The "humanized" N-glycosylation pathway in CRISPR/Cas9-edited Nicotiana benthamiana significantly enhances the immunogenicity of a S/preS1 Hepatitis B Virus antigen and the virus-neutralizing antibody response in vaccinated mice.

The recent SARS-CoV-2 pandemic has taught the world a costly lesson about the devastating consequences of viral disease outbreaks but also, the remarkable impact of vaccination in limiting life and economic losses. Vaccination against human Hepatitis B Virus (HBV), a major human pathogen affecting 290 million people worldwide, remains a key action towards viral hepatitis elimination by 2030. To meet this goal, the development of improved HBV antigens is critical to overcome non-responsiveness to standard vaccines based on the yeast-produced, small (S) envelope protein. We have recently shown that combining relevant immunogenic determinants of S and large (L) HBV proteins in chimeric antigens markedly enhances the anti-HBV immune response. However, the demand for cost-efficient, high-quality antigens remains challenging. This issue could be addressed by using plants as versatile and rapidly scalable protein production platforms. Moreover, the recent generation of plants lacking ß-1,2-xylosyltransferase and α-1,3-fucosyltransferase activities (FX-KO), by CRISPR/Cas9 genome editing, enables production of proteins with "humanized" N-glycosylation. In this study, we investigated the impact of plant N-glycosylation on the immunogenic properties of a chimeric HBV S/L vaccine candidate produced in wild-type and FX-KO Nicotiana benthamiana. Prevention of ß-1,2-xylose and α-1,3-fucose attachment to the HBV antigen significantly increased the immune response in mice, as compared with the wild-type plant-produced counterpart. Notably, the antibodies triggered by the FX-KO-made antigen neutralized more efficiently both wild-type HBV and a clinically relevant vaccine escape mutant. Our study validates in premiere the glyco-engineered Nicotiana benthamiana as a substantially improved host for plant production of glycoprotein vaccines.

Hepatitis C virus E2 envelope glycoprotein produced in Nicotiana benthamiana triggers humoral response with virus-neutralizing activity in vaccinated mice.

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.

Improving the production of AHL lactonase AiiO-AIO6 from Ochrobactrum sp. M231 in intracellular protease-deficient Bacillus subtilis.

Quorum quenching (QQ) blocks bacterial cell-to-cell communication (i.e., quorum sensing), and is a promising antipathogenic strategy to control bacterial infection via inhibition of virulence factor expression and biofilm formation. QQ enzyme AiiO-AIO6 from Ochrobactrum sp. M231 has several excellent properties and shows biotherapeutic potential against important bacterial pathogens of aquatic species. AiiO-AIO6 can be secretory expressed in Bacillus subtilis via a non-classical secretion pathway. To improve AiiO-AIO6 production, four intracellular protease-deletion mutants of B. subtilis 1A751 were constructed by individually knocking out the intracellular protease-encoding genes (tepA, ymfH, yrrN and ywpE). The AiiO-AIO6 expression plasmid pWB-AIO6BS was transformed into the B. subtilis 1A751 and its four intracellular protease-deletion derivatives. Results showed that all recombinant intracellular protease-deletion derivatives (BSΔtepA, BSΔymfH, BSΔyrrN and BSΔywpE) had a positive impact on AiiO-AIO6 production. The highest amount of AiiO-AIO6 extracellular production of BSΔywpE in shake flask reached 1416.47 U/mL/OD600, which was about 121% higher than that of the wild-type strain. Furthermore, LC-MS/MS analysis of the degrading products of 3-oxo-C8-HSL by purification of AiiO-AIO6 indicated that AiiO-AIO6 was an AHL-lactonase which hydrolyzes the lactone ring of AHLs. Phylogenetic analysis showed that AiiO-AIO6 was classified as a member of the α/ß hydrolase family with a conserved "nucleophile-acid-histidine" catalytic triad. In summary, this study showed that intracellular proteases were responsible for the reduced yields of heterologous proteins and provided an efficient strategy to enhance the extracellular production of AHL lactonase AiiO-AIO6.

In-depth characterization of Trichoderma reesei cellobiohydrolase TrCel7A produced in Nicotiana benthamiana reveals limitations of cellulase production in plants by host-specific post-translational modifications.

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.

Antibiotic Application and Resistance in Swine Production in China: Current Situation and Future Perspectives.

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.

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development.

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 humoural 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.

Transcriptome and metabolome analyses provide insights into root and root-released organic anion responses to phosphorus deficiency in oat.

Roots and root-released organic anions play important roles in uptake of phosphorus (P), an essential macronutrient for food production. Oat, ranking sixth in the world's cereal production, contains valuable nutritional compounds and can withstand poor soil conditions. Our aim was to investigate root transcriptional and metabolic responses of oat grown under P-deficient and P-sufficient conditions. We conducted a hydroponic experiment and measured root morphology and organic anion exudation, and analysed changes in the transcriptome and metabolome. Oat roots showed enhanced citrate and malate exudation after 4 weeks of P deficiency. After 10 d of P deficiency, we identified 9371 differentially expressed transcripts with a 2-fold or greater change (P<0.05): 48 sequences predicted to be involved in organic anion biosynthesis and efflux were consistently up-regulated; 24 up-regulated transcripts in oat were also found to be up-regulated upon P starvation in rice and wheat under similar conditions. Phosphorylated metabolites (i.e. glucose-6-phosphate, myo-inositol phosphate) were reduced dramatically, while citrate and malate, some sugars and amino acids increased slightly in P-deficient oat roots. Our data are consistent with a strategy of increased organic anion efflux and a shift in primary metabolism in response to P deficiency in oat.