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.

Figure 1. Expression of N protein in GS115, KM71 and SMD1168 P. pastoris strains.

Concentrations of Geneticin in selection plates for the specific clones, and clone numbers, are indicated. Yeast lysates were diluted 1/600 before loading on western blot.

Figure 2. Expression of N-PbCS in P. pastoris.

(A) Schematic representation of N-PbCS fusion protein. MV-N (dark grey) is composed of a core domain in N-terminal and a unstructured tail domain in C-terminal . The GAAGAGA linker is in black. PbCS (light grey) corresponds the central repeat region flanked by major portions of the N-terminal and C-terminal domains of the protein . Amino acids numbering are given according to N from the MV Schwarz vaccine strain and PbCS from the Pb ANKA strain. For sequence details, see Figure S1. (B) Quantitative western blot analysis of SMD1168 expressing N-PbCS or (C) N. In (B) and (C), yeast lysates were diluted as indicated, the MV-N protein was used as a standard with increasing concentrations and western blots were probed with an anti-N antibody.

Figure 3. ELISA quantification of N or PbCS proteins in ultracentrifugation (U) fractions and pellets of SMD1168 lysates expressing N alone at 871 ng/YU (A) or N-PbCS at 12 ng/YU (B).

Yeast cultures, lysates and ultracentrifugations were performed in duplicate (3 U and 4 U for N expressing yeast, and 5 U and 6 U for N-PbCS yeast). Values correspond to optical densities at OD_{450 nm} (taking OD_{620 nm} as reference) multiplied by sample dilutions. SB: suspension buffer.

Figure 4. Electron microscopy analysis of yeast lysates from SMD1168 P. pastoris expressing N (A) or N-PbCS (B).

Scale bars are indicated. Black arrows highlight RNP rod and ring structures.

Figure 5. Immunofluorescence analysis of N or N-PbCS expression in yeasts (N staining in green, PbCS staining in red and nuclei in blue).

Each image is the maximal intensity projection of three consecutive focal planes spaced 0.5 µm apart.

Figure 6. Schematic representation of the immunization protocol.

Immunization and challenge schedule is given in days (d) and bleeding time points in weeks.

Figure 7. Humoral responses elicited in mice after immunization.

(A) Kinetics of anti-PbCS IgG responses in mice immunized with N-PbCS yeasts. OD_{450 nm} are expressed in log₁₀ scale. Black arrows indicate immunization schedule. (B) Isotyping of humoral IgG responses at day 42 in mice immunized with N-PbCS. The bars correspond to median values per group. Asterisks (*) indicate significant median differences (p<0.05; Mann-Whitney nonparametric test). (C) Anti-N IgG titers in mice serums collected at day 42 after immunization with WT yeast or yeasts expressing N, PbCS or N-PbCS. Median values were compared by the Wicoxon Two Sample Test (p = 0.4558). (D) Antibody titers of N-PbCS mice are compared (see panels B and C). The lines associate titers from the same mouse. (E) Anti-P. pastoris IgG responses towards whole yeast. (F) Anti-P. pastoris IgG responses towards lysed yeast. The bars in (E) and (F) correspond to mean values per group. Hash sign (#) indicates anti-N antibody-negative mice from the N-PbCS group (see panel C).

Figure 8. Experimental challenge of immunized mice.

(A) Mean and standard deviations log₁₀ values of parasitemia in mice immunized by N-PbCS, PbCS, N or WT yeast, and in non-immunized mice following infection with 6,000 GFP⁺ Pb sporozoites. Blood parasitemia is expressed in log₁₀ scale as the percentage of infected red blood cells (iRBCs) out of total RBCs along the first 7 days follow up. Asterisks (*) indicate the significance level of the Mann-Whitney nonparametric test: two symbols correspond to p<0.005 and three to p<0.0005. (B) Parasitemia at day 5 post-challenge. Bars correspond to medians. Asterisks (*) indicate significant median differences (one symbol for p<0.05 and two for p<0.005; Mann-Whitney nonparametric test). (C) Inverse correlation between the day of death (x axis) and the percentage of iRBCs per total RBCs (y axis; arithmetic scale) per mouse. The cause of death is given in the upper part of the graph. (D) Survival curves of immunized mice after challenge with 6,000 GFP⁺ Pb sporozoites.