The stage-specific in vitro efficacy of a malaria antigen cocktail provides valuable insights into the development of effective multi-stage vaccines

Multicomponent vaccines targeting different stages of Plasmodium falciparum represent a promising, holistic concept towards better malaria vaccines. Additionally, an effective vaccine candidate should demonstrate cross-strain specificity because many antigens are polymorphic, which can reduce vaccine efficacy. A cocktail of recombinant fusion proteins (VAMAX-Mix) featuring three diversity-covering variants of the blood-stage antigen PfAMA1, each combined with the conserved sexual-stage antigen Pfs25 and one of the pre-erythrocytic-stage antigens PfCSP TSR or PfCelTOS, or the additional blood-stage antigen PfMSP1 19, was produced in Pichia pastoris and used to immunize rabbits. The immune sera and purified IgG were used to perform various assays determining antigen specific titers and in vitro efficacy against different parasite stages and strains. In functional in vitro assays we observed robust inhibition of blood-stage (up to 90%), and sexual-stage parasites (up to 10 0%) and biased inhibition of pre-erythrocytic parasites (0-40%). Cross-strain blood-stage efficacy was observed in erythrocyte invasion assays using four different P. falciparum strains. The quantification of antigen-specific IgGs allowed the determination of specific IC50 values. The significant difference in antigen-specific IC50 requirements, the direct correlation between antigen-specific IgG and the relative quantitative representation of antigens within the cocktail, provide valuable implementations for future multi-stage, multi-component vaccine designs

Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites

We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2x10(6)). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules

RP-HPLC analysis of individual and mixed PfAMA1-DiCo1, PfAMA1-DiCo2 and PfAMA1-DiCo3 samples.

<p>Upper panel: Injection of the individual PfAMA1 DiCo proteins to establish the chromatographic profile of each of these proteins. PfAMA1 DiCo1 in black, PfAMA1 DiCo2, in blue and PfAMA1 DiCo3 in red. Lower panel: Chromatogram of the drug product containing the three PfAMA1 DiCo proteins at equimolar ratios, in green. For chromatographic details see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164053#sec002" target="_blank">Methods</a> Section.</p

Analysis of experimental excipients mixed with PfAMA1 DiCo Drug Substances.

<p>Analysis of experimental excipients mixed with PfAMA1 DiCo Drug Substances.</p

Fermentation profiles for the GMP runs for PfAMA1-DiCo Drug Substances.

<p>Upper panel. Base consumption and pH. Base consumption (NH₄OH addition) is related to growth and metabolic activity, since NH₄⁺ is the single source of nitrogen. Addition of base compensates the acidification during aerobic growth of microbes in mineral media. A pH setpoint of pH = 6.0 was used throughout the fermentations. Lower panel. Biomass and dissolved oxygen. The offline determined OD₆₀₀ was taken as a measure for biomass (1 OD₆₀₀ ~ 1 g/L w/w). The curves show the typical batch, glycerol fed-batch, methanol adaptation and methanol induction phases of Mut^s Pichia strain fermentations. Black lines; PfAMA1 DiCo1; blue lines, PfAMA1 DiCo2; red lines, PfAMA1 DiCo3.</p

Seroconversion as a function of dose.

<p>Groups of 11 mice were immunized with indicated amounts of Drug Product formulated in either Rehydragel (Panels A, C and E) or GLA-SE (Panels B, D and F). On day 28 they were immunized again. Serum was collected on day 42 and ELISAs were performed using either PfAMA1 DiCo1 (Panels A,B), PfAMA1 DiCo2 (Panels C,D) or PfAMA1 DiCo3 (Panels E,F) as coating antigen. Individual mice within a treatment group are indicated by symbols, where the same symbol within a treatment groups represents the same animal for PfAMA1 DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3, respectively.</p

Signature peptide MS analysis of pre-GMP produced PfAMA1 DiCo Drug substance intermediates.

<p>Panel A. Overloaded SDS-Gel of HIC eluate fractions with excised bands in marked boxes. Panel B. Examples of signature peptides identified in the 21 excised protein bands for PfAMA1-DiCo preparations. Key to preparations: PfAMA1 DiCo1 (Green), PfAMA1 DiCo2 (Orange) and PfAMA1 DiCo3 (Blue). In yellow the signature amino acids in these peptides, allowing for the differentiation between individual PfAMA1 DiCo proteins. Numbering of the amino acids is as in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164053#pone.0164053.ref015" target="_blank">15</a>].</p

SDS-PAGE, Western Blotting and SEC profiles of the Drug substances PfAMA1 DiCo1, PfAMA1 DiCo2 and PfAMA1 DiCo3.

<p>Panel A: Reducing SDS-PAGE, silver-stained. Panel B: Non-reducing SDS-PAGE, silver-stained. Panel C: Non-reducing SDS-PAGE and Western-Blot, immune-staining using the reduction-sensitive monoclonal anti-PfAMA1 antibody 4G2. Panel D: SE-Chromatography of each of the PfAMA1 Drug Substances. PfAMA1 DiCo1 (black), PfAMA1 DiCo2 (blue) and PfAMA1 DiCo3 (red) was carried out on a Superdex200 10/300 GL prepacked column (GE Healthcare), using an Åkta Purifier Basic (GE Healthcare). PBS (140 mM NaCl, 10 mM sodium phosphate, 3 mM KCl, pH 7.4, Calbiochem) was used as running buffer at 0.5 mL.min^-1.</p

Batch analysis and stability data for PfAMA1-DiCo Drug Product.

<p>Batch analysis and stability data for PfAMA1-DiCo Drug Product.</p