Establishment of a yeast-based VLP platform for antigen presentation

Abstract

BackgroundChimeric virus-like particles (VLP) allow the display of foreign antigens on their surface and have proved valuable in the development of safe subunit vaccines or drug delivery. However, finding an inexpensive production system and a VLP scaffold that allows stable incorporation of diverse, large foreign antigens are major challenges in this field.ResultsIn this study, a versatile and cost-effective platform for chimeric VLP development was established. The membrane integral small surface protein (dS) of the duck hepatitis B virus was chosen as VLP scaffold and the industrially applied and safe yeast Hansenula polymorpha (syn. Pichia angusta, Ogataea polymorpha) as the heterologous expression host. Eight different, large molecular weight antigens of up to 412 amino acids derived from four animal-infecting viruses were genetically fused to the dS and recombinant production strains were isolated. In all cases, the fusion protein was well expressed and upon co-production with dS, chimeric VLP containing both proteins could be generated. Purification was accomplished by a downstream process adapted from the production of a recombinant hepatitis B VLP vaccine. Chimeric VLP were up to 95% pure on protein level and contained up to 33% fusion protein. Immunological data supported surface exposure of the foreign antigens on the native VLP. Approximately 40 mg of chimeric VLP per 100 g dry cell weight could be isolated. This is highly comparable to values reported for the optimized production of human hepatitis B VLP. Purified chimeric VLP were shown to be essentially stable for 6 months at 4 °C.ConclusionsThe dS-based VLP scaffold tolerates the incorporation of a variety of large molecular weight foreign protein sequences. It is applicable for the display of highly immunogenic antigens originating from a variety of pathogens. The yeast-based production system allows cost-effective production that is not limited to small-scale fundamental research. Thus, the dS-based VLP platform is highly efficient for antigen presentation and should be considered in the development of future vaccines.

Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced inHansenula polymorpha

“…Heterologous yeast strains were stored as glycerol stocks at − 80 °C. Recombinant H. polymorpha strains co-producing the dS and a fusion protein were generated by the “staggered transformation approach” and screened as previously described [44].…”

“…Pfs25-dS/dS VLP were purified from strain RK#097 in preparative manner as described before [44, 51]. Briefly, cells were disrupted by six cycles of high pressure homogenization (∼1500 bar, APV 2000, SPX Flow Technology, Unna, Germany) and the cell homogenate was adjusted to 4.5 % (w/w) PEG 6000 and 0.45 M NaCl.…”

“…Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as previously described [44]. In short: The Criterion TM system from BioRad (München, Germany) was used to run SDS-PAGE.…”

“…Coomassie staining of polyacrylamide (PAA) gels were also done as previously described [54] . N -Glycosylation of the heterologous target proteins was analyzed by treatment with an endoglycosidase H (EndoH) prior to SDS-PAGE [44].…”

“…In contrast to previous VLP platforms, the dS-based VLP scaffold allows the stable incorporation of a variety of large molecular weight (MW) foreign antigens. In combination with the yeast expression system, this technology is highly productive and not limited to small scale fundamental research [44]. Thus, the key challenges in the field of chimeric VLP development are met [13,14,45] which makes this platform an attractive and competitive alternative to previously described VLP platforms [30–33].…”