Investigating the feasibility of generating chimeric intergroup rotavirus triple-layered virus-like particles on SA11 double-layered particles

Abstract

There remains a need for safer and more effective human rotavirus (RV) vaccines, despite the availability of several commercial rotavirus vaccines, especially given that RV is a leading cause of diarrhoea-related deaths worldwide among children younger than 5 years old. After the first 12 months following vaccination, the vaccine efficacy of the main commercially available RV vaccines was 44% in the high-mortality settings of developing countries of Africa and Asia which is lower than the 77% to 98% efficacy of the medium- to low-mortality settings of developed countries. This may also be due to the different strains of rotavirus present in developing countries of Africa and Asia compared to those strains upon which these vaccines are based. Therefore, non-replicating regional-specific vaccines may be needed in populations with partial immunity to overcome this gap in vaccine efficacy. Baculovirus-expressed RV VP2/4/6/7 triple-layered particles (TLPs) are promising candidate dead subunit vaccines that have been shown to protect against RV infections in several animal models. However, producing these multicomponent particles using a co-infection strategy is difficult and ineffective. Our previous co-infection results visualised by transmission electron microscopy (TEM) revealed problems with the assembly of RV-TLPs. Less than 30% were fully assembled RV-TLPs when co-infected with dualcistronic recombinant baculoviruses (rBVs) expressing VP2/6 and VP4/7. It was hypothesized that the partial assembly of RV-TLPs may have been due to a lack of calcium during assembly. Therefore, in this study, we attempted to generate fully assembled RV-TLPs using dualcistronic rBVs expressing RVA/Human-wt/ZAF/GR10924/1999/G9P[6] VP2 and VP6 co-infected with dualcistronic rBVs expressing either genotype G12P[4], G12P[6], or G12P[8] VP4 and VP7 in media supplemented to 20mM calcium. However, only RV-DLPs were formed when using a co-infection strategy despite the additional calcium supplementation. To overcome the inherent disadvantage of co-infections, where it is impossible to control the ratios at which individual cells are infected, multi-gene baculovirus donor plasmids have previously been developed, but they have not been widely implemented.

In this study, we further investigated the feasibility of generating a single recombinant baculovirus expressing all four RVA/Simian-tc/ZAF/SA11-N5/1958/G3P5B[2] TLP proteins (VP2/6/4/7). A pFastBACquad donor plasmid containing insect cell codon-optimised open reading frames (ORFs) of all four SA11 TLP proteins was designed, purchased and used in a Bac-to-Bac Baculovirus vector expression system (BVES) to generate quadcistronic rBVs expressing SA11 VP2/6/7/4 simultaneously in the same cell. Using these quadcistronic rBVs, fully assembled SA11 RV-TLPs were generated and visualized using TEM. Therefore, using quadcistronic rBVs in a Bac-to-Bac BVES presents a better and more cost-effective platform to investigate the formation of RV virus-like particles, than using a co-infection approach using dualcistronic rBVs, and was successful in generating fully assembled SA11 VP2/6/4/7 TLPs. The results presented herein serve as proof of principle that quadcistronic rBVs in a Bac-to-Bac BVES may be used to investigate the extent to which SA11 VP2/6 DLPs may serve as a universal backbone for generating heterologous VP2/6/4/7 RV-TLPs with a variety of regional-specific VP4/7 outer capsids as regional-specific subunit vaccine candidates against RV. Further, chimeric RV-TLPs may be instrumental in evaluating RV structural compatibility and stability by using recombinant baculoviruses as a vector, thereby avoiding any possible known, or unknown, genetic restrictions that may affect the generation of chimeric rescued RVs generated by a reverse genetics system. Baculovirus-generated TLPs may also aid basic research into the start of the RV replication cycle by investigating viral entry into host cells.