Hepatitis B remains a major global health problem despite the availability of a safe and effective vaccine. Segments of the population lack access to or respond poorly to the parenteral vaccine, perpetuating the infection-transmission cycle. A low cost, orally-delivered vaccine has the potential to alleviate many of these problems. Here we describe the expression of a bioencapsulated hepatitis B surface antigen (HBsAg) in maize and its immunogenicity, demonstrating for the first time a commercially feasible oral subunit vaccine production system for a major disease. This work surmounts previous barriers to plant-produced vaccines by expressing HBsAg at much higher levels and retaining antigen immunogenicity post-processing: factors which facilitated a robust immune response in mice without the need for an adjuvant. This method provides a practical solution to the delivery of a low-cost, stable oral vaccine.
Summary
Using plants as biofactories for industrial enzymes is a developing technology. The application of this technology to plant biomass conversion for biofuels and biobased products has potential for significantly lowering the cost of these products because of lower enzyme production costs. However, the concentration of the enzymes in plant tissue must be high to realize this goal. We describe the enhancement of the accumulation of cellulases in transgenic maize seed as a part of the process to lower the cost of these dominant enzymes for the bioconversion process. We have used breeding to move these genes into elite and high oil germplasm to enhance protein accumulation in grain. We have also explored processing of the grain to isolate the germ, which preferentially contains the enzymes, to further enhance recovery of enzyme on a dry weight basis of raw materials. The enzymes are active on microcrystalline cellulose to release glucose and cellobiose.
Porcine Epidemic Diarrhea Virus (PEDV) causes severe diarrhea and mortality in piglets. Robust immunity may break the transmission cycle. Expression of antigens in maize grains is a promising method for producing low-cost vaccines. As a first step, we expressed maize constructs containing PEDV S1 spike protein targeted to various cellular locations including the cell wall, endoplasmic reticulum, and vacuole, and fused to carrier proteins E. coli heat labile subunit (LTB) and a dendritic cell (DC) binding peptide, and obtained sufficient antigen for oral immunization. Constructs targeting S1 to the ER or fused to carrier proteins produced high levels of antigen of greater than 20 mg/kg. Oral administration to pigs elicited serum neutralizing antibodies, supporting oral immunization as a practical and cost-effective PEDV vaccine.
Key MessageTransgenic expression of spike protein antigen S1 from Porcine Epidemic Diarrhea Virus in maize accumulated sufficient antigen for oral immunization and elicited serum neutralizing antibodies in pigs.
a b s t r a c tEfficient hydrolysis of cellulosic biomass into free sugars for the production of bioethanol would allow utilization of an abundant and renewable natural resource. However, complex microcrystalline cellulose in biomass resists digestion. A preferred method to deconstruct cellulose is using enzyme cocktails, but the presence of multiple enzymes with synergistic outcomes for this process complicates direct biochemical analysis as a reliable indicator of deconstruction. Methods to detect release of free sugars rather than direct measurement of enzymatic activity are available but are time-consuming, and do not reflect the production of metabolic inhibitors of microbial growth which may interfere with the subsequent fermentation of sugars to ethanol. The work presented here describes an automated method for detecting the release of free sugars from enzymatic digestion of cellulosic biomass that allows for realtime measurements during the enzyme treatments and provides an indication of metabolic inhibitors that can interfere with subsequent growth of microbes during fermentation.
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