OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.

METHODSThe rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.

RESULTSThe optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.

CONCLUSIONA large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; Antigens, Surface ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Toxoplasma ; immunology

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.
Animals ; Antigens, Protozoan/*biosynthesis ; Brain/parasitology ; Female ; *Gene Expression ; Heat-Shock Proteins/*biosynthesis ; Immunocompromised Host ; Life Cycle Stages ; Lung/parasitology ; Mice ; Protozoan Proteins/*biosynthesis ; Real-Time Polymerase Chain Reaction ; Toxoplasma/*genetics/physiology ; Toxoplasmosis, Animal

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.
Protozoan Proteins/immunology/*isolation & purification ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Life Cycle Stages/immunology ; Leishmaniasis, Cutaneous/immunology ; Leishmania major/*immunology ; Immunoglobulin G/*biosynthesis/blood ; Humans ; Female ; Blotting, Western/methods ; Antigens, Protozoan/immunology/*isolation & purification ; Animals

In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.
Recombinant Proteins/biosynthesis/genetics/*immunology ; Plasmodium vivax/isolation & purification ; Plasmodium falciparum/*immunology ; Molecular Sequence Data ; Malaria, Falciparum/blood/*diagnosis ; Humans ; Genes, Protozoan/genetics/immunology ; Fluorescent Antibody Technique, Direct/methods ; Escherichia coli/genetics ; Enzyme-Linked Immunosorbent Assay/methods ; Early Diagnosis ; DNA, Protozoan/chemistry ; DNA Primers/chemistry ; Cloning, Molecular/methods ; Base Sequence ; Antigens, Protozoan/biosynthesis/chemistry/genetics/*immunology ; Animals ; Amino Acid Sequence

The LACK gene from Leishmania, an analogue of the receptor of activated protein kinase C, was discovered recently. In this study, the LACK gene of Leishmania donovani was obtained from the recombinant plasmid T-LACK by PCR. The gene was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid then was transfected into the eukaryotic cell COS-7, and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction, thus indicating that the recombinant plasmid pcDNA3-LACK can express LACK protein in euka ryotic cell COS-7.
Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; COS Cells ; Cloning, Molecular ; DNA, Recombinant ; biosynthesis ; genetics ; Eukaryotic Cells ; metabolism ; Genetic Vectors ; Leishmania donovani ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vaccines, DNA