Unstructured proteins of the malaria parasite Plasmodium falciparum as vaccine candidates

Malaria vaccine research has been battling with persistent challenges, including polymorphisms of vaccine antigens, difficulties with production processes, and limited immune protection against the disease. Intrinsically unstructured proteins (IUPs) are a fairly newly classified group of proteins that have no stable 3D structure and are generally heat-resistant. They usually contain low complexity regions and repetitive sequences, both of which are distinct characteristics of the malaria proteome. Surprisingly, some of the vaccine candidates that have been extensively studied were later reported to have unstructured regions, some of which serve as targets of protective immunity. In keeping with their interesting immunological profiles and their unique properties, which are exceptionally beneficial for vaccine production, malarial IUP antigens may be good vaccine candidates. This PhD project has the following aims:- 1) to develop a synthetic unstructured protein antigen based on the Block 2 region of MSP-1, named the MSP-1 hybrid 2) to characterize a novel vaccine antigen derived from the MSP-3.3 protein, namely an IUP region of PF10_0347 gene product, for its potential as a vaccine candidate 3) to develop a second-generation vaccine by combining the MSP-1 hybrid, with two allelic variants of MSP-2, to overcome antigenic polymorphism and strain-specific immune responses 4) to validate protocols for IUP identification from proteins extracted from the malaria parasite. This study showed that 1) MSP-1 hybrid production was scalable, yielding high protein yields with comparable immunological properties to small-scale production. MSP-1 hybrid was shown to be compatible with different adjuvants, and elicited specific antibodies covering the whole range of Block 2 allelic diversities. 2) A novel antigen, MSP-3.3C, an IUP based on the 3’ region of the PF10_0347 gene, was cloned, expressed and purified. Anti-MSP3.3C antibodies showed very strong parasite growth inhibitory effects in vitro. 3) The MSP-multihybrid antigen was expressed using simple techniques, but only at low levels. It contains epitopes from all three parasite antigen components, and is recognized by specific naturally acquired antibodies. 4) an unconventional 2D gel technique was tested as a method of malaria parasite IUP identification. Plans for further validation of this technique were discussed

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

Nonspecific Immunoglobulin M Binding and Chondroitin Sulfate A Binding Are Linked Phenotypes of Plasmodium falciparum Isolates Implicated in Malaria during Pregnancy

Binding of immunoglobulin M (IgM) antibodies from normal human serum to the surface of Plasmodium falciparum-infected red blood cells (iRBC) has previously been demonstrated only in parasites that form rosettes with uninfected red cells. We show that natural, nonspecific IgM but not IgG, IgA, IgD, or IgE also binds to the surface of iRBC selected for adhesion to chondroitin sulfate A (CSA), a placental receptor for parasites associated with malaria in pregnancy. The protease sensitivity of IgM-binding appears to match that of CSA binding, suggesting that the two phenotypes may be mediated by the same parasite molecule. We also show that a wide range of mouse monoclonal antibodies of the IgM class bind nonspecifically to CSA-selected iRBC, an important consideration in the interpretation of immunological assays performed on these parasite lines

Uniparental inheritance of the mitochondrial gene cytochrome b in Plasmodium falciparum

The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite

Comparative Testing of Six Antigen-Based Malaria Vaccine Candidates Directed Toward Merozoite-Stage Plasmodium falciparum▿

Immunogenicity testing of Plasmodium falciparum antigens being considered as malaria vaccine candidates was undertaken in rabbits. The antigens compared were recombinant baculovirus MSP-119 and five Pichia pastoris candidates, including two versions of MSP-119, AMA-1 (domains I and II), AMA-1+MSP-119, and fused AMA-1/MSP-119). Animals were immunized with equimolar amounts of each antigen, formulated in Montanide ISA720. The specificities and titers of antibodies were compared using immunofluorescence assays and enzyme-linked immunosorbent assay (ELISA). The antiparasite activity of immunoglobulin G (IgG) in in vitro cultures was determined by growth inhibition assay, flow cytometry, lactate dehydrogenase assay, and microscopy. Baculovirus MSP-119 immunizations produced the highest parasite-specific antibody titers in immunofluorescence assays. In ELISAs, baculovirus-produced MSP-119 induced more antibodies than any other single MSP-119 immunogen and three times more MSP-119 specific antibodies than the AMA-1/MSP-119 fusion. Antibodies induced by baculovirus MSP-119 gave the highest levels of growth inhibition in HB3 and 3D7 parasite cultures, followed by AMA-1+MSP-119 and the AMA-1/MSP-119 fusion. With the FCR3 isolate (homologous to the AMA-1 construct), antibodies to the three AMA-1-containing candidates gave the highest levels of growth inhibition at high IgG concentrations, but antibodies to baculovirus MSP-119 inhibited as well or better at lower IgG concentrations. The two P. pastoris-produced MSP-119-induced IgGs conferred the lowest growth inhibition. Comparative analysis of immunogenicity of vaccine antigens can be used to prioritize candidates before moving to expensive GMP production and clinical testing. The assays used have given discriminating readouts but it is not known whether any of them accurately reflect clinical protection

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

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