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

BACKGROUND: Yeast 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. RESULTS: When 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. CONCLUSIONS: This 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.

The ABCA1 domain responsible for interaction with HIV-1 Nef is conformational and not linear.

HIV-1 Nef is an accessory protein responsible for inactivation of a number of host cell proteins essential for anti-viral immune responses. In most cases, Nef binds to the target protein and directs it to a degradation pathway. Our previous studies demonstrated that Nef impairs activity of the cellular cholesterol transporter, ABCA1, and that Nef interacts with ABCA1. Mutation of the (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 disrupted interaction with Nef. Here, we tested Nef interaction with the ABCA1 C-terminal cytoplasmic fragment using yeast 2-hybrid system assay and co-immunoprecipitation analysis in human cells. Surprisingly, analysis in a yeast 2-hybrid system did not reveal any interaction between Nef and the C-terminal cytoplasmic fragment of ABCA1. Using co-immunoprecipitation from HEK 293T cells expressing these polypeptides, only a very weak interaction could be detected. The (2226)DDDHLK motif in the C-terminal cytoplasmic tail of ABCA1 found previously to be essential for interaction between ABCA1 and Nef is insufficient to bestow strong binding to Nef. Molecular modeling suggested that interaction with Nef may be mediated by a conformational epitope composed of the sequences within the cytoplasmic loop of ABCA1 and the C-terminal cytoplasmic domain. Studies are now underway to characterize this epitope.

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

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.