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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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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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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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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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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.