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Abstract

Cardiomyopathy syndrome (CMS) is a severe cardiac disease occurring in the grow-out sea phase of farmed Atlantic salmon with approximately 100 outbreaks annually in Norway. Piscine myocarditis virus (PMCV) is believed to be the causative agent of CMS. There is no vaccine available to control CMS, partially because PMCV withstands propagation in known cell cultures. In the present study, we selected the putative capsid protein of PMCV as the candidate antigen for immunization experiments and produced it in the plant Nicotiana benthamiana by transient expression. The recombinant PMCV antigen formed virus-like particles (VLPs). To evaluate the efficacy of the plant made VLP vaccine, a PMCV infection model was established. In an experimental salmon vaccination trial, the VLP vaccine triggered innate immunity, and indicative but not significant inhibition of viral replication in heart, spleen and kidney tissues was observed. Similarly, a reduction of inflammatory lesions in cardiomyocytes and subendocardial infiltration by mononuclear leukocytes were observed. Therefore, there was no difference in efficacy or immune response observed post the plant made PMCV VLP antigen vaccination. Taken together, this study has demonstrated that plant made VLP antigens should be investigated further as a possible platform for the development of PMCV antigens for a CMS vaccine.

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Abstract

Chronic infection with hepatitis C virus (HCV) remains a leading cause of liver-related pathologies and a global health problem, currently affecting more than 71 million people worldwide. The development of a prophylactic vaccine is much needed to complement the effective antiviral treatment available and achieve HCV eradication. Current strategies focus on increasing the immunogenicity of the HCV envelope glycoprotein E2, the major target of virus-neutralizing antibodies, by testing various expression systems or manipulating the protein conformation and the N-glycosylation pattern. Here we report the first evidence of successful production of the full-length HCV E2 glycoprotein in Nicotiana benthamiana, by using the Agrobacterium-mediated transient expression technology. Molecular and functional analysis showed that the viral protein was correctly processed in plant cells and achieved the native folding required for binding to CD81, one of the HCV receptors. N-glycan analysis of HCV-E2 produced in N. benthamiana and mammalian cells indicated host-specific trimming of mannose residues and possibly, protein trafficking. Notably, the plant-derived viral antigen triggered a significant immune response in vaccinated mice, characterized by the presence of antibodies with HCV-neutralizing activity. Together, our study demonstrates that N. benthamiana is a viable alternative to costly mammalian cell cultures for the expression of complex viral antigens and supports the use of plants as cost-effective production platforms for the development of HCV vaccines.

Abstract

Aquaculture has undergone rapid development in the past decades. It provides a large part of high-quality protein food for humans, and thus, a sustainable aquaculture industry is of great importance for the worldwide food supply and economy. Along with the quick expansion of aquaculture, the high fish densities employed in fish farming increase the risks of outbreaks of a variety of aquatic diseases. Such diseases not only cause huge economic losses, but also lead to ecological hazards in terms of pathogen spread to marine ecosystems causing infection of wild fish and polluting the environment. Thus, fish health is essential for the aquaculture industry to be environmentally sustainable and a prerequisite for intensive aquaculture production globally. The wide use of antibiotics and drug residues has caused intensive pollution along with risks for food safety and increasing antimicrobial resistance. Vaccination is the most effective and environmentally friendly approach to battle infectious diseases in aquaculture with minimal ecological impact and is applicable to most species of farmed fish. However, there are only 34 fish vaccines commercially available globally to date, showing the urgent need for further development of fish vaccines to manage fish health and ensure food safety. Plant genetic engineering has been utilized to produce genetically modified crops with desirable characteristics and has also been used for vaccine production, with several advantages including cost-effectiveness, safety when compared with live virus vaccines, and plants being capable of carrying out posttranslational modifications that are similar to naturally occurring systems. So far, plant-derived vaccines, antibodies, and therapeutic proteins have been produced for human and animal health. However, the development of plant-made vaccines for animals, especially fish, is still lagging behind the development of human vaccines. The present review summarizes the development of fish vaccines currently utilized and the suitability of the plant-production platform for fish vaccine and then addresses considerations regarding fish vaccine production in plants. Developing fish vaccines by way of plant biotechnology are significant for the aquaculture industry, fish health management, food safety, and human health.

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Abstract

The demand for animal protein has increased considerably worldwide, especially in China, where large numbers of livestock and poultry are produced. Antibiotics have been widely applied to promote growth and prevent diseases. However, the overuse of antibiotics in animal feed has caused serious environmental and health risks, especially the wide spread of antimicrobial resistance (AMR), which seriously affects animal and human health, food safety, ecosystems, and the sustainable future development of animal protein production. Unfortunately, AMR has already become a worldwide challenge, so international cooperation is becoming more important for combatting it. China’s efforts and determination to restrict antibiotic usage through law enforcement and effective management are of significance. In this review, we address the pollution problems of antibiotics; in particular, the AMR in water, soil, and plants caused by livestock and poultry manure in China. The negative impact of widespread and intensive use of antibiotics in livestock production is discussed. To reduce and mitigate AMR problems, we emphasize in this review the development of antibiotic substitutes for the era of antibiotic prohibition.

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Abstract

To meet increasing demand for animal protein, swine have been raised in large Chinese farms widely, using antibiotics as growth promoter. However, improper use of antibiotics has caused serious environmental and health risks, in particular Antimicrobial resistance (AMR). This paper reviews the consumption of antibiotics in swine production as well as AMR and the development of novel antibiotics or alternatives in China. The estimated application of antibiotics in animal production in China accounted for about 84240 tons in 2013. Overuse and abuse of antibiotics pose a great health risk to people through food-borne antibiotic residues and selection for antibiotic resistance. China unveiled a national plan to tackle antibiotic resistance in August 2016, but more support is needed for the development of new antibiotics or alternatives like plant extracts. Antibiotic resistance has been a major global challenge, so international collaboration between China and Europe is needed.

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Abstract

Sustainable production of biofuels from lignocellulose feedstocks depends on cheap enzymes for degradation of such biomass. Plants offer a safe and cost‐effective production platform for biopharmaceuticals, vaccines and industrial enzymes boosting biomass conversion to biofuels. Production of intact and functional protein is a prerequisite for large‐scale protein production, and extensive host‐specific post‐translational modifications (PTMs) often affect the catalytic properties and stability of recombinant enzymes. Here we investigated the impact of plant PTMs on enzyme performance and stability of the major cellobiohydrolase TrCel7A from Trichoderma reesei, an industrially relevant enzyme. TrCel7A was produced in Nicotiana benthamiana using a vacuum‐based transient expression technology, and this recombinant enzyme (TrCel7Arec) was compared with the native fungal enzyme (TrCel7Anat) in terms of PTMs and catalytic activity on commercial and industrial substrates. We show that the N‐terminal glutamate of TrCel7Arec was correctly processed by N. benthamiana to a pyroglutamate, critical for protein structure, while the linker region of TrCel7Arec was vulnerable to proteolytic digestion during protein production due to the absence of O‐mannosylation in the plant host as compared with the native protein. In general, the purified full‐length TrCel7Arec had 25% lower catalytic activity than TrCel7Anat and impaired substrate‐binding properties, which can be attributed to larger N‐glycans and lack of O‐glycans in TrCel7Arec. All in all, our study reveals that the glycosylation machinery of N. benthamiana needs tailoring to optimize the production of efficient cellulases.

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Abstract

Hepatitis B Virus (HBV) infection can be prevented by vaccination. Vaccines containing the small (S)envelope protein are currently used in universal vaccination programs and achieve protective immuneresponse in more than 90% of recipients. However, new vaccination strategies are necessary for successfulimmunization of the remaining non- or low-responders. We have previously characterized a novel HBVchimeric antigen, which combines neutralization epitopes of the S and the preS1 domain of the large (L)envelope protein (genotype D). The S/preS121–47chimera produced in mammalian cells and Nicotianabenthamiana plants, induced a significantly stronger immune response in parenterally vaccinated micethan the S protein. Here we describe the transient expression of the S/preS121–47antigen in an edibleplant, Lactuca sativa, for potential development of an oral HBV vaccine. Our study shows that oral admin-istration of adjuvant-free Lactuca sativa expressing the S/preS121–47antigen, three times, at 1lg/dose,was sufficient to trigger a humoral immune response in mice. Importantly, the elicited antibodies wereable to neutralize HBV infection in an NTCP-expressing infection system (HepG2-NTCP cell line) moreefficiently than those induced by mice fed on Lactuca sativa expressing the S protein. These results sup-port the S/preS121–47antigen as a promising candidate for future development as an edible HBV vaccine.

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Abstract

Dengue fever is a mosquito (Aedes aegypti) ‐transmitted viral disease that is endemic in more than 125 countries around the world. There are four serotypes of the dengue virus (DENV 1‐4) and a safe and effective dengue vaccine must provide protection against all four serotypes. To date, the first vaccine, Dengvaxia (CYD‐TDV), is available after many decades’ efforts, but only has moderate efficacy. More effective and affordable vaccines are hence required. Plants offer promising vaccine production platforms and food crops offer additional advantages for the production of edible human and animal vaccines, thus eliminating the need for expensive fermentation, purification, cold storage and sterile delivery. Oral vaccines can elicit humoral and cellular immunity via both the mucosal and humoral immune systems. Here, we report the production of tetravalent EDIII antigen (EDIII‐1‐4) in stably transformed lettuce chloroplasts. Transplastomic EDIII‐1‐4‐expressing lettuce lines were obtained and homoplasmy was verified by Southern blot analysis. Expression of EDIII‐1‐4 antigens was demonstrated by immunoblotting, with the EDIII‐1‐4 antigen accumulating to 3.45% of the total protein content. Immunological assays in rabbits showed immunogenicity of EDIII‐1‐4. Our in vitro gastrointestinal digestion analysis revealed that EDIII‐1‐4 antigens are well protected when passing through the oral and gastric digestion phases but underwent degradation during the intestinal phase. Our results demonstrate that lettuce chloroplast engineering is a promising approach for future production of an affordable oral dengue vaccine.

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Abstract

The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases ( e.g . cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV-related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca s ativa ) using Agrobacterium - mediated transient expression technology. The wild-type dimer (E1E2) and a variant without an N-glycosylation site in the E2 polypeptide (E1E2 Δ N6) were expressed, and appropriate N-glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell-expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti-HCV IgM for both antigens; however, the E1E2 Δ N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N-glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti-HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low-cost oral vaccine development.