DNA vaccination with a gene encoding Toxoplasma gondii Deoxyribose Phosphate Aldolase (TgDPA) induces partial protective immunity against lethal challenge in mice

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite that causes a pathological status known as toxoplasmosis, which has a huge impact on human and animal health. Currently, the main control strategy depends on the usage of drugs that target the acute stage of the infection, however, drawbacks were encountered while applying this method; therefore, development of an alternative effective method would be important progress. Deoxyribose Phosphate Aldolase (TgDPA) plays an important role supporting cell invasion and providing energy for the parasite. METHODS: TgDPA was expressed in Escherichia coli and the purified recombinant protein was used to immunize rats. The antibodies obtained were used to verify in vitro expression of TgDPA. The vector pVAX1 was utilized to formulate a DNA vaccine designated as pTgDPA, which was used to evaluate the immunological changes and the level of protection against challenge with the virulent RH strain of T. gondii. RESULTS: DNA vaccine, TgDPA revealed that it can induce a strong humoral as well as cellular mediated response in mice. These responses were a contribution of T(H)1, T(H)2 and T(H)17 type of responses. Following challenge, mice immunized with TgDPA showed longer survival rates than did those in control groups. CONCLUSIONS: Further investigation regarding TgDPA is required to shed more light on its immunogenicity and its possible selection as a vaccine candidate

Cellular Immune Response and Abomasum worm burden in Goats Vaccinated with HC58cDNA Vaccine against H. contortus Infection

Vaccination with DNA vaccines derived from adult H. contortus induces significant level of protection against homologous infection in goat. To date however, mechanism of protection is not well understood, especially in goat. In this study, HC58 DNA vaccinated goats were artificially infected with 5, 000 dose of infective H. contortus L3 (third larval stage), and cellular immune responses and abomasum worm burden examined. The results showed that peripheral CD4+, CD8+ T and B lymphocytes for nematode challenged Groups 1, 2 and 4 increased subsequent to L3 infection compared to negative control Group 3. Likewise, the mean eosinophil and lymphocyte counts increased substantially after vaccination and L3 challenge. On the contrary, circulating neutrophil and white blood cells reduced under similar experimental conditions in goats carrying an equal L3 nematode burden. These findings suggested that regulation of H. contortus expulsion in goat is a complex mechanism orchestrated by CD4+ and CD8+T cells, recruitment of eosinophil and lymphocytes and inclined towards development of Th2 responses. Keywords: Haemonchus contortus; goat; HC58DNA vaccine; cellular immune responses

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite

The Serine/Threonine-Protein Phosphatase 1 From Haemonchus contortus Is Actively Involved in Suppressive Regulatory Roles on Immune Functions of Goat Peripheral Blood Mononuclear Cells

Serine/threonine-protein phosphatases (STPs), as integral constituents of parasitic excretory/secretory proteins, are assumed to be released during the host–parasite interactions. However, knowledge about these phosphatases and their immunoregulatory and immune protective efficiencies with host peripheral blood mononuclear cells (PBMCs) is scant. In this study, an open reading frame of STP from Haemonchus contortus designated as HcSTP-1 was amplified and cloned using reverse-transcription-polymerase chain reaction (RT-PCR) method. The 951-bp nucleotides sequence was encoded to a protein of 316 amino acid residues, conserved in characteristics motifs GDXHG, GDYVDRG, GNHE, HGG, RG, and H. The HcSTP-1 protein was detected at approximately 35 kDa as recombinant protein fused in an expression vector system and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunohistochemically, HcSTP-1 was found to be localized in both male and female adult worm sections. Using immunofluorescence assay, the binding activity of rHcSTP-1 was confirmed on surface of goat PBMCs, which resulted in expression of multiple cytokines and various immunoregulatory activities in vitro. The RT-PCR results showed that mRNA level of interleukin-2, TGF-β1, IFN-γ, and IL-17 (with 10 µg/ml) was upregulated and IL-10 was decreased. However, IL-6 showed no change after PBMCs incubated with rHcSTP-1 protein. Further functional analysis showed that migratory activity of cells, intracellular nitrite production (NO), and apoptotic efficiency of PBMCs were elevated at significant level, whereas the proliferation of goat PBMCs and monocytes-associated major histocompatibility complex (MHC)-I and MHC-II expressions were decreased significantly at concentration-dependent fashion. Our results showed that the HcSTP-1 protein engaged in vital suppressive regulatory roles on host immune cells, which might represent a potential molecular target for controlling H. contortus infection in future

Cloning and characterization of a selenium-independent glutathione peroxidase (HC29) from adult Haemonchus contortus

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus

<i>Eimeria maxima</i> Rhomboid-like Protein 5 Provided Partial Protection against Homologous Challenge in Forms of Recombinant Protein and DNA Plasmid in Chickens

Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX–EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX–EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX–EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX–EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX–EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX–EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis

Transmembrane protein 63A is a partner protein of Haemonchus contortus galectin in the regulation of goat peripheral blood mononuclear cells

Abstract Background Hco-gal-m and -f were two isoforms of galectin cloned from male and female Haemonchus contortus, respectively, and it was demonstrated that recombinant Hco-gal-m and -f could act as immune suppressors. However, little is known about the receptors or binding partners of these galectins in the host. The research of the molecular mechanisms that govern the interactions between these galectins and host molecules will fill a gap in our understanding how parasite galectins interact with host cells. Methods A yeast two-hybrid system was used to identify the binding partners of Hco-gal-m and -f in this research. The interaction between rHco-gal-m and candidate binding protein was validated by co-immunoprecipitation. The localization of transmembrane protein 63A (TMEM63A) in peripheral blood mononuclear cells (PBMCs) was detected by immunofluorescence. The distribution of TMEM63A in T cells, B cells and monocytes in PBMCs was detected by flow cytometry. The immunomodulatory effects of Hco-gal-m and TMEM63A on cell proliferation, migration, apoptosis, nitric oxide production and cytokine secretion were observed by co-incubation of rHco-gal-m and TMEM63A-siRNA with goat PBMCs and monocytes. Results We found that TMEM63A, a functionally unknown protein, from goat PBMCs could bind to Hco-gal-m and -f. Immunofluorescence showed that TMEM63A was localized to the cell membrane. Flow cytometric analysis revealed that TMEM63A was expressed in the majority of goat PBMCs. After using RNA interference to knockdown expression of TMEM63A, the PBMC proliferation and migration were significantly increased, while the influence of rHco-gal-m on monocyte phagocytosis, PBMC nitric oxide production and migration were potently blocked. In addition, the production of IL-10, IFN-γ and TGF-β induced by rHco-gal-m were also altered. Conclusions Our results show that TMEM63A is a binding partner of Hco-gal-m/f, and involved in the immune responses of host PBMCs induced by Hco-gal-m for the first time

Proteomic analysis of protein interactions between Eimeria maxima sporozoites and chicken jejunal epithelial cells by shotgun LC-MS/MS

Abstract Background Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this parasitic pathogen. Methods In the present study, chicken jejunal epithelial cells were isolated and cultured in vitro. Western blot was employed to analyze the soluble proteins of E. maxima sporozoites that bound to chicken jejunal epithelial cells. Co-immunoprecipitation (co-IP) assay was used to separate the E. maxima proteins that bound to chicken jejunal epithelial cells. Shotgun LC-MS/MS technique was used for proteomics identification and Gene Ontology was employed for the bioinformatics analysis. Results The results of Western blot analysis showed that four proteins bands from jejunal epithelial cells co-cultured with soluble proteins of E. maxima sporozoites were recognized by the positive sera, with molecular weights of 70, 90, 95 and 130 kDa. The co-IP dilutions were analyzed by shotgun LC-MS/MS. A total of 204 proteins were identified in the E. maxima protein database using the MASCOT search engine. Thirty-five proteins including microneme protein 3 and 7 had more than two unique peptide counts and were annotated using Gene Ontology for molecular function, biological process and cellular localization. The results revealed that of the 35 annotated peptides, 22 (62.86%) were associated with binding activity and 15 (42.86%) were involved in catalytic activity. Conclusions Our findings provide an insight into the interaction between E. maxima and the corresponding host cells and it is important for the understanding of molecular mechanisms underlying E. maxima invasion