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Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

ABSTRACT: Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the circumsporozoite protein (CS) of Plasmodium, the etiologic agent of malaria. The CS protein from Plasmodium berghei (Pb) was selected given the availability of the stringent C57Bl/6 mouse model of infection by Pb sporozoites, allowing the evaluation of vaccine efficacy in vivo. PbCS was multimerized by fusion to the measles virus (MV) nucleoprotein (N) known to auto-assemble in yeast in large-size ribonucleoprotein rods (RNPs). Expressed in P. pastoris, the N-PbCS protein generated highly multimeric and heterogenic RNPs bearing PbCS on their surface. Electron microscopy and immunofluorescence analyses revealed the shape of these RNPs and their localization in peripheral cytoplasmic inclusions. Subcutaneous immunization of C57Bl/6 mice with heat-inactivated whole P. pastoris expressing N-PbCS RNPs provided significant reduction of parasitemia after intradermal challenge with a high dose of parasites. Thus, in the absence of accessory adjuvants, a very low amount of PbCS expressed in whole yeast significantly decreased clinical damages associated with Pb infection in a highly stringent challenge model, providing a proof of concept of the intrinsic adjuvancy of this vaccine strategy. In addition to PbCS multimerization, the N protein contributed by itself to parasitemia delay and long-term mice survival. In the future, mixtures of whole recombinant yeasts expressing relevant Plasmodium antigens would provide a multivalent formulation applicable for antigen combination screening and possibly for large-scale production, distribution and delivery of a malaria vaccine in developing countries.

SUBMITTER: Jacob D

PROVIDER: S-EPMC3903550 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

Jacob Daria D Ruffie Claude C Dubois Myriam M Combredet Chantal C Amino Rogerio R Formaglio Pauline P Gorgette Olivier O Pehau-Arnaudet Gérard G Guery Charline C Puijalon Odile O Barale Jean-Christophe JC Ménard Robert R Tangy Frédéric F Sala Monica M

PloS one 20140127 1

Yeasts are largely used as bioreactors for vaccine production. Usually, antigens are produced in yeast then purified and mixed with adjuvants before immunization. However, the purification costs and the safety concerns recently raised by the use of new adjuvants argue for alternative strategies. To this end, the use of whole yeast as both production and delivery system appears attractive. Here, we evaluated Pichia pastoris yeast as an alternative vaccine production and delivery system for the ci ...[more]

PMID: 24475165

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Project description:BackgroundCurrently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy.ResultsA scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen.ConclusionsFor the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Project description:BackgroundYeast cells represent an established bioreactor to produce recombinant proteins for subunit vaccine development. In addition, delivery of vaccine antigens directly within heat-inactivated yeast cells is attractive due to the adjuvancy provided by the yeast cell. In this study, Pichia pastoris yeast lysates carrying the nucleoprotein (N) from the measles vaccine virus were evaluated as a novel subunit vaccine platform to deliver the circumsporozoite surface antigen (CS) of Plasmodium. When expressed in Pichia pastoris yeast, the N protein auto-assembles into highly multimeric ribonucleoparticles (RNPs). The CS antigen from Plasmodium berghei (PbCS) was expressed in Pichia pastoris yeast in fusion with N, generating recombinant PbCS-carrying RNPs in the cytoplasm of yeast cells.ResultsWhen evaluated in mice after 3-5 weekly subcutaneous injections, yeast lysates containing N-PbCS RNPs elicited strong anti-PbCS humoral responses, which were PbCS-dose dependent and reached a plateau by the pre-challenge time point. Protective efficacy of yeast lysates was dose-dependent, although anti-PbCS antibody titers were not predictive of protection. Multimerization of PbCS on RNPs was essential for providing benefit against infection, as immunization with monomeric PbCS delivered in yeast lysates was not protective. Three weekly injections with N-PbCS yeast lysates in combination with alum adjuvant produced sterile protection in two out of six mice, and significantly reduced parasitaemia in the other individuals from the same group. This parasitaemia decrease was of the same extent as in mice immunized with non-adjuvanted N-PbCS yeast lysates, providing evidence that the yeast lysate formulation did not require accessory adjuvants for eliciting efficient parasitaemia reduction.ConclusionsThis study demonstrates that yeast lysates are an attractive auto-adjuvant and efficient platform for delivering multimeric PbCS on measles N-based RNPs. By combining yeast lysates that carry RNPs with a large panel of Plasmodium antigens, this technology could be applied to developing a multivalent vaccine against malaria.

Dataset Information

Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

Publications

Whole Pichia pastoris yeast expressing measles virus nucleoprotein as a production and delivery system to multimerize Plasmodium antigens.

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