Expression of rotavirus capsid protein, VP6, in various yeasts

[摘要] Rotavirus infection is one of the six leading causes of death among children under the age of five years. Globally it causes more than 215 000 deaths annually of which 65% occur in low- and/or middle-income countries. The two licenced live-attenuated vaccines (Rotarix�?and RotaTeq�? tend to have a lower efficacy in low- and/or middle-income countries. The lower efficacy of rotavirus live-attenuated vaccines could be due to maternal antibodies and oral polio vaccines interfering with rotavirus vaccine uptake. Rotavirus live-attenuated vaccines have been associated with reassortment with circulating genotype strains. As alternative, subunit vaccines such as viral proteins can be considered. Rotavirus VP6 protein is considered as a candidate for subunit vaccine development. Rotavirus VP6 antibodies are responsible for long lasting immunity and the antibodies against VP6 block the release of the viral mRNA. Previous studies showed rotavirus VP6 provide heterologous protection by a significant reduction of virus shedding in mice and gnotobiotic pigs.Various recombinant yeasts were engineered by a previous MSc student, Mr M.S. Makatsa, using a unique yeast expression vector (pKM177). The recombinant yeasts contained an open reading frame (ORF) encoding rotavirus VP6 for the RVA/Human wt/ZAF/GR10924/1999/G9P[6] strain. The ORF was codon optimised to favour expression in Arxula adeninivorans (AO), Kluyveromyces lactis (KO) and Pichia pastoris/Pichia angusta (PO). However, there was no expression of VP6 optimised for expression in A. adeninivorans and K. lactis due to an additional out-of-frame ATG in the promoter region of the expression vector. In this study, the additional ATG was successfully removed by site-directed mutagenesis and the Kozak sequence was optimised to produce modified delATG_pKM177_AOVP6 and delATG_pKM177_KOVP6 constructs.The modified delATG_pKM177_AOVP6, delATG_pKM177_KOVP6 as well as delATG_pKM177_POVP6, the modified plasmid containing the VP6 ORF codon optimised for expression in P. pastoris/P angusta and obtained from a colleague, were transformed into 14 different yeast strains. Partial PCR amplification of the VP6 ORFs was conducted to screen for integration of the expression cassettes into the yeast genomes. Debaryomyces hansenii UFS0610 and Yarrowia lipolytica UFS2415 resulted in no colony formation. Integration into the P. angusta genome was efficient for all the VP6 optimised ORFs.Integration of the VP6 ORFs in the Y. lipolytica PO 1F, UFS0097 and UFS2221 strains were relatively poor as only 16% of the clones screened showed integrated into the genome. The delATG_pKM177_AOVP6 and delATG_pKM177_POVP6 had 75-80% integration in the various yeast genomes, while delATG_pKM177_KOVP6 integration was relatively low at 46%. Expression of VP6 from all the ORFs was effective in P. angusta strains followed by A.adeninivorans strains from the UNESCO-MIRCEN yeast culture collection (A. adeninivorans UFS1219 and A. adeninivorans UFS1220), S. cerevisiae and K. lactis. High expression of VP6 in P. angusta and P. pastoris has been reported in previous studies. In this study, almost all P. angusta colonies screened, expressed VP6, while only 13% of P. pastoris colonies screened expressed VP6. There was relatively low expression of VP6 in Y. lipolytica strains as well as the prototype A. adeninivorans strain, LS3.Six yeasts were identified that successfully expressed rotavirus VP6. Rotavirus VP6 has a unique feature of assembling into oligomeric structures depending on the pH and ionic strength. Assembly of nanotubes or nanosphere have only been reported for VP6 produced by insect cells and E. coli, but not in yeast cells. A simple method was adapted to purify and allowed VP6 to assemble in oligomeric structures. Only VP6 produced in A. adeninivorans UFS1219 was able to assemble in both nanotubes and nanospheres, while VP6 produced in Y. lipolytica only assembled in nanospheres. The recombinant A. adeninivorans 1219 strain shows great potential as producer of a rotavirus subunit vaccine candidate.

[发布机构] University of the Free State