Bioencapsulation of the hepatitis B surface antigen and its use as an effective oral immunogen

Abstract

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.

Figure 1

Construct design for expression of HBsAg in Zea mays. UBI, Ubiquitin promoter; Glob1, globulin1 promoter; BAASS, barley alpha amylase signal sequence; KDEL, ER targeting sequence; PinII, potato proteinase inhibitor II termination sequence. All constructs also contained an herbicide resistance gene following the PinII termination sequence.

Figure 2

Expression from single seed in the first generation after transformation (T1), as determined by a sandwich ELISA. Each bar represents the highest expressing seed from a given ear. The top ten expressing ears are displayed for each construct. Expression levels are reported as % total soluble protein (%TSP).

Figure 3

Increases in HBsAg %TSP gained from the production of hybrid grain. Expression is reported as the average of all positive single seeds from the ear chosen for subsequent breeding (T1 seed) or as the highest expressing ear (50-seed bulk) from a given generation. T1 seed was used to generate homozygous (hmz) lines in the SP122 and SP114 genetic backgrounds, and these homozygotes were used to produce hybrid seed (from SP122×SP114) and hybrid grain (from selfed hybrid plants).

Figure 4

Fecal anti-HBsAg sIgA levels in mice as determined by a sandwich ELISA. Mean O.D. values were determined for each treatment of 8 mice. Black arrows indicate the initiation of oral boosting.

Figure 5

Serum anti-HBsAg a) IgG and b) IgA response as determined by a sandwich ELISA.. Normalized O.D. values were calculated for each mouse relative to its own value just prior to oral boost (O.D. at weekn/O.D. at week12), and geometric means of normalized values were calculated for each group of 8 mice. White arrows represent primary injection of Recombivax and black arrows indicate oral boosts.

Figure 6

Total change in serum anti-HBsAg Ig in mice fed control versus HBE germ with or without the LT(R192G/L211A) adjuvant. Values were determined using serum Ig levels in week12 (pre-boost) and week19 (post-boost).