Plant-expressed Hepatitis B core antigen virus-like particles: Characterization and investigation of their stability in simulated and pig gastro-intestinal fluids

Abstract

Virus-like particles (VLPs) are potential oral vaccine candidates, as their highly compact structure may allow them to withstand the harsh conditions of the gastro-intestinal (GI) environment.Hepatitis B core antigen (HBcAg) is an immunogenic protein that assembles into 30 or 34 nm diameter VLPs. Here, the stabilities of both the HBcAg polypeptide itself and the threedimensional structure of the VLPs upon exposure to in vitro and ex vivo simulated gastric and intestinal fluids were investigated. Plant-expressed HBcAg VLPs were efficiently purified by sucrose density gradient and characterized. The purified VLPs did not show major chemical or physical instability upon exposure to the low pH conditions typically found in the stomach; however, they completely agglomerated upon acidification and subsequent pH neutralization. The HBcAg polypeptide was highly digested upon exposure to pepsin in simulated gastric fluids.HBcAg appeared more stable in both simulated and ex vivo intestinal fluids, where despite a partial digestion of the HBcAg polypeptide, the VLPs maintained their most immunogenic epitopes and their particulate conformation. These results suggest that HBcAg VLPs are likely to be unstable in gastric fluids, yet if the gastric instability could be bypassed, they could maintain their particulate structure and immunogenicity in intestinal fluids.

Stability of plant virus-based nanocarriers in gastrointestinal fluids

“…The gradients were centrifuged at 31 200 rpm for 2.5 hours at 4 °C in a TH-641 rotor. 23 Following centrifugation, fractions were collected from the gradients, and analysed by SDS-PAGE and by UV spectrophotometry.…”

“…Therefore, if orally administered, their stability could be also compromised by physical denaturation of their quaternary structure at gastric pH and possible proteolysis by digestive enzymes. 23 …”

Parenteral–Oral Immunization with Plant-Derived HBcAg as a Potential Therapeutic Vaccine against Chronic Hepatitis B

“…Microscopic immunochemistry showed a high intracellular accumulation of the HBcAg CLPs. Such bioencapsulation may subsequently provide additional protection from degradation in the digestive tract, even though HBcAg is relatively stable in the acidic environment of the stomach [24]. Hence, considering both findings, orally administered plant-associated HBcAg passes through the intestine, activating Gut Associated Lymphoid Tissue (GALT), as also described for other antigens [25,26].…”

Virus-like particle vaccines: immunology and formulation for clinical translation

“…Nicotiana benthamiana tobacco are also used as an efficient plant expression system for production of VLP. HBV VLP purified from N. benthamiana are capable of withstanding a simulation of the acidic environment within the stomach [175]; however, the HBcAg polypeptide of HBV VLP was digested when exposed to porcine pepsin. The protection of protein-based VLP from enzymatic digestion is a significant hurdle for oral delivery, despite the promise of this vaccination route.…”