The sodium hydrogen exchanger 1 (NHE1), which functions in maintaining the ratio of Na+ and H+ ions, is widely distributed in cell plasma membranes. It plays a prominent role in pH balancing, cell proliferation, differentiation, adhesion, and migration. However, its exact subcellular location and biological functions in Toxoplasma gondii are largely unclear. In this study, we cloned the C-terminal sequence of T. gondii NHE1 (TgNHE1) incorporating the C-terminal peptide of NHE1 (C-NHE1) into the pGEX4T-1 expression plasmid. The peptide sequence was predicted to have good antigenicity based on the information obtained from an immune epitope database. After induction of heterologous gene expression with isopropyl-b-D-thiogalactoside, the recombinant C-NHE1 protein successfully expressed in a soluble form was purified by glutathione sepharose beads as an immunogen for production of a rabbit polyclonal antiserum. The specificity of this antiserum was confirmed by western blotting and immunofluorescence. The antiserum could reduce T. gondii invasion into host cells, indicated by the decreased TgNHE1 expression in T. gondii parasites that were pre-incubated with antiserum in the process of cell entry. Furthermore, the antiserum reduced the virulence of T. gondii parasites to host cells in vitro, possibly by blocking the release of Ca2+. In this regard, this antiserum has potential to be a valuable tool for further studies of TgNHE1.
Animals ; Cell Line ; Immune Sera/genetics/immunology/*metabolism ; Male ; Mice ; Protozoan Proteins/genetics/*metabolism ; Rabbits ; Recombinant Proteins/immunology ; Sheep ; Sodium-Hydrogen Antiporter/genetics/immunology/*metabolism ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/parasitology/prevention & control

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.
Epitopes, B-Lymphocyte/*chemistry/genetics/*immunology ; Female ; Humans ; Immunodominant Epitopes/chemistry/genetics/*immunology ; Malaria, Vivax/immunology/*parasitology ; Middle Aged ; Plasmodium vivax/chemistry/genetics/*immunology ; Protein Structure, Tertiary ; Protozoan Proteins/chemistry/genetics/*immunology ; Reticulocytes/*parasitology

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.
Amino Acid Sequence ; Antibodies, Protozoan/*blood/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Base Sequence ; Humans ; India ; Malaria, Vivax/*diagnosis/*epidemiology/immunology ; Merozoite Surface Protein 1/genetics/*immunology ; Plasmodium vivax/genetics/immunology ; Protozoan Proteins/genetics/*immunology ; Reagent Kits, Diagnostic ; Recombinant Proteins/diagnostic use/immunology ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Seroepidemiologic Studies ; Uganda

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis

Toxoplasma gondii is an apicomplexan parasite with a broad host range of most warm-blooded mammals including humans, of which one-thirds of the human population has been infected worldwide which can cause congenital defects, abortion, and neonatal complications. Here, we developed a rapid diagnostic test (RDT) for T. gondii infection. Antigenic N-terminal half of the major surface antigen (SAG1) was linked with intrinsically unstructured domain (IUD) of dense granule protein 2 (GRA2). The recombinant GST-GRA2-SAG1A protein was successfully expressed and purified as 51 kDa of molecular weight. Furthermore, antigenicity and solubility of the rGST-GRA2-SAG1A protein were significantly increased. The overall specificity and sensitivity of GST-GRA2-SAG1A loaded RDT (TgRDT) were estimated as 100% and 97.1% by comparing with ELISA result which uses T. gondii whole cell lysates as the antigen. The TgRDT tested with Uganda people sera for field trial and showed 31.9% of seroprevalence against T. gondii antibody. The TgRDT is proved to be a kit for rapid and easy to use with high accuracy, which would be a suitable serodiagnostic tool for toxoplasmosis.
Adolescent ; Adult ; Amino Acid Sequence ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/genetics/*immunology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Molecular Sequence Data ; Protozoan Proteins/genetics/*immunology ; Recombinant Fusion Proteins ; Reproducibility of Results ; Republic of Korea/epidemiology ; Sensitivity and Specificity ; Serologic Tests ; Time Factors ; Toxoplasma/genetics/*immunology/isolation & purification ; Toxoplasmosis/*diagnosis/epidemiology/parasitology ; Uganda/epidemiology ; Young Adult

A 12-year-old spayed female mixed-bred dog presented with nasal bleeding of 2 days duration and a skin nodule in the left flank. No abnormalities were found in coagulation profiles and blood pressure. Cytological evaluation of the nodule revealed numerous characteristic round organisms having a nucleus and a bar within macrophages and in the background, consistent with leishmaniasis. In vitro culture was unsuccessful but PCR of the nodular aspirate identified the organisms as Leishmania infantum, and the final diagnosis was canine leishmaniasis. No history of travel to endemic countries was noted. Because the dog had received a blood transfusion 2 years before the illness, serological screening tests were performed in all donor dogs of the commercial blood bank using the commercial Leishmania ELISA test kit, and there were no positive results. Additional 113 dogs with hyperglobulinemia from Seoul were also screened with the same kits but no positive results were obtained. To the best of the author's knowledge this is the first autochthonous case of canine leishmaniasis in Korea.
Animals ; Base Sequence ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Dog Diseases/*diagnosis/epidemiology ; Dogs ; Enzyme-Linked Immunosorbent Assay/veterinary ; Female ; Giant Cells/pathology ; Leishmania infantum/genetics/immunology/*isolation & purification ; Leishmaniasis, Visceral/diagnosis/*veterinary ; Molecular Sequence Data ; Polymerase Chain Reaction/veterinary ; Protozoan Proteins/genetics ; Republic of Korea ; Sequence Analysis, DNA/veterinary ; Serologic Tests/veterinary

The precise diagnosis of the acute toxoplasmosis in pregnant women and immunocompromsied patients has critical importance. Most of the commercially available assays use the whole Toxoplasma soluble extract as the antigen. However, the assays currently available for the detection of specific anti-Toxoplasma antibodies may vary in their abilities to detect serum immunoglobulins, due to the lack of a purified standardized antigen. The aim of this study was production and evaluation of the usefulness of the recombinant Toxoplasma gondii GRA7 antigen for the serodiagnosis of Toxoplasma gondii IgM and IgG by ELISA. A total of 70 T. gondii IgM positive sera, 74 T. gondii IgG positive sera, and 60 sera from subjects who were not infected with T. gondii were examined. These sera were shown different absorbance values in ELISA test. To control the specificity of the rGRA7 other parasitic diseases, for example, echinococcosis, malaria, leishmaniasis, fascioliasis, and strongyloidiasis were tested of which none showed positive results. Sensitivity and specificity of the generated recombinant IgG ELISA in comparison with commercial ELISA (com ELISA) were 89% and 90%, and the sensitivity and specificity of the generated recombinant IgM ELISA were 96% and 90%, respectively. The results obtained here show that this antigen is useful for diagnostic purposes.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/*diagnostic use/genetics/*isolation & purification ; Enzyme-Linked Immunosorbent Assay/methods ; Female ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Protozoan Proteins/*diagnostic use/genetics/*isolation & purification ; Recombinant Proteins/diagnostic use/genetics/isolation & purification ; Sensitivity and Specificity ; Toxoplasma/*immunology ; Toxoplasmosis/*diagnosis

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.
Antibodies, Protozoan/blood/immunology ; Antigens, Protozoan/*blood/genetics/immunology ; Diagnostic Tests, Routine/*methods ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Humans ; Iran ; Malaria, Vivax/blood/*diagnosis/immunology/*parasitology ; Male ; Membrane Proteins/blood/genetics/immunology ; Plasmodium vivax/isolation & purification/*physiology ; Protozoan Proteins/blood/genetics/immunology ; Sensitivity and Specificity

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8+ T-cells and CD4+ T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8+ cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.
Animals ; Antigens, Protozoan/administration & dosage/genetics/*immunology ; CD8-Positive T-Lymphocytes/*immunology ; COS Cells ; Cercopithecus aethiops ; Humans ; Lymphocyte Activation ; Malaria, Vivax/*immunology/parasitology ; Membrane Proteins/administration & dosage/genetics/*immunology ; Mice ; Mice, Inbred BALB C ; Plasmodium vivax/genetics/*immunology ; Protozoan Proteins/administration & dosage/genetics/*immunology ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Vaccines, DNA/administration & dosage/genetics/*immunology