Assembly and Characterization of HBc Derived Virus-like Particles with Magnetic Core

Core-virus like particles (VLPs) assembly is a kinetically complex cascade of interactions between viral proteins, nanoparticle’s surface and an ionic environment. Despite many in silico simulations regarding this process, there is still a lack of experimental data. The main goal of this study was to investigate the capsid protein of hepatitis B virus (HBc) assembly into virus-like particles with superparamagnetic iron oxide nanoparticles (SPIONs) as a magnetic core in relation to their characteristics. The native form of HBc was obtained via agroinfection of Nicotiana benthamiana with pEAQ-HBc plasmid. SPIONs of diameter of 15 nm were synthesized and functionalized with two ligands, providing variety in ζ-potential and hydrodynamic diameter. The antigenic potential of the assembled core-VLPs was assessed with enzyme-linked immunosorbent assay (ELISA). Morphology of SPIONs and core-VLPs was evaluated via transmission electron microscopy (TEM). The most successful core-VLPs assembly was obtained for SPIONs functionalized with dihexadecyl phosphate (DHP) at SPIONs/HBc ratio of 0.2/0.05 mg/mL. ELISA results indicate significant decrease of antigenicity concomitant with core-VLPs assembly. In summary, this study provides an experimental assessment of the crucial parameters guiding SPION-HBc VLPs assembly and evaluates the antigenicity of the obtained structures

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

Chronic hepatitis B (CHB) is the cause of severe liver damage, cirrhosis, and hepatocellular carcinoma for over 240 million people worldwide. Nowadays, several types of treatment are being investigated, including immunotherapy using hepatitis B core antigen (HBcAg) assembled into highly immunogenic capsid-like particles (CLPs). Immunogenicity of plant-produced and purified HBcAg, administered parenterally or intranasally, was previously reported. In this study, a novel parenteral–oral vaccination scheme is proposed using plant-derived HBcAg preparations. The antigen for injection was obtained via transient expression in Nicotiana benthamiana. HBcAg-producing transgenic lettuce was lyophilized and used as an orally delivered booster. The intracellular location of plant-produced HBcAg CLPs implies additional protection in the digestive tract during oral immunization. BALB/c mice were intramuscularly primed with 10 µg of the purified antigen and orally boosted twice with 5 or 200 ng of HBcAg. A long-lasting and significant systemic response after boosting with 200 ng HBcAg was induced, with anti-HBc titer of 25,000. Concomitantly, an insignificant mucosal response was observed, with an S-IgA titer of only 500. The profile of IgG isotypes indicates a predominant Th1 type of immune response, supplemented by Th2, after injection–oral vaccination. The results demonstrate that a low dose of parenteral–oral immunization with plant-derived HBcAg can elicit a specific and efficient response. This study presents a potential new pathway of CHB treatment