuous nature of the world's vaccine supply and the potential for manufacturing issues to severely disrupt vital access to important vaccines. The application of new technologies to the discovery, assessment, development and production of vaccines has the potential to prevent such occurrences and enable the introduction of new vaccines. Gene-based vaccines, virus-like particles, plant-derived vaccines and novel adjuvants and delivery systems represent promising approaches to creating safer, more potent vaccines. As a consequence, more people will have faster access to more effective vaccines against a broader spectrum of infectious diseases. However, the increased cost of producing new vaccines and regulatory uncertainty remain challenges for vaccine manufacturers.     

Inducible expression of Norwalk virus capsid protein gene in plant cell suspension cultures

Plant cell suspension cultures can be used to make safe vaccines at a lower cost than conventional procedures. An inducible gene expression system provides an opportunity to optimize the conditions of vaccine production in a plant system. In this investigation, a dexamethasone-inducible Norwalk virus capsid protein (NVCP) gene expression system has been developed in cell suspension cultures for four different plant species: tobacco (Nicotiana tabacum), rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and slash pine (Pinus elliottii Engelm.) via Agrobacterium-mediated transformation. Resulting transgenic cell lines were confirmed by Southern blot analyses and NVCP gene expression was confirmed by Northern blot analysis. NVCP gene expression was observed in all 24 cell lines tested, but there were minor differences in transgene expression among the transgenic cell lines. The highest level of NVCP gene expression was observed 48 h after addition of the glucocorticoid hormone dexamethasone (10 mg/l), for all transgenic cell lines derived from four different plant species. This investigation demonstrated that expression of NVCP in different transgenic cell lines and in different species was tightly controlled by the inducer, and the inducible gene expression system could be useful in controlling expression of NVCP or similar proteins for production of vaccines in cultured plant cells.     

Plants as bioreactors for the production of vaccine antigens

Plants have been identified as promising expression systems for commercial production of vaccine antigens. In phase I clinical trials several plant-derived vaccine antigens have been found to be safe and induce sufficiently high immune response. Thus, transgenic plants, including edible plant parts are suggested as excellent alternatives for the production of vaccines and economic scale-up through cultivation. Improved understanding of plant molecular biology and consequent refinement in the genetic engineering techniques have led to designing approaches for high level expression of vaccine antigens in plants. During the last decade, several efficient plant-based expression systems have been examined and more than 100 recombinant proteins including plant-derived vaccine antigens have been expressed in different plant tissues. Estimates suggest that it may become possible to obtain antigen sufficient for vaccinating millions of individuals from one acre crop by expressing the antigen in seeds of an edible legume, like peanut or soybean. In the near future, a plethora of protein products, developed through ‘naturalized bioreactors’ may reach market. Efforts for further improvements in these technologies need to be directed mainly towards validation and applicability of plant-based standardized mucosal and edible vaccines, regulatory pharmacology, formulations and the development of commercially viable GLP protocols. This article reviews the current status of developments in the area of use of plants for the development of vaccine antigens.     

HPV‐16 targeted DNA vaccine expression: The role of purification

DNA vaccines have come to light in the last decades as an alternative method to prevent many infectious diseases, but they can also be used for the treatment of specific diseases, such as cervical cancer caused by Human Papillomavirus (HPV). This virus produces E6 and E7 oncoproteins, which alter the cell cycle regulation and can interfere with the DNA repairing system. These features can ultimately lead to the progression of cervical cancer, after cell infection by HPV. Thus, the development of a DNA vaccine targeting both proteins arises as an interesting option in the treatment of this pathology. Nonetheless, before evaluating its therapeutic potential, the purity levels of a biopharmaceutical must meet the regulatory agency specifications. Previously, our research group successfully purified the supercoiled isoform of the recombinant HPV‐16 E6/E7 DNA vaccine with virtual 100% purity by affinity chromatography. The present work was designed to evaluate the effect that pDNA sample purity levels may exert in the expression of a target protein. Thus, in vitro studies were performed to assess the vaccine ability to produce the target proteins and to compare the expression efficiency between the pDNA sample obtained by affinity chromatography, which only presents the sc isoform and fulfils the regulatory agency recommendations, and the same DNA vaccine retrieved by a commercial purification kit, which contains different pDNA isoforms. Our achievements suggest that the E6/E7 DNA vaccine purified by affinity chromatography promotes higher E6 and E7 mRNA and protein expression levels than the DNA vaccine purified with the commercial kit. Overall, these results underline the importance that a purification strategy may present in the therapeutic outcome of recombinant DNA vaccines, envisaging their further application as biopharmaceuticals. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:546–551, 2018     

The quality standardization of a national vaccine against yellow fever

One of the commercial lots of yellow fever vaccine has been attested as the National Branch Standard (NBS) of yellow fever vaccine. This NBS has been studied in all tests required by the regulations for standard vaccines. The NBS has been found to meet the necessary requirements in all its characteristics. The study of the thermostability of the NBS at temperatures of 4-10 degrees C, 20-22 degrees C, 37 degrees C during storage for 24 hours to 1 year has revealed the rapid loss of the infectious capacity of the virus at the above temperatures and its high stability during storage at -20 degrees C. Thus, the NBS has been found to retain the required level of immunizing potency for 3 months at a temperature of 4-10 degrees C, for 1 month at 20-22 degrees C and for 2 weeks (the term of observation) at 37 degrees C. The heat resistance of the NBS of yellow fever vaccine corresponds to the WHO requirements. The newly developed NBS has been used as the standard preparation for controlling 27 lots of commercial yellow fever vaccine.     

Improving Quality Control of Contagious Caprine Pleuropneumonia Vaccine with Tandem Mass Spectrometry

Vaccines to protect livestock against contagious caprine pleuropneumonia (CCPP) consist of inactivated, adjuvanted antigens. Quality control of these vaccines is challenging as total protein quantification provides no indication of protein identity or purity, and culture is not an option. Here, a tandem mass spectrometry approach is used to identify the mycoplasma antigen contained in reference samples and in commercial CCPP vaccines. By the same approach, the relative amounts of mycoplasma antigen and residual proteins originating from the production medium are determined. Mass spectrometry allows easy and rapid identification of the peptides present in the vaccine samples. Alongside the most probable mycoplasma species effectively present in the vaccines, a very high proportion of peptides from medium constituents are detected in the commercial vaccines tested.     

The purification and characterization of an acellular pertussis vaccine

An acellular pertussis vaccine manufactured by Biken was investigated for purity, potency and toxicity. The vaccine was composed of almost equal proportions of pertussis toxin (PT) and filamentous hemagglutinin (FHA). The purity of the vaccine was 97-99%. The protective effects of component vaccines containing various ratios of PT and FHA were tested and it was found that the ratio of 1:1 provided the most effective vaccine.     

High performance anion exchange chromatography purification of probiotic bacterial extracellular vesicles enhances purity and anti-inflammatory efficacy

Bacterial extracellular vesicles (BEVs), including outer membrane vesicles, have emerged as a promising new class of vaccines and therapeutics to treat cancer and inflammatory diseases, among other applications. However, clinical translation of BEVs is hindered by a current lack of scalable and efficient purification methods. Here, we address downstream BEV biomanufacturing limitations by developing a method for orthogonal size- and charge-based BEV enrichment using tangential flow filtration (TFF) in tandem with high performance anion exchange chromatography (HPAEC). The data show that size-based separation coisolated protein contaminants, whereas size-based TFF with charged-based HPAEC dramatically improved purity of BEVs produced by probiotic Gram-negative Escherichia coli and Gram-positive lactic acid bacteria (LAB). Escherichia coli BEV purity was quantified using established biochemical markers while improved LAB BEV purity was assessed via observed potentiation of anti-inflammatory bioactivity. Overall, this work establishes orthogonal TFF + HPAEC as a scalable and efficient method for BEV purification that holds promise for future large-scale biomanufacturing of therapeutic BEV products.     

A review of the effectiveness of vaccine potency control testing

The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.     

Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants

The use of genetically modified (GM) plants to synthesize proteins that are subsequently processed, regulated and sold as pharmaceuticals challenges two very different established regulatory frameworks, one concerning GM plants and the other covering the development of biotechnology-derived drugs. Within these regulatory systems, specific regulations and guidelines for plant-made pharmaceuticals (PMPs) - also referred to as plant-derived pharmaceuticals (PDPs) - are still evolving. The products nearing commercial viability will ultimately help to road test and fine-tune these regulations, and might help to reduce regulatory uncertainties. In this review, we summarize the current state of regulations in different countries, discuss recent changes and highlight the need for further regulatory development in this burgeoning, new industry. We also make the case for the harmonization of international regulations.     

The development and use of vaccine adjuvants

Interest in vaccine adjuvants is intense and growing, because many of the new subunit vaccine candidates lack sufficient immunogenicity to be clinically useful. In this review, I have emphasized modern vaccine adjuvants injected parenterally, or administered orally, intranasally, or transcutaneously with licensed or experimental vaccines in humans. Every adjuvant has a complex and often multi-factorial immunological mechanism, usually poorly understood in vivo. Many determinants of adjuvanticity exist, and each adjuvanted vaccine is unique. Adjuvant safety is critical and can enhance, retard, or stop development of an adjuvanted vaccine. The choice of an adjuvant often depends upon expensive experimental trial and error, upon cost, and upon commercial availability. Extensive regulatory and administrative support is required to conduct clinical trials of adjuvanted vaccines. Finally, comparative adjuvant trials where one antigen is formulated with different adjuvants and administered by a common protocol to animals and humans can accelerate vaccine development.     

Comparision of a serological potency assay for furunculosis vaccines (Aeromonas salmonicida subsp. salmonicida) to intraperitoneal challenge in Atlantic salmon (Salmo salar L.)

Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed.The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination.There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge.This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines.     

A banana or a syringe: journey to edible vaccines

Constant emergence of diseases, along with the expanding size of world population creates demands for newer vaccines which can meet the challenges that conventional vaccines have not been able to overcome. The application of transgenic plants in the production of pharmaceuticals has led to the new approach of plant-based, orally-delivered vaccines. In recent years a number of recombinant vaccine antigens have been expressed in different plant tissues. The review highlights the generation of edible vaccines, their mode of action and their clinical application in various human diseases. Though the road ahead seems promising, there are several constraints which restrict the success and public acceptability of these vaccines. These include problems of choice of plants, storage, delivery, dosage, safety, public perception, quality control and licensing.     

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

Gene therapy is a promising technology for the treatment of several acquired and inherited diseases. However, for gene therapy to be a commercial and clinical success, scalable cell culture processes must be in place to produce the required amount of viral vectors to meet market demand. Each type of vector has its own distinct characteristics and consequently its own challenges for production. This article reviews the current technology that has been developed for the efficient, large-scale manufacture of retrovirus, lentivirus, adenovirus, adeno-associated virus and herpes simplex virus vectors.     

Regulation and standardization of IPV and IPV combination vaccines.

Inactivated poliovirus vaccine (IPV) is not only increasingly used on a global basis but also is a component of many combination vaccines. Standardization and control of IPV continues to be a challenge for manufacturers and regulators. A rat immunogenicity assay is currently recommended by many authorities, including WHO, as the definitive in vivo potency. Alternative in vitro assays to determine D-antigen content have been developed and are routinely used in some countries to assess IPV potency assay. However, the other less reliable in vivo immunogenicity assays are also used (e.g. monkey, chick). We review some of the regulatory challenges facing current and future IPV assessment, with a focus on the relevance of in vivo and in vitro tests, considerations for Sabin derived IPV and discussion of future efforts for standardization.     

Plant cell factories and mucosal vaccines

Many advances continue to be made in the field of plant-derived vaccines. Plants have been shown capable of expressing a multicomponent vaccine that when orally delivered induces a T-helper cell subset 1 response and enables passive immunization. Furthermore, a plant-derived vaccine has been shown to protect against challenge in the target host. Increased antigen expression levels (up to 4.1% total soluble protein) have been obtained through transformation of the chloroplast genome. In view of these findings, plant-derived vaccines have been proved as valuable commodities to the world's health system; however, before their application, studies need to focus on optimization of immunization strategies and to investigate antigen stability.     

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.     

Comparative efficacy of live replicating sheeppox vaccine strains in Ovines

In the present study, two sheeppox vaccines made from strains [sheeppox virus-Srinagar (SPPV-Srin) and Ranipet (SPPV-R)] indigenous to India and adapted to Vero cells were compared in terms of their safety, potency, efficacy and antigenic value with the commercial in-use Roumanian Fanar (SPPV-RF) vaccine, a foreign strain adapted in primary lamb testes cells. The safety test indicated that the SPPV (Sri and RF) vaccines were safe while SPPV-R was not completely attenuated and caused excessive adverse reactions at the passage level tested. The immunized animals showed DTH reaction and resisted virulent SPPV challenge, while control animals developed disease. Specific virus could be detected in the controls and animals immunized with lower dilutions of vaccines after challenge but not in any of the sheep immunized with 1 and 100 doses of each vaccine. All vaccines were found potent and the PD₅₀ was highest for SPPV (Srin and R) followed by RF. The immunized animals were seroconverted following vaccination with sustained antibody responses after challenge. In conclusion, indigenous SPPV-Srin vaccine was found to be as efficacious as SPPV-R and SPPV-RF vaccines. Thus, there is potential benefit in replacing the currently used commercial vaccine SPPV-RF with indigenous SPPV-Srin vaccine for use in India.     

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.