The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
*Adjuvants, Immunologic ; Animals ; Antibodies, Protozoan/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Cytokines/genetics/immunology ; Euphorbiaceae/*chemistry ; Hypersensitivity, Delayed/immunology ; Immunization ; Immunoglobulin G/immunology ; Latex/chemistry ; Leishmania/immunology ; Leishmaniasis, Cutaneous/*immunology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/genetics/immunology ; Plant Lectins/*immunology/isolation & purification ; Protozoan Vaccines/immunology/pharmacology ; Skin/pathology

OBJECTIVETo identify and clone a new full ORF gene of PyDyn (Plasmodium yoelii dynamin-like protein), and examine the protection of their expression products.

METHODUsing the P. yoelii Genome technology and RT-PCR.

RESULTSThe full ORF gene of PyDyn was amplified from mRNA of the erythrocytic stage of P. yoelii., three domains of PyDyn were expressed in E. coli., and the fairly positive immunogenicity of them was showed by IFA. The full ORF gene of PyDyn was 2,433 bp and encode 811 amino acids. Its Gene Bank access number is AF458071. PyDyn belongs to the dynamin-like protein family according to its property.

CONCLUSIONThe new full ORF gene of PyDyn is obtained and identified; their expressed domains are probably new candidates for malaria vaccine.

Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Dynamins ; genetics ; immunology ; Escherichia coli ; genetics ; Genes, Protozoan ; genetics ; immunology ; Malaria Vaccines ; immunology ; Molecular Sequence Data ; Plasmodium yoelii ; chemistry ; genetics ; Protozoan Proteins ; genetics ; immunology ; Vaccines, Synthetic ; immunology

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

OBJECTIVETo test the immunity of peritoneal monocytes against Plasmodium yoelii infected red blood cells (target cells).

METHODSSaponinized Plasmodium yoelii infected red blood cells (SPRBC, Ghost erythrocyte) were used to immunize mice i.p twice. Three weeks later, the infected red blood cells were injected i.p.; 90 min later, the total peritoneal cells were isolated and washed for scanning electromicroscopy to observe the effects of the peritoneal monocyte to the target cell.

RESULTSThe peritoneal cells of the immunized mice were activated after 90 min of the challenge of target cells. The size of the cell was not even and the pili on the cell surface turned to be long and densed. Cell interconnections were found among the cells. In some peritoneal monocytes, their cell plasma were scattered (omlette-like) or with the shape as "cellular bomb". The scattered or the sheeted pili and spredding cell plasma could adhere to the target cells which were perforated densely and damaged.

CONCLUSIONThe protective adaptive immunity exists in the peritoneal monocytes of immunized mice.

Animals ; Antibodies, Protozoan ; immunology ; Erythrocyte Membrane ; parasitology ; Female ; Malaria Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Monocytes ; immunology ; ultrastructure ; Peritoneum ; cytology ; Plasmodium yoelii ; immunology ; ultrastructure

Rhesus monkeys (5 in each group) were inoculated with recombinant fusion protein of cholera toxin B subunit and multi-valent epitopes of Plasmodium falciparum intranasal or intramuscular (i.m.). Immune-responses and protective effect were evaluated. The antibody titer (Geometry mean) against CTB reached 1:512 (intranasal) and 1:10000 (i.m.) 14 day after 3rd immunization, and antibodies against P. falciparum were also elucidated, the titers in i.m. group were also significantly higher than that in intranasal group. The monkeys were challenged with 1.25 x 10(8) sporozoites of P. cynomolgi, Patent infection was observed in all 5 monkeys in control group inoculated with PBS in 10 - 14 days after challenge. Patent infection was also observed in 5 animals inoculated via intranasal and 2 animals in intramuscular group 19th days after challenge, But the infection last only 4 days in 3 animals in intranasal group and 2 animals in intramuscular group. The results demonstrated that the vaccine candidate could induce protective immune-responses in rhesus monkey against the challenge of P. cynomolgi.
Animals ; Antibodies, Bacterial ; blood ; Antibodies, Protozoan ; blood ; Cholera Toxin ; genetics ; immunology ; Erythrocytes ; parasitology ; Macaca mulatta ; Malaria ; prevention & control ; veterinary ; Malaria Vaccines ; immunology ; Monkey Diseases ; prevention & control ; Plasmodium cynomolgi ; Plasmodium falciparum ; immunology ; Recombinant Fusion Proteins ; immunology ; Vaccines, Synthetic ; immunology

OBJECTIVETo explore the characteristics of protective immunity against Plasmodium yoelii (P.y.) infection by asexual blood-stages cellular vaccine.

METHODSThe particulate vaccines were constructed by saponin or double-distilled-water lysed parasitic red blood cells and inoculated into BALB/c mice by intraperitoneal injection (i.p.). Each group was challenged by the lethal erythrocytic P.y. parasites, and then their parasitemia and survival rates were detected. Expressions of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were detected by RT-PCR. ELISA showed the serum antibodies against the malaria challenge and their-subclasses. Special membrane protein was recognized by immunofluorescence assay.

RESULTSThe vaccination with saponified erythrocytic parasites protected the immunized mice against P.y. challenge, while double-distilled-water lysed vaccine did not (P < 0.01). This protection was characterized by the increase of both IFN-gamma/IgG2a and IL-4/IgG1. Meanwhile, MHC class I alpha chain molecule was recognized on the membrane of infected-erthythrocyte.

CONCLUSIONSaponified P.y. asexual blood-stage cellular vaccine has a significantly high protective immunity against this lethal P.y. malaria, and the immunity may be associated with the expression levels of IgG2a and IFN-gamma. MHC class I alpha chain on infected erythrocytes may play an important role in the successful immunization.

Animals ; Antibodies, Protozoan ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Histocompatibility Antigens Class I ; blood ; Immunoglobulin G ; blood ; Malaria ; prevention & control ; Malaria Vaccines ; immunology ; Mice ; Mice, Inbred BALB C ; Plasmodium yoelii ; immunology ; Random Allocation ; Vaccination

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

To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 microg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5x10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.
Animals ; Antibodies, Protozoan/blood ; Antigens, Protozoan/genetics/*immunology ; Cell Proliferation ; Chickens ; Coccidiosis/immunology/pathology/*prevention & control ; Disease Models, Animal ; Eimeria/genetics/*immunology ; Injections, Intramuscular ; Lymphocytes/immunology ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Vaccination/methods ; Vaccines, DNA/administration & dosage/genetics/*immunology

Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against N. caninum infection was evaluated in vitro and in vivo. Two major immunodominant surface antigens (NcSAG1 and NcSRS2) and two dense granule proteins (NcDG1 and NcDG2) of N. caninum tachyzoites were expressed in E. coli, respectively. An in vitro neutralization assay using polyclonal antisera raised against each recombinant antigen showed inhibitory effects on the invasion of N. caninum tachyzoites into host cells. Separate groups of gerbils were immunized with the purified recombinant proteins singly or in combinations and animals were then challenged with N. caninum. Following these experimental challenges, the protective efficacy of each vaccination was determined by assessing animal survival rate. All experimental groups showed protective effects of different degrees against experimental infection. The highest protection efficacy was observed for combined vaccination with NcSRS2 and NcDG1. Our results indicate that combined vaccination with the N. caninum recombinant antigens, NcSRS2 and NcDG1, induces the highest protective effect against N. caninum infection in vitro and in vivo.
Animals ; Antibodies, Protozoan/immunology ; Antigens, Protozoan/immunology ; Cercopithecus aethiops ; Coccidiosis/prevention & control ; Dose-Response Relationship, Immunologic ; Gene Expression ; Gerbillinae ; Neospora/*immunology ; Protozoan Vaccines/*immunology ; Research Support, Non-U.S. Gov't ; Vaccines, Synthetic/immunology ; Vero Cells

OBJECTIVE: To analyze the RNA binding protein of Toxoplasma gondii (TgDDX39) using bioinformatics technology, and to evaluate the immunogenicity of TgDDX39, so as to provide insights into development of toxoplasmosis vaccines. METHODS: The amino acid sequences of TgDDX39 were retrieved from the ToxoDB database, and the physicochemical properties, transmembrane structure domain, signal peptide sites, post-translational modification sites, coils, secondary and tertiary structures, hydrophobicity, and antigenic epitopes of the TgDDX39 protein were predicted using online bioinformatics tools, incluiding ProtParam, TMHMM 2.0, SignalP 5.0, NetPhos 3.1, COILS, SOPMA, Phyre2, ProtScale, ABCpred, SYFPEITHI and DNA-STAR. RESULTS: TgDDX39 protein was predicted to be an unstable hydrophilic protein with the molecular formula of C₂₁₇₃H₃₄₅₈N₅₉₈O₆₆₁S₁₈, which contained 434 amino acids and had an estimated molecular weight of 49.1 kDa and a theoretical isoelectric point of 5.55. The protein was predicted to have an extremely low possibility of signal peptides, without transmembrane regions, and contain 27 phosphorylation sites. The β turn and random coils accounted for 39.63% of the secondary structure of the TgDDX39 protein, and a coiled helix tended to produce in one site. In addition, the TgDDX39 protein contained multiple B and T cell antigenic epitopes. CONCLUSIONS Bioinformatics analyses predict that TgDDX39 protein has high immunogenicity and contains multiple antigenic epitopes. TgDDX39 protein is a potential candidate antigen for vaccine development.
Humans ; Toxoplasma/metabolism* ; Toxoplasmosis/prevention & control* ; Vaccines ; Epitopes, T-Lymphocyte ; Computational Biology ; Protozoan Proteins/chemistry*