Expression and Purification of Porcine Rotavirus Structural Proteins in Silkworm Larvae as a Vaccine Candidate

Abstract

In this study, silkworm larvae were used for expression of porcine rotavirus A (KS14 strain) inner capsid protein, VP6, and outer capsid protein, VP7. Initially, VP6 was fused with Strep-tag II and FLAG-tag (T-VP6), and T-VP6 was fused further with the signal peptide of Bombyx mori 30k6G protein (30k-T-VP6). T-VP6 and 30 k-T-VP6 were then expressed in the fat body and hemolymph of silkworm larvae, respectively, with respective amounts of 330 μg and 50 μg per larva of purified protein. Unlike T-VP6, 30k-T-VP6 was N -glycosylated due to attached signal peptide. Also, VP7 was fused with PA-tag (VP7-PA). Additionally, VP7 was fused with Strep-tag II, FLAG-tag, and the signal peptide of Bombyx mori 30k6G protein (30k-T-ΔVP7). Both VP7-PA and 30k-T-ΔVP7 were expressed in the hemolymph of silkworm larvae, with respective amounts of 26 μg and 49 μg per larva of purified protein, respectively. The results from our study demonstrated that T-VP6 formed nanoparticles of greater diameter compared with the ones formed by 30k-T-VP6. Also, higher amount of VP6 expressed in silkworm larvae reveal that VP6 holds the potential for its use in vaccine development against porcine rotavirus with silkworm larvae as a promising host for the production of such multi-subunit vaccines.

Display of multiple proteins on engineered canine parvovirus-like particles expressed in cultured silkworm cells and silkworm larvae

“…However, there was a slight decrease in final yields for two loop variants (∼2.3 mg for SpTL2, ∼1.1 mg for SpTLx), possibly due to the relatively higher loss during Strep-column purification. To confirm whether the purified VP2 proteins could form VLPs correctly and efficiently, we conducted gentle purification of formed VLP from purified VP2 monomers where a standard 20% sucrose cushion was employed ( Huhti et al, 2010 ; Kato et al, 2022 ). After ultracentrifuge at 122,000 × g , the fractions from the supernatant (monomers and nonVLPs) and precipitation (VLPs) were loaded to SDS-PAGE.…”

“…Other VLPs of interest already developed in BEVS could also take advantage of our strategy for plug-and-displayable VLP purposes. It would be exciting for those needing more than one structural protein to assemble VLPs correctly, either non-enveloped or enveloped viruses, such as Rotavirus and Rous sarcoma virus ( Minkner and Park, 2018 ; Kato et al, 2022 ). As shown in Figure 4 , we also successfully purified EGFP-displayed VLPs from fusion tag-free and the SpT/SnT double-tagged VP2 variant.…”

Equine Rotavirus A under the One Health Lens: Potential Impacts on Public Health

“…Thus, alternatives to generate safer and more efficacious vaccines than current live-attenuated vaccines are critically needed. Experimental vaccines against porcine RVA have not been widely developed and tested and have been limited to subunit and virus-like particle approaches with limited success [ 232 , 233 , 234 , 235 ]. More recently, vaccine manufacturers such as Merck Animal Health (Rahway, NJ, USA) and Harrisvaccines (Ames, IA, USA) have utilized an RNA particle technology for the development of either prescription vaccines based on farm-specific VP7 sequences from circulating RVA strains (Sequivity ® ) or a porcine rotavirus group C (RVC) vaccine (using SirraVax SM RNA Particle Technology, Harrisvaccines) to circumvent challenges in isolating and propagating RVC for conventional vaccine design.…”