William D. Morgan

Suramin and suramin analogues inhibit merozoite surface protein-1 secondary processing and erythrocyte invasion by the malaria parasite Plasmodium falciparum

Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 μm) and to Plasmodium vivax MSP142 (Kd = 0.3 μm) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 μm) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd ∼ 15 mm) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays, whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor binding to a macromolecular substrate to prevent its proteolysis by a protease.

Malaria Vaccine-Related Benefits of a Single Protein Comprising Plasmodium falciparum Apical Membrane Antigen 1 Domains I and II Fused to a Modified Form of the 19-Kilodalton C-Terminal Fragment of Merozoite Surface Protein 1

ABSTRACT We show that the smallest module of Plasmodium falciparum AMA1 (PfAMA1) that can be expressed in the yeast Pichia pastoris while retaining the capacity to induce high levels of parasite-inhibitory antibodies comprises domains I and II. Based on this, two fusion proteins, differing in the order of the modules, were developed. Each comprised one module of PfAMA1 (FVO strain, amino acids [aa] 97 to 442) (module A) and one module of PfMSP119 (Wellcome strain, aa 1526 to 1621) (module Mm) in which a cystine had been removed to improve immune responses. Both fusion proteins retained the antigenicity of each component and yielded over 30 mg/liter purified protein under fed-batch fermentation. Rabbits immunized with purified fusion proteins MmA and AMm had up to eightfold-higher immune responses to MSP119 than those of rabbits immunized with module Mm alone or Mm mixed with module A. In terms of parasite growth inhibition, fusion did not diminish the induction of inhibitory antibodies compared with immunization with module A alone or module A mixed with module Mm, and fusion outperformed antibodies induced by immunization with module M or Mm alone. When tested against parasites expressing AMA1 heterologous to the immunogen, antibodies to the fusion proteins inhibited parasite growth to a greater extent than did antibodies either to the individual antigens or to the mixture. These results suggest that compared with the individual modules delivered separately or as a mixture, fusion proteins containing these two modules offer the potential for significant vaccine-related advantages in terms of ease of production, immunogenicity, and functionality.

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

ABSTRACT 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.

Plasmodium falciparum infection of the placenta impacts on the T helper type 1 (Th1)/Th2 balance of neonatal T cells through CD4+CD25+ forkhead box P3+ regulatory T cells and interleukin-10.

Placental malaria infection affects the T helper type 1 (Th1)/Th2 balance in neonatal children. We investigated a potential role of regulatory T cells in this balance by comparing T cell responses of cord blood mononuclear cells (CBMC) from parasitized and non‐parasitized placenta of Gambian women. CBMC were depleted of CD4+CD25+ forkhead box P3 (FoxP3)+ regulatory T cells and analysed in vitro for their ability to produce interferon (IFN)‐γ, sCD30 and interleukin (IL)‐10 in response to phytohaemagglutinin (PHA), live Plasmodium falciparum, schizont extracts and the recombinant P. falciparum blood stage antigen merozoite surface protein 1 (MSP119). As expected, lower IFN‐γ and higher sCD30 responses were observed for the cells from the parasitized group. In addition, higher IL‐10 levels were produced by CBMC from the parasitized group. Depletion of regulatory T cells decreased IL‐10 production, which resulted in a restoration of IFN‐γ expression in response to all stimuli. The Th2 marker sCD30 remained significantly higher in the parasitized group in response to malaria protein antigens while similar levels were recovered between both groups in response to live P. falciparum. Similar effects were observed by adding an antibody that blocks IL‐10 function. These results suggest that the impact of P. falciparum infection on Th1 differentiation of neonatal T cells can be ascribed to regulatory T cells through production of IL‐10.

Correlated bond rotations in interactions of arginine residues with ligand carboxylate groups in protein ligand complexes.

© 1997 Federation of European Biochemical Societies.

Antigenic and sequence diversity at the C-terminus of the merozoite surface protein-1 from rodent malaria isolates, and the binding of protective monoclonal antibodies.

Merozoite surface protein-1 (MSP-1) is a major candidate in the development of a vaccine against malaria. Immunisation with a recombinant fusion protein containing the two Plasmodium yoelii MSP-1 C-terminal epidermal growth factor-like domains (MSP-1(19)) can protect mice against homologous but not heterologous challenge, and therefore, antigenic differences resulting from sequence diversity in MSP-1(19) may be crucial in determining the potential of this protein as a vaccine. Representative sequence variants from a number of distinct P. yoelii isolates were expressed in Escherichia coli and the resulting recombinant proteins were screened for binding to a panel of monoclonal antibodies (Mabs) capable of suppressing a P. yoelii YM challenge infection in passive immunisation experiments. The sequence polymorphisms affected the binding of the antibodies to the recombinant proteins. None of the Mabs recognised MSP-1(19) of P. yoelii yoelii 2CL or 33X or P. yoelii nigeriensis N67. The epitopes recognised by the Mabs were further distinguished by their reactivity with the other fusion proteins. The extent of sequence variation in MSP-1(19) among the isolates was extensive, with differences detected at 35 out of the 96 positions compared. Using the 3-dimensional structure of the Plasmodium falciparum MSP-1(19) as a model, the locations of the amino acid substitutions that may affect Mab binding were identified. The DNA sequence of MSP-1(19) from two Plasmodium vinckei isolates was also cloned and the deduced amino acid sequence compared with that in other species.

Interaction of malaria parasite-inhibitory antibodies with the merozoite surface protein MSP1(19) by computational docking

Merozoite surface protein 1 (MSP1) of the malaria parasite Plasmodium falciparum is an important vaccine candidate antigen. Antibodies specific for the C‐terminal maturation product, MSP119, have been shown to inhibit erythrocyte invasion and parasite growth. Specific monoclonal antibodies react with conformational epitopes contained within the two EGF‐like domains that constitute the antigen MSP119. To gain greater insight into the inhibitory process, the authors selected two strongly inhibitory antibodies (designated 12.8 and 12.10) and modeled their structures by homology. Computational docking was used to generate antigen–antibody complexes and a selection filter based on NMR data was applied to obtain plausible models. Molecular Dynamics simulations of the selected complexes were performed to evaluate the role of specific side chains in the binding. Favorable complexes were obtained that complement the NMR data in defining specific binding sites. These models can provide valuable guidelines for future experimental work that is devoted to the understanding of the action mechanism of invasion‐inhibitory antibodies. Proteins 2007.

An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria

The 19‐kDa C‐terminal region of merozoite surface protein 1 (MSP119), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild‐type MSP119‐derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)‐γ] and Th0/Th2 [interleukin (IL)‐13 and sCD30] responses to the wild‐type MSP119 and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP119 (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild‐type, included higher IFN‐γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL‐13 production than the wild‐type, while the conserved peptides P16 and P19 induced the highest IFN‐γ and IL‐13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D‐related (HLA‐DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild‐type or modified protein in combination with their peptides.