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.

Edible Vaccines: Promises and Challenges

“…The capacity of whole-cell yeast-oriented vaccines to generate an immune response has been studied [59,90]. Notable evidence showing that this system can induce a protection of the mucosa is found from several preclinical studies based on orally administered Saccharomyces cerevisiae and developed for various influenza agents such as HPV, Actinobacillus pleuropneumoniae [91,92].…”

Immunoprevention of KRAS-driven lung adenocarcinoma by a multipeptide vaccine

“…GI-4000 are intact, heat-inactivated yeast containing known KRAS mutations, and have been shown to be well tolerated and able to stimulate mutation-specific T-cell responses, the cytotoxic response in multiple clinical and preclinical tests, which ultimately lead to favorable overall survival trend [ 12 – 14 ]. However, whole organism vaccines are still limited by the difficulties in large-scale manufacturing, as well as repeat dosing to enhance vaccine efficiency [ 15 ]. Compare to conventional vaccines, advantages of immunoprevention using peptide vaccines include minimal toxicity, immediate and long-term memory, ease of delivery and cost effectiveness [ 16 ].…”

Yeast lysates carrying the nucleoprotein from measles virus vaccine as a novel subunit vaccine platform to deliver Plasmodium circumsporozoite antigen

“…Subcutaneous immunizations with whole recombinant Pichia pastoris yeasts expressing such RNPs induced a strong anti-PbCS humoral response, and upon challenge, delayed the onset of red blood cell infection by P. berghei and prolonged mouse survival [6]. Notably, these results were achieved in the C57Bl/6 mouse model, which is highly susceptible to P. berghei infection (ANKA strain) and subsequent development of cerebral malaria [17, 18].…”