The assembly of hepatitis B virus core particles in transgenic tobacco, carrot and rice plants

The spread amongst humans of viral diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis and severe acute respiratory syndrome (SARS) is alarming. A plant-based high fidelity production system is being developed with emphasis on producing antigens capable of being orally delivered to humans in plant packets. To test whether transgenic tobacco, carrot and rice plants can correctly process and assemble the hepatitis-B virus (HBV) core particle/antigen (HBcAg), they were transformed with a C-terminal truncated version of the HBcAg subunit coding sequence. Transgenic tobacco, carrot and rice plants processed the HBV subunits accurately indicating that these recombinant expression systems can be extended to produce other proteins at reduced costs.;Western blotting analysis showed the presence of the HBcAg in transgenic tobacco, carrot and rice plants. The HBcAg levels increased from the H1 to the H4 transgenic tobacco lines. Plant codon-optimization of the HBcAg sequence and addition of the KDEL encoded sequence led to higher levels of HBcAg. The most effective modification was observed when the extensin signal sequence replaced the AMV-RNA4 translation enhancer sequence resulting in the highest observed yields of HBcAg in both the leaves and seeds of the best H4 tobacco plant. In edible plants, higher levels of HBcAg were observed in carrot roots as opposed to carrot leaves and in rice seeds as opposed to rice leaves. Further analyses via electron microscopy indicated that the HBV subunits had assembled into virus-like particles of 25--30 nm diameter in all three plant systems. Therefore, these studies may aid in the global quest to develop cheap, safe and effective vaccines.;In the wild-type expression construct (H1); the enhanced cauliflower mosaic virus double 35S (CaMV-d35S) promoter was fused to the alfalfa mosaic virus RNA 4 (AMV-RNA4) sequence to achieve greater translation of a C-terminal truncated HBV core particle subunit. A second expression construct (H2) was plant-codon optimized to match the Arabidopsis thaliana plant genome codon preferences. A third codon-optimized expression construct (H3) had a KDEL (lysyl-aspartyl-glutamyl-leucine) encoded sequence. While a fourth expression construct (H4) included an extensin signal sequence in place of the AMV-RNA4 sequence.