Cloning and expression of virus-like particles (VLPs) in microalgal expression systems

Virus-like particles (VLPs) can be constructed from self-assembled viral capsid proteins. While preserving structural and antigenic similarities to the authentic native viruses, they are non-infectious and non-replicating molecules due to the lack of genetic material. Additionally, the highly repetitive and organised display of epitopes on the VLP surface promotes strong immunogenic responses. Some VLPs can also serve as particulate carriers with linked heterologous epitopes, forming so-called chimeric VLPs. Hepatitis B core antigen (HBcAg) VLPs are one of the most promising and well-documented particulate carriers for heterologous epitope presentation. The formation of HBcAg VLPs has previously been demonstrated in various prokaryotic and eukaryotic hosts. However, the development of chimeric HBcAg VLP-based vaccine candidates will require an inexpensive, safe and robust production platform. The transgenic green alga Chlamydomonas reinhardtii is examined here as an alternative expression system due to its ability to fold complex protein structures, relatively low production costs and the absence of viral toxins or human pathogens giving it GRAS (Generally Recognised as Safe) status. In this work, the codon-optimised nucleotide sequence of the truncated HBcAg monomer (HBc150_HA) was inserted into the C. reinhardtii chloroplast genome and HBc150_HA expression was confirmed by western blot analysis. Subsequently, both bioprocess and genetic engineering strategies were assessed to boost HBc150_HA production. These resulted in a 26-fold increase in HBc150_HA production which accumulated up to 2.34% (w/w) of total soluble protein (TSP) mainly as consequence of using a stronger 16S ribosomal RNA (rRNA) promoter. Based on the densitometric analysis, the yield of HBc150_HA (4.9 mg per g dry cell weight (DCW)) was almost four-fold higher than the yeast-derived tandem-core HBcAg (1.3 mg per g DCW) in which two monomers were genetically fused. Despite low abundancy, isometric particles of 25-30 nm in diameter and with a morphology typical of HBcAg VLPs were observed in transmission electron microscopy (TEM) after partial purification by either sucrose gradient ultracentrifugation or size exclusion chromatography (SEC). The results of immunogold labelling further confirmed the presence of capsid assembly in the HBc150_HA-expressing microalgal samples. This is the first study to show that HBcAg VLPs can be synthesized and correctly assembled in microalgal chloroplasts. It therefore enables further exploration of the potential of C. reinhardtii as a versatile platform for low-cost production of recombinant therapeutic proteins.