Trials for the co-expression of the merozoite surface protein-1 and circumsporozoite protein genes of Plasmodium vivax

Merozoite surface protein-1 (MSP-1), a major asexual blood stage antigen, and circumsporozoite protein (CSP), a component of sporozoites that includes a Plasmodium vivax B-cell epitope, are strong candidates for use in a malaria vaccine. A chimeric recombinant gene containing portions of both msp-1 and csp from P. vivax separated by Pro-Gly linker motif was generated. The construct gene was named mlc (msp-1, linker, and csp). The MLC chimeric recombinant protein had a molecular weight of approximately 25 kDa when expressed in Escherichia coli, as determined with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis. The purified chimeric protein reacted with the sera of patients infected with P. vivax but not with the sera of uninfected patients according to western blot analysis. The chimeric protein reacted well with sera of malaria patients (109/115, 94.78%) as assessed with enzyme-linked immunosorbent assay (ELISA). BALB/c mice that were orally immunized with the MLC chimeric recombinant protein successfully produced antigen-specific antibodies. Additionally, levels of the Th1-associated cytokines IL-12(p40), TNF-α, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Therefore, the E. coli-expressed MLC chimeric recombinant protein might be used as a valuable vaccine candidate for oral immunization against vivax malaria.     

Structure and development of the surface coat of erythrocytic merozoites of Plasmodium knowlesi

Summary The surface of extracellular merozoites of P. knowlesi is covered with a coat 15–20 nm thick, made up of clusters of filaments standing erect on the plasma membrane. Filaments have stems 2 nm thick, the peripheral ends of which are complex, branching or ending in long trailing threads. Coat filaments occur on the surface of the parasite in regular rows at an early schizont stage, and persist until well after merozoite release. They are sensitive to trypsin and papain, and bind ethanolic phosphotungstate, indicating a proteinaceous nature. They are also removed by exposure to phosphate-buffered saline. Filaments bear negative charges, binding cationised ferritin throughout the depth of the coat and staining with ruthenium red. They cover the whole merozoite surface and mediate intercellular adhesion at distances of 15–150 nm, membrane to membrane. It is suggested that these filaments correspond to a major merozoite surface protein, and are important in the initial capture of red cells.     

Plasmodium knowlesi as a Threat to Global Public Health

Malaria is a tropical disease caused by protozoans of the Plasmodium genus. Delayed diagnosis and misdiagnosis are strongly associated with higher mortality. In recent years, a greater importance is attributed to Plasmodium knowlesi, a species found mainly in Southeast Asia. Routine parasitological diagnostics are associated with certain limitations and difficulties in unambiguous determination of the parasite species based only on microscopic image. Recently, molecular techniques have been increasingly used for predictive diagnosis. The aim of the study is to draw attention to the risk of travelling to knowlesi malaria endemic areas and to raise awareness among personnel involved in the therapeutic process.     

In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.     

Changing the N-terminal sequence protects recombinant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> circumsporozoite protein from degradation in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Proteolytic degradation is the primary obstacle in the use of the yeast Pichia pastoris for the expression of recombinant proteins. During the production of a recombinant Plasmodium falciparum circumsporozoite protein in this system, the (NANP) _n repeats region at the N-terminus were completely proteolytically degraded. To remove the potential proteolytic site within the recombinant protein, different strategies were tried, including adjusting the cultivation conditions and mutating the sequence at the junction of the repeat domain and C-terminal region, but the degradation continued. However, modification of the N-terminal sequence by adding an epitope-based peptide to the N-terminus not only protected the repeat domain from cleavage by native proteases during longer induction in the yeast host and purification process, but also stabilized this recombinant protein emulsified with adjuvant ISA720 for at least 6 months. The results showed that proteolytic degradation of the recombinant circumsporozoite protein produced in P. pastoris was amino acid sequence (NANP)-specific, and that this effect was likely dependent on the conformation of the recombinant protein.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

Influence of medium osmolality on the in vitro growth ofPlasmodium falciparum: A morphologic and radioisotopic study

Summary The study of the growth rate and incorporation of [³H]hypoxanthine and [¹⁴C]isoleucine showed that in vitro variations ofPlasmodium falciparum parasitemia levels and incorporation rates of the two radiolabeled molecules have been correlated. In our experimental conditions,P. falciparum blood forms in vitro tolerate osmolalities ranging from 180 to 360 mOsm. A weak hypo-osmolality (241 mOsm) favored the development of the parasite. The highest sensitivity of the parasite to osmotic variations was observed during schizogony. The merozoite stage and reinvasion process seemed less affected by hypo-osmolalities than by hyperosmolalities. The minor alterations in morphology of the parasites in hypo- and hyperosmotic media suggested thatP. falciparum may have efficient osmoregulatory power.     

The accumulation of lactic acid and its influence on the growth ofPlasmodium falciparum in synchronized cultures

Summary Synchronization ofPlasmodium falciparum cultured in vitro results in a one-step growth pattern that allows the study of stage-specific metabolic activities of the parasites. Lactic acid (LA) was selected as a metabolic marker, and the concentration of this end product found in spent media was correlated with the different erythrocytic stages of the parasites. When the medium was changed at 12 h intervals, cultures containing predominantly trophozoites produced 3.66±0.55 μmol LA per 12 h per 10⁷ parasitized cells (n=26), an amount of LA that is about 8 to 20 times higher than that found in corresponding cultures containing predominantly ring forms. Depending on the stage of development, parasitized red blood cells produced between 5 and 100 times more LA than uninfected erythrocytes (3.72±0.62 μmol LA per 12 hours per 10⁹ red blood cells) (n=41) when cultured under identical conditions. The intraerythrocytic development of the parasites was not impaired by exposure to extracellular concentrations of LA up to 12 mM over a 12 h period. The growth resulting in such cultures was described as uninhibited and was characterized by a multiplication index of 10 or higher. Above the threshold of 12 mM of LA, progressive inhibition of parasite development occurred. The stage-specific LA production reported can be used to predict the amount of LA that will have accumulated at the end of a subsequent 12 h incubation period during synchronized in vitro growth ofPlasmodium falciparum. Using these values, it is possible to establish an optimal medium exchange schedule, thereby assuring uninhibited growth and a correspondingly high parasite yield. J. W. Z. was supported during part of this study by a long-term fellowship of the European Molecular Biology Organization, Heidelberg, West Germany, followed by a Research Associateship from the National Research Council, Washington, D.C. The project was supported by grants from the Medical Research Council to J. G. S. and by the Naval Research and Development Command, Work Unit No. 3M 162 770 A871 AE 312. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the U.S. Navy Department or the naval service at large.     

Circulating immune complexes in rodent and simian malaria

The circulating immune complexes have been detected in the sera of albino rats infected withPlasmodium berghei and rhesus monkeys infected with P.knowlesi by (i) quantitative cryoprecipitation assay and (ii) polyethylene glycol assay. In the rodent model, the levels of circulating immune complexes increased during infection and decreased considerably in the post-infection period. In the simian system, high levels were detected during peak parasitaemia. Polyethylene glycol precipitate obtained from the sera during acuteP. knowlesi infection when analysed by Immunoelectrophoresis was found to contain (i) monkey IgG, (ii) four other components of monkey plasma, (iii) two components of normal monkey erythrocytes and (iv) antigen(s) ofP. knowlesi.     

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.     

Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein

Three long synthetic peptides corresponding to amino (N), repeat (R) and carboxyl (C) regions of the Plasmodium vivax circumsporozoite (CS) protein were synthesised and used to assess their potential as vaccine candidates. Antigenicity studies were carried out using human blood samples from residents of a malaria-endemic area of Colombia, and immunogenicity was tested in Aotus monkeys. The N and C peptides spanned the total native amino and carboxyl flanking regions, whereas the R peptide corresponded to a construct based on the first central nona-peptide repeated in tandem three times and colinearly linked to a universal T-cell epitope (ptt-30) derived from tetanus toxin. All three peptides had been shown previously to contain several B-, T-helper (Th) and Cytotoxic T Lymphocytes (CTL) epitopes. Sixty-one percent of the human sera reacted with the R region, whereas 35 and 39% of the samples had antibodies against the N and C peptides, respectively. Human Peripheral Blood Mononuclear Cells (PBMC) showed higher levels of IFN-γ than IL-4 when stimulated with peptides containing Th epitopes. Aotus monkeys immunised with the peptides formulated in either Montanide ISA720 or Freund's adjuvants produced strong antibody responses that recognised the peptide immunogens and the native circumsporozoite protein on sporozoites. Additionally, high IFN-γ production was induced when Aotus lymphocytes were stimulated in vitro with each of the three peptides. We observed boosting of antibody responses and IFN-γ production by exposure to live sporozoites. These results confirm the high antigenicity and immunogenicity of such synthetic polypeptides and underline their vaccine potential.     

The cultivation of the exoerythrocytic stages ofPlasmodium berghei from sporozoites

Summary Cultures of embryonic rat brain and liver, and embryonic turkey brain were inoculated with sporozoites ofPlasmodium berghei. Sporozoites succeeded in establishing exoerythrocytic infections in approximately 10% of the cultures. The exoerythrocytic parasites developed to a late schizont stage with some showing early segmentation although free merozoites were not observed. The morphology and rate of development of the exoerythrocytic parasites in culture appear similar to that seen in vivo. This work was supported by ONR Contract No. N00014-76-C-1132 and Naval Medical Research and Development Command, Research Work Unit No. M0095PN.002.5058. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in theGuide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.     

Cultivation of the exoerythrocytic stage ofPlasmodium berghei in primary cultures of mouse hepatocytes and continuous mouse cell lines

Summary Plasmodium berghei exoerythrocytic (EE) stages have been cultured in vitro in human continuous cell lines and primary cultures of both human and rat hepatocytes. Although the predominant experimental model of irradiated sporozoite-induced protective immunity is the mouse,P. berghei has not been cultivated in primary mouse hepatocytes or in continuous mouse lines. Because of this, target cells are not available for determining if these immunized mice produce cytotoxic T lymphocytes (CTLs) that recognizeP. berghei antigens expressed on hepatocytes in the context of class I major histocompatability (MHC) antigens. We report the development of methods for cultivating the (EE) stage ofP. berghei in murine hepatocytes and in two cell lines derived from the livers of BALB/c mice; one line produced from a primary hepatocyte culture and the other produced by fusion of mouse hepatocytes with a continuous rat liver line. Mature parasites were detected by microscopy and by DNA probe in both cell lines, each of which supported complete development ofP. berghei liver stages and production of infectious merozoites. Since class I MHC antigens are present on the surface of primary hepatocytes and the mouse X rat hybrid line, these cells can be used to detect cytotoxic T cells against liver stage parasites. This work was supported by the Naval medical Research and Development Command, Bethesda, MD, work unit no. 3M161102B510AK111, ONR contract N00014-83-C-0355, and by contract DPE-0453-C-00-3051-00 of the U.S. Agency for International Development, Washington, D.C. The opinions and assertions herein are not to be construed as official or as reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in the current edition of the “Guide for the Care and Use of Laboratory Animals”, Institute of Laboratory Animal Resources, National Research Council, DHHS, Pub. no. (NIH)85-23     

Functional domains of colicin M

The structure of colicin M of Escherichia coli was studied with regard to its organization into functional domains. A proteolytic fragment with an M_r of 24,000 was isolated which comprised the carboxyterminal portion of the protein. It adsorbed to the outer membrane receptor protein and inhibited killing of cells by colicin M and by phage T5 that uses the same receptor. The fragment killed cells when the outer membrane was rendered permeable to macromolecules for a short time by the osmotic shock procedure. It is concluded that the fragment contains the receptor binding site and the active center but is lacking the sequence required for transport into cells.The carboxy-terminal amino acid sequence-Lys-Arg of the fragment was identical to that obtained from colicin M. Release of lysine and arginine led to inactivation of colicin M. The sequence of the first 39 amino acids of the amino terminal end of colicin M was determined.Abbreviation EGTA ethylene glyco-bis (-aminoethyl ether)-N-N-tetraacetate     

Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

Malaria, one of the world''s most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.     

An assembly zone antigen of the insect cuticle

The assembly zone is a morphologically distinct region in the insect integument that lies between the epidermis and its principal secretory product, the lamellate cuticle. Despite its central location in the process of cuticle formation, little is known about its structure or function. Using various antisera we have shown that in Drosophila melanogaster larvae and pupae the assembly zone is antigenically distinct from the overlying lamellate cuticle. This observation suggests that this region does not contain lamellae in the process of assembling but rather is a stable and permeable matrix through which lamellar components travel in the process of cuticle formation. Curiously an antigen present in the assembly zone was also contained in the moulting gel, indicating a heretofore unsuspected chemical relationship between these two materials.     

Expression and characterization of a parasite-specific antigen on macrophages after infection withLeishmania donovani

A rabbit polyclonal antibody to crude soluble antigen ofLeishmania donovani promastigotes recognized a determinant expressed on the surface membrane of mouse peritoneal macrophages and human monocyte derived macrophages infectedin vitro. The determinant was recognized on infected macrophage surface only when F (ab)₂ fragments of anti-leishmanial antiserum was employed in immunofluorescence. F(ab)₂ fragments of human patient sera also could recognize the determinant. The expression of this antigen was not stage-specific for the parasite. Immunochemical analyses revealed this antigen to be of 51 kDa protein. Specific leaching of membrane proteins by trypsin showed three bands of expressed antigens of 26, 11 and 10 kDa, which in all likelihood might be arising from the 51 kDa antigen. The antigen was not expressed until 12 h of post infection, reached a maximum level at 24 h and thereafter attained a steady state level as studied upto 96 h of post infection. This typc of antigen might have a great potential in immunodiagnostics and site-specific drug targeting.