Herstellung eines Produktionsstammes zur optimierten Expression von rekombinanten Proteinen aus Plasmodium falciparum in Tetrahymena thermophila

Im Rahmen der Arbeit „Herstellung eines Produktionsstammes zur optimierten Expression von rekombinanten Proteinen aus Plasmodium falciparum in Tetrahymena thermophila“ konnte erfolgreich die innovative Produktionstechnologie Tetrahymena thermophila zur Expression und Sekretion plasmodialer Oberflächen‑Antigene eingesetzt werden. Hierzu wurden Vakzinkandidaten aus dem parasitären Blutstadium von Plasmodium falciparum gewählt. Bei den Malaria-Antigenen handelte es sich zum einen um neuartige, multiallelische Hybridkonstrukte auf der Grundlage des Hauptoberflächenantigens des Merozoiten, MSP-1, und zum anderen um die duffy binding like Domänen, die auf dem gut charakterisierten Zytoadhärenzprotein der Trophozoiten und Gametozyten der maternalen Malaria PfEMP1/Var2CSA basieren (DBL2 und DBL3). Hierzu erfolgte die Generierung eines Protease-knock-out Stamms und die Anpassung des Kultivierungsprozesses des generierten Protease-knock-out Stamms in der Kolbenanzucht sowie in der 0,5l Fermentation

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens

Schematic diagram of the MSP-1-BBM construct which encodes the N-terminal MSP-1 Block 1 region, the K1 Block 2 synthetic sequence, the RO33 Block 2 sequence, the MAD20 Block 2 synthetic sequence [31] and a C-terminal fragment encoding residues 1571 to 1702 of MSP-1 precursor protein of <i>P. falciparum</i>.

<p>The Block 2 region is arranged similar to natural alleles. Synthetic Block 2 repeat sequences of the K1 and MAD20 serotypes are indicated by vertical and diagonal hatched markings, respectively.</p

Analysis of purified MSP-1-BBM protein by SDS-PAGE and Western blotting.

<p>A. Coomassie-stained gel of purified MSP-1-BBM protein produced in <i>T. thermophila</i>. Lane 1. Molecular weight markers. Lane 2. 0.5 µg purified MSP-1-BBM protein. B. Western blot of MSP-1-BBM protein and MSP-1 hybrid probed with with mAb 12.2, (specific for repeat sequences present in the K1 serotype of MSP-1 Block 2). Lane 1. Molecular weight markers. Lane 2. 0.5 µg of <i>Tetrahymena</i>-derived MSP-1-BBM protein. Lane 3. Negative control. Lane 4. 0.5 µg MSP-1 Block 2 hybrid protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Galamo1" target="_blank">[30]</a> (positive control).</p

Immunogenicity in mice of recombinant MSP-1-BBM protein from <i>T. thermophila</i>[49].

<p>A group of five MF1 mice were immunized s.c. three times, at 2 week intervals with MSP-1-BBM protein formulated in CoVaccine HT as described. Twelve days after the last immunization (d40), serum samples from each animal were tested by ELISA for antibody responses against the MSP-1 Block 2 hybrid protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cowan1" target="_blank">[31]</a> K1-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a>, MAD20-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a>, RO33-type Block 2 protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Cavanagh2" target="_blank">[54]</a> and MSP-1₁₉ protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087198#pone.0087198-Burghaus1" target="_blank">[53]</a>. Titers were calculated as outlined in materials and methods and expressed as arbitrary units (AU). Data is shown on a natural logarithmic scale as dotplots of serum reactivity for individual animals with the median level of Ab reactivity indicated by the solid horizontal line.</p

Indirect immunofluorescence assay (IFA) of sera from MSP-1-BBM immunized mice against three strains of <i>P. falciparum</i>.

<p>A. Representative micrograph of IFA assay with sera from MSP-1-BBM immunized mice. DAPI staining of parasite nuclei is shown in blue and fluorescence from the FITC-conjugated secondary antibody is shown in green. B. IFA titers of sera from mice immunized with MSP-1-BBM protein. Sera were tested by IFA against the 3D7 (K1 serotype), MAD20 and RO33 strains of <i>P. falciparum</i>, as described in materials and methods. IFA endpoint data is shown on a log₁₀ scale on the Y axis. Each symbol represents the serum reactivity for an individual animal, with the geometric mean of Ab reactivity against each parasite strain indicated by the solid line. C. Western blot of MSP-1 Block 2 hybrid and MSP1₁₉ proteins probed with pooled serum from mice immunized with MSP-1-BBM protein. Lane 1: Molecular weight markers, Lane 2: MSP-1 block 2 hybrid protein, Lane 3: MSP1₁₉-GST fusion protein.</p

Epitope mapping of sera from MSP-1-BBM immunized mice by recognition of peptide epitopes within the MSP-1 Block 2 region of the MSP-1-BBM construct.

<p>A series of 133-terminally biotinylated dodecapeptides, representing the sequence diversity of all three Block 2 serotypes were used in ELISA to map the antibody specificities present in the sera of immunized animals. Reactivity with individual peptides is shown as shaded boxes, with the depth of shading of each box representing the strength of reactivity of a 1∶500 dilution of sera with each peptide. The sequences and Block 2 serotype (K1, MAD20 and RO33) of each peptide are indicated down the right hand side of the diagram.</p