Biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken.

To evaluate the biosafety of the plasmid pcDNA3-1E of Eimeria acervulina in chicken, two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E at dose of 50 µg/chicken. At the 15 days post-injection, the tissue samples were collected, the total DNA was extracted, and the 3-1E gene was amplified by PCR. Genomic DNA was first purified away from free plasmid using gel electrophoresis, and then assayed for integrated plasmid using PCR amplification of the 3-1E gene. Simultaneously, the environmental dejection samples were collected, the total bacterial DNA was extracted and then transfer of the pcDNA3-1E gene was detected by PCR amplification of the 3-1E gene. Two-week-old chickens were injected intramuscularly with the plasmid pcDNA3-1E with three dosage groups of 100 µg, 500 µg and 2500 µg/chicken for 14 days respectively, and with physiological saline at dose of 2500 µL/chicken as control group for acute toxicity test. A target band of 583 bp was obtained by PCR with chicken genomic DNA as template. If the chicken genomic DNA was purified, no target band could be obtained. It showed that the recombinant plasmid pcDNA3-1E existed in tissues, and no genomic integration of DNA plasmid was detected in the immunized chickens. No target band was found by PCR with environmental dejection bacteria genomic DNA as template. It showed that integration and transfer phenomenon did not exist in environment. The acute toxicity results showed the typical clinical symptoms did not occur in the inoculated chickens, the blood biochemical indices and viscera configuration were not affected significantly in the inoculated group and control group (P>0.05). The results showed that the plasmid pcDNA3-1E was safe and suitable for chicken clinical trials.

Toxoplasma gondii: bioinformatics analysis, cloning and expression of a novel protein TgIMP1.

Toxoplasma gondii is an obligate intracellular protozoan parasite, infecting a large variety of animals and human beings. In recent years, the study of DNA vaccine against T. gondii has made a great progress; however, few vaccines have completely controlled toxoplasmosis. Thus people started to look for more effective antigenic proteins. Here we report a novel T. gondii protein termed immune mapped protein 1 (TgIMP1). We used multiple bioinformatics approaches to predict the physical and chemical characters, signal peptide, transmembrane domain, epitope, topological structure and function of the protein, and we theoretically determined that the TgIMP1 has multiple epitopes, and with immunogenicity, suggesting that the TgIMP1 may be a vaccine candidate against toxoplasmosis. Then the gene coding TgIMP1 was obtained by PCR and connected with cloning vector. Recombinant plasmid was identified by PCR, double digestion and sequencing analysis. Then the TgIMP1 gene was directly inserted into the eukaryotic expression vector pBudCE4.1, so that the recombinant eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed. After identification by PCR and restriction enzyme digestion, the recombinant plasmid pBudCE4.1-TgIMP1 was transfected into cells of HFF, and then identified by RT-PCR. The results showed that the eukaryotic expression plasmid pBudCE4.1-TgIMP1 was constructed and was transfected to the HFF cells successfully.

Comparative efficacy of immunization with inactivated whole tachyzoites versus a tachyzoite-bradyzoite mixture against neosporosis in mice.

The worldwide economic impact of Neospora caninum infection has caused the development of effective vaccines to become one of the main goals in the field of neosporosis research. In this study, the protection conferred by antigens from inactivated whole tachyzoites (TZ) and a tachyzoite-bradyzoite mixture (TZ-BZ) of N. caninum (Nc-Spain7 isolate) incorporated into a water-in-oil emulsion (W/O) and aluminium hydroxide-ginseng extract (Al/G) was evaluated in mouse models of congenital and cerebral N. caninum infection. Immunization with TZ-BZ induced congenital and cerebral neosporosis exacerbation that was mainly characterized by reduced neonatal median survival time and increased parasite presence in adult mouse brains. The immune response of mice immunized with TZ-BZ was characterized by an increase in IFN-γ expression prior to challenge and an increase in IL-4 expression accompanied with significantly higher levels of antibodies against 2 recombinant bradyzoite-specific proteins (rNcSAG4 and rNcBSR4) after challenge. Immunization with TZ in W/O significantly reduced neonatal mortality, vertical transmission as well as parasite presence in adult mouse brains and induced a strong humoral immune response. The current study demonstrates the critical role of stage-specific antigens and adjuvants on the development of effective inactivated vaccines for the prevention of N. caninum infection.

Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi.

Cytotoxic CD8(+) T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8(+) T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8(+) T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4(+) T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8(+) T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

Toxoplasma rhoptries: unique secretory organelles and source of promising vaccine proteins for immunoprevention of toxoplasmosis.

Toxoplasma gondii is an obligate intracellular protozoan parasite classified in the phylum Apicomplexa, which includes numerous notable human and animal pathogens (Plasmodium species, Cryptosporidium species, Neospora caninum, etc.). The invasive stages of apicomplexans are characterized by the presence of an apical complex composed of specialized cytoskeletal and secretory organelles, including rhoptries. Rhoptries, unique apical secretory organelles shared exclusively by all apicomplexan parasites, are known to be involved in an active parasite's penetration into the host cell associated with the biogenesis of specific intracellular compartment, parasitophorous vacuole in which the parasite multiplies intensively, avoiding intracellular killing. Due to the key biological role of rhoptries, rhoptry proteins have recently become vaccine candidates for the prevention of several parasitoses, toxoplasmosis among them. The article presents current data on T. gondii rhoptries biology and new approaches to the development of effective vaccines against toxoplasmosis using rhoptry antigens.

Molecular docking of immunogenic peptide of Toxoplasma gondii and encapsulation with polymer as vaccine candidate.

Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. T. gondii causes primary, chronic infection and mortality. Major surface antigen 1 is the most abundant tachyzoite surface protein and highly conserved between species and causes strong humoural response. Some studies showed that the peptide sequence of surface antigen has immunity. Therefore, tachyzoite surface antigenic peptide sequence is one of the good candidates for vaccine development. However, conformational information and delivery systems are very important parameters for vaccine development. Computational chemistry which is used as an effective method to perform drug or vaccine design provides important information on structure-activity relationship, biological effects of functional groups, molecular geometry, design of enzyme inhibitors and antagonists. The interaction of immunological peptides with protein systems was carried out by means of computing the free energy of binding using the molecular docking technique. Due to the major histocompatibility complex (MHC), proteins play a substantial role for adaptive immunity, the crystal structure of a MHC class I, which plays a pivotal role in the adaptive branch of the immune system, was preferred for docking calculations. A delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles and peptide loaded PLGA nanoparticles was prepared in this study to improve the bioavailability of tachyzoite surface antigenic peptide sequence. Double emulsion method (water-in-oil-in-water or w/o/w) was used for synthesis of PLGA and peptide loaded PLGA nanoparticles. The average particle size, polydispersity index and zeta potential values of PLGA and peptide loaded PLGA nanoparticles were measured with zeta-sizer by using dynamic light scattering (DLS) technique. The scanning electron microscope (SEM) (Zeiss Supra 50 V) was used for imagining the peptide loaded PLGA nanoparticles. Cell toxicity of nanoparticles was assayed on AGS (gastric adenocarcinoma) cell line. To evaluate mitochondrial activity of cells and toxicity studies, XTT methods were carried out. In this study, we aimed to obtain specific immunological peptide loaded PLGA nanoparticles and characterize the formation with FTIR, zeta sizer and SEM imaging, and evaluate cytotoxicity and carry out molecular docking calculations of peptide-MHC protein in order to enlight in vivo events as vaccine candidate against T. gondii.

Vaccination with microneme protein NcMIC4 increases mortality in mice inoculated with Neospora caninum.

NcMIC4 is a Neospora caninum microneme protein that has been isolated and purified on the basis of its unique lactose-binding properties. We have shown that this protein binds to galactosyl residues of lactose; antibodies directed against NcMIC4 inhibit host cell interactions in vitro, thus making it a vaccine candidate. Because of this feature, NcMIC4 was first purified on a larger scale in its native, functionally active form using lactose-agarose affinity chromatography. Second, NcMIC4 was expressed in Escherichia coli as a histidine-tagged recombinant protein (recNcMIC4) and purified through Ni-affinity chromatography. Third, NcMIC4 cDNA was cloned into the mammalian pcDNA3.1 DNA vector and expression was confirmed upon transfection of Vero cells in vitro. For vaccination studies, we employed the murine cerebral infection model based on C57Bl/6 mice, employing experimental groups of 10 mice each. Two groups were injected intraperitoneally with purified native NcMIC4 and recNcMIC4, respectively, employing RIBI adjuvant. The third group was vaccinated intramuscularly with pcDNA-NcMIC4. Control groups included an infection control, an adjuvant control, and a pcDNA3.1 control group. Following 3 injections at 4-wk intervals, mice were challenged by i.p. inoculation of 2 x 10(6) N. caninum tachyzoites (Nc-1 isolate). During the course of parasite challenge (3 wk), mice from the 3 different test groups showed varying degrees of symptoms bearing a semblance to neosporosis, i.e., walking disorder, rounded back, apathy, and paralysis of the hind limbs. Control groups showed no symptoms at all. Most notably, vaccination with pcDNA-MIC4 proved antiprotective, with 60% of mice succumbing to infection within 3 wk, and all mice lacking a measurable anti-NcMIC4 IgG response. NcMIC4 in its native form elicited a substantial humoral IgG1 immune response and a reduction in cerebral parasite load compared to the controls, but 20% of mice succumbed to infection. Vaccination with recNcMIC4 also resulted in 20% of mice dying; however, in this group, cerebral parasite load was similar to the controls, and recNcMIC4 vaccination elicited a mixed IgG1/IgG2 response. In conclusion, vaccines based on NcMIC4, especially pcDNA-NcMIC4, render mice more susceptible to cerebral disease upon challenge with N. caninum tachyzoites.

Immune Response and Protective Efficacy of a Heterologous DNA-Protein Immunization with Leishmania Superoxide Dismutase B1.

Growing evidence shows that antioxidant proteins of Leishmania could be used as vaccine candidates. In this study, we report the efficacy of Leishmania donovani iron superoxide dismutase B1 (LdFeSODB1) as a vaccine antigen in BALB/c mice in a DNA-protein prime-boost immunization regimen in the presence or absence of murine granulocyte macrophage colony stimulating factor (mGMCSF) DNA adjuvant. The expression study confirmed that LdFeSODB1 is expressed in mammalian cells and mGMCSF fusion mediates the secretion of the recombinant protein. Heterologous immunization with LdFeSODB1 induced a strong antibody- and cell-mediated immune response in mice. Immunization triggered a mixed Th1/Th2 response as evidenced by the ratio of IgG2a to IgG1. Antigen-stimulated spleen cells from the immunized mice produced high level IFN-γ. Multiparametric flow cytometry data showed that immunization with LdFeSODB1 induced significantly higher expression of TNF-α or IL-2 by antigen-stimulated T cells. Eight weeks after L. major infection, immunization with the antigen shifted the immune response to a more Th1 type than the controls as demonstrated by IgG2a/IgG1 ratio. Moreover, IFN-γ production by antigen-stimulated spleen cells from immunized mice remained high. The footpad swelling experiment showed that immunization with LdFeSODB1 resulted in partial protection of mice from a high dose L. major infection.

Molecular characterization of a new Babesia bovis thrombospondin-related anonymous protein (BbTRAP2).

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite's growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

Expression, purification and antigenicity of Neospora caninum-antigens using silkworm larvae targeting for subunit vaccines.

Infection of Neospora caninum causes abortion in cattle, which has a serious worldwide impact on the economic performance of the dairy and beef industries. Now, inexpensive and efficacious vaccines are required to protect cattle from neosporosis in livestock industry. In this study, N. caninum surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2) were expressed in hemolymph of silkworm larvae as a soluble form. Expressed SAG1 and SRS2 clearly showed antigenicity against N. caninum-positive sera of cow. SAG1 and SRS2 were purified to near homogeneity from hemolymph of silkworm larvae using anti-FLAG M2 antibody agarose: approximately 1.7 mg of SAG1 from 10 silkworm larvae and 370 µg of SRS2 from 17 silkworm larvae. Mice that were injected by antigens induced antibodies against SAG1 and SRS2. This study indicates that it is possible that this silkworm expression system leads to a large-scale production of N. caninum-antigens with biological function and low production cost. Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system paves the way to produce largely and rapidly these recombinant antigens for its application to subunit vaccines against neosporosis in cattle.

Potency, efficacy and durability of DNA/DNA, DNA/protein and protein/protein based vaccination using gp63 against Leishmania donovani in BALB/c mice.

BACKGROUND: Visceral leishmaniasis (VL) caused by an intracellular protozoan parasite Leishmania, is fatal in the absence of treatment. At present there are no effective vaccines against any form of leishmaniasis. Here, we evaluate the potency, efficacy and durability of DNA/DNA, DNA-prime/Protein-boost, and Protein/Protein based vaccination against VL in a susceptible murine model. METHODS AND FINDINGS: To compare the potency, efficacy, and durability of DNA, protein and heterologous prime-boost (HPB) vaccination against Leishmania donovani, major surface glycoprotein gp63 was cloned into mammalian expression vector pcDNA3.1 for DNA based vaccines. We demonstrated that gp63 DNA based vaccination induced immune responses and conferred protection against challenge infection. However, vaccination with HPB approach showed comparatively enhanced cellular and humoral responses than other regimens and elicited early mixed Th1/Th2 responses before infection. Moreover, challenge with parasites induced polarized Th1 responses with enhanced IFN-γ, IL-12, nitric oxide, IgG2a/IgG1 ratio and reduced IL-4 and IL-10 responses compared to other vaccination strategies. Although, vaccination with gp63 DNA either alone or mixed with CpG- ODN or heterologously prime-boosting with CpG- ODN showed comparable levels of protection at short-term protection study, DNA-prime/Protein-boost in presence of CpG significantly reduced hepatic and splenic parasite load by 107 fold and 10¹° fold respectively, in long-term study. The extent of protection, obtained in this study has till now not been achieved in long-term protection through HPB approach in susceptible BALB/c model against VL. Interestingly, the HPB regimen also showed marked reduction in the footpad swelling of BALB/c mice against Leishmania major infection. CONCLUSION/SIGNIFICANCE: HPB approach based on gp63 in association with CpG, resulted in robust cellular and humoral responses correlating with durable protection against L. donovani challenge till twelve weeks post-vaccination. These results emphasize the potential of DNA-prime/Protein-boost vaccination over DNA/DNA and Protein/Protein based vaccination in maintaining long-term immunity against intracellular pathogen like Leishmania.

IL-12 signaling drives CD8+ T cell IFN-gamma production and differentiation of KLRG1+ effector subpopulations during Toxoplasma gondii Infection.

IFN-gamma-producing CD8(+) T lymphocytes are essential effector cells that mediate protective immunity during murine toxoplasmosis, and yet their effector development remains poorly characterized. Vaccination with the carbamoyl phosphate synthase (CPS) mutant strain of Toxoplasma gondii was used to examine the CD8(+) T cell response in the peritoneal effector site. Four CTL subpopulations with varying effector potentials were defined based on the expression of effector molecules and the cell surface activation markers CD62L and killer cell lectin-like receptor G1 (KLRG1). Further phenotypic analysis revealed that the acquisition of KLRG1 among effector subpopulations correlated with the down-regulation of both IL-7R and CD27, suggesting that KLRG1 marks dominant, end-stage effector cells. Using gene-targeted mice, we tested the in vivo requirements of key IL-12 signaling components for effector CTL differentiation. Contrary to established models of viral and bacterial infection, CD8(+) T cell-intrinsic IL-12 signaling was required for the generation of IFN-gamma-producing CTLs in response to T. gondii. Importantly, the development of the KLRG1(+) effector subpopulations, but not the memory precursor-containing KLRG1(-) effector subset, was critically reliant on IL-12. Furthermore, IL-12 signaling-dependent T-bet expression was also found to be important for differentiation of KLRG1(+) effectors. Our results underscore a vital role for IL-12 in not only the induction of IFN-gamma expression but also in the development of heterogeneous subpopulations of effector CD8(+) T cells generated in response to the intracellular parasite T. gondii.

Toxoplasma gondii: isolation of tachyzoites rhoptries and incorporation into Iscom.

Rhoptries have been isolated from Toxoplasma gondii tachyzoites by subcellular fractionation in isopynic density sucrose gradient. Five bands were observed, and transmission electron microscopy of these indicated that rhoptries were in band 3. This band had a density of 1.17 g/cm(3). Fraction 1 had membrane structures of the parasite. Fraction 2 contained membranes and mitochondria. Fraction 4 had mostly conoid structure and fraction 5 showed ghosts. The electrophoretic and Western blotting analysis of the fractions indicated the presence of a number of proteins. Iscoms were constructed from band 3, which contained the rhoptry structures. Iscom showed a only protein incorporated of 55 kDa. Isolation of the parasite organelles has got in this work is necessary to identification, characterization, and function elucidation of the organelle proteins.

Theileria annulata-infected cells produce abundant proteases whose activity is reduced by long-term cell culture.

Lysates of Theileria annulata-infected bovine lymphoblastoid cells and their uninfected counterparts were tested for protease activity using gelatin substrate SDS-PAGE. The infected cells produced a number of extra activities at pH 8.0 in the presence of Ca2+. Calcium was found to enhance the activities but was not an absolute requirement. Studies using inhibitors, including E64, 3,4-dichloroisocoumarin, pepstatin and 1, 10-phenanthroline suggested that the activities were metalloproteases. We analysed two vaccine lines; the Ode line from India and the Ankara Pendik line from Turkey. In the Ode line the later passage had very much reduced levels of the enzyme activities. In the case of the Ankara Pendik line both stages analysed had very low protease activities, but a reduction from the early to late passage was also observed. The reduction in the level of protease activity was also observed as a gradual process during on-going culture of lines derived from the Hissar and Ode stocks. In the Ode line we demonstrated a parallel decrease in the production of microschizonts upon temperature shift in vitro.

Immune responses and protective effect in mice vaccinated orally with surface sporozoite protein of Eimeria falciformis in ISCOMs.

Immunostimulating complexes (ISCOMs) were built after treatment of a purified surface protein from Eimeria falciformis sporozoites with a palmitic acid derivation, leading to a high ratio (33-64%) of P27 incorporation in these cage-like structures. P27 kept its antigenicity after incorporation in ISCOMs, which induced, after iterative intubations by the oral route to groups of mice, a systemic IgG response, a local IgA response, and a local enhanced cellular response as demonstrated by lymphoproliferation of mesenteric lymph node cells upon in vitro stimulation with antigen. This immunization (120 micrograms in six oral doses at 2-day intervals) afforded mice a partial protection (60%) against a subsequent 400 oocyst challenge. The reduction in daily oocyst excretion was corroborated by significantly different weight losses between immunized and control mice on days 9 and 10 postinfection and the subsequent death of these control mice. These observations provide the first application of ISCOMs to parasitic intestinal diseases.