Identification of engineered yeast strains for the production of rotavirus VP6-based vaccine candidates

Abstract

Despite two live-attenuated oral rotavirus vaccines (Rotarix®, RotaTeq®) having been used for a decade in more than 96 countries, rotavirus continues to be the major etiological agent of severe gastroenteritis and diarrhoea-related hospitalisation in infants and children less than 5 years old. Two additional live-attenuated oral vaccines (Rotavac® and RotaSIIL®) were pre-qualified for global use recently which should help expand the vaccine coverage. The highest incidence of rotavirus infection remains in low-income countries in sub-Saharan Africa and Asia. The efficacies of Rotarix® and RotaTeq® are very high in the high- to middle-income countries, however, lower efficacies have been observed in the low-income countries of sub-Saharan Africa and Asia. Notwithstanding this lower efficacy, many lives have been saved, which means a significant impact in terms of public health benefits in these two regions.

Apart from the efficacies of these vaccines, certain other shortcomings have been associated with the live-attenuated rotavirus vaccines. Intussusception, genome reassortment and viral shedding are some of the risks associated with these vaccines. Furthermore, the vaccines are quite expensive and require a cold-chain. Most of the affected, low-income countries could not implement these vaccines but for the help of GAVI (Global Alliance for Vaccines and Immunization). All these associated problems, therefore, call for the development of alternative, cost-effective and non-replicating rotavirus vaccine candidates.

Morphologically, rotavirus is an icosahedral, concentric particle with three layers of viral proteins, which encapsulate the viral genome. Capsid protein, VP6, is the most abundant and immunodominant viral protein, and together with other capsid proteins, are candidates of sub-unit, non-replicating vaccines. Rotavirus virus-like particles, either as a single- or multi-layer, have been used together with adjuvants to induce protective immune responses in animal models. Recombinantly expressed rotavirus VP6 protein has attracted attention as the safest parenterally administered vaccine candidate capable of inducing an immune response in animals. Rotavirus VP6 can induce a mucosal IgA response against rotavirus infections with heterotypic protection. Identifying a scalable expression system for production of the VP6 protein has been a major setback for the advancement of this immunoreactive protein as a vaccine candidate.

In this study, a wide-range yeast expression system was used to screen sixteen yeast strains in order to identify strain(s) capable of expressing rotavirus VP2 and VP6 encoded by three different yeast codon-optimised open reading frames (ORFs), which had been further optimised for expression by improving the Kozak consensus sequence. Hansenula polymorpha (UOFS_Y0915) was identified as the only yeast capable of expressing these proteins simultaneously. Furthermore, after negative staining with uranylacetate solution, a particle of 58 nm in size was observed with transmission electron microscopy. This particle has a rough, spherical shape with a distinct single-shelled layer of rotavirus VP6 capsid enclosing the core protein, VP2.

Production of rotavirus VP6 protein in yeasts has thus far been by the intracellular expression, but to reduce the costs of downstream processing in a production facility, it would be better to produce this protein by extracellular secretion. Therefore, a recombinant wide-range expression plasmid was modified by promoter and signal sequence optimisations to enable, respectively, the expression and secretion in engineered Kluyveromyces lactis (UOFS_Y1167). A deep-well bioreactor screen coupled with ELISA testing evaluated VP6 expression in the growth medium by approximately one hundred K. lactis colonies for the first time. Therefore, as proof-of-concept, a screening protocol was developed that would allow for the identification of more yeast strains with the potential to secrete rotavirus VP6 protein. Identification of such yeasts will enable scaled-up production of this protein in bioreactors. This should, in turn, facilitate cost-effective production of rotavirus VP6 protein as a potential next generation, non-replicating rotavirus vaccine candidate.