Identification of a novel methyltransferase-type 12 protein from Haemonchus contortus and its effects on functions of goat PBMCs.

BACKGROUND: Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host-parasite interaction has not been addressed yet. RESULTS: An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-ß1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. CONCLUSIONS: Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

Molecular characterisation and the protective immunity evaluation of Eimeria maxima surface antigen gene.

BACKGROUND: Coccidiosis is recognised as a major parasitic disease in chickens. Eimeria maxima is considered as a highly immunoprotective species within the Eimeria spp. family that infects chickens. In the present research, the surface antigen gene of E. maxima (EmSAG) was cloned, and the ability of EmSAG to stimulate protection against E. maxima was evaluated. METHODS: Prokaryotic and eukaryotic plasmids expressing EmSAG were constructed. The EmSAG transcription and expression in vivo was performed based on the RT-PCR and immunoblot analysis. The expression of EmSAG in sporozoites and merozoites was detected through immunofluorescence analyses. The immune protection was assessed based on challenge experiments. Flow cytometry assays were used to determine the T cell subpopulations. The serum antibody and cytokine levels were evaluated by ELISA. RESULTS: The open reading frame (ORF) of EmSAG gene contained 645 bp encoding 214 amino acid residues. The immunoblot and RT-PCR analyses indicated that the EmSAG gene were transcribed and expressed in vivo. The EmSAG proteins were expressed in sporozoite and merozoite stages of E. maxima by the immunofluorescence assay. Challenge experiments showed that both pVAX1-SAG and the recombinant EmSAG (rEmSAG) proteins were successful in alleviating jejunal lesions, decreasing loss of body weight and the oocyst ratio. Additionally, these experiments possessed anticoccidial indices (ACI) of more than 170. Higher percentages of CD4+ and CD8+ T cells were detected in both EmSAG-inoculated birds than those of the negative control groups (P < 0.05). The EmSAG-specific antibody concentrations of both the rEmSAG and pVAX1-EmSAG groups were much higher than those of the negative controls (P < 0.05). Higher concentrations of IL-4, IFN-γ, TGF-ß1 and IL-17 were observed more in both the rEmSAG protein and pVAX1-SAG inoculated groups than those of negative controls (P < 0.05). CONCLUSIONS: Our findings suggest that EmSAG is capable of eliciting a moderate immune protection and could be used as an effective vaccine candidate against E. maxima.

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

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.

Protective immunity against Eimeria maxima induced by vaccines of Em14-3-3 antigen.

Eimeria maxima 14-3-3 (Em14-3-3) open reading frame (ORF) which consisted of 861 bp encoding a protein of 286 amino acids was successfully amplified and sequenced. Subsequently, the Em14-3-3 ORF was subcloned into pET-32a (+) and pVAX1, respectively. RT-PCR and immunoblot analyses confirmed that the target gene was successfully transcribed and expressed in vivo. Immunofluorescence analysis showed that Em14-3-3 was expressed in both the sporozoites and merozoites. The animal experiments demonstrated that both rEm14-3-3 and pVAX1-14-3-3 could clearly alleviate jejunum lesions and body weight loss. The Em14-3-3 vaccines could increase oocyst decrease ratio, as well as produce an anticoccidial index of more than 165. The percentages of CD4+ in both the Em14-3-3 immunized groups were much higher, when compared with those of PBS, pET32a (+), and pVAX1 controls (P < 0.05). Similarly, the anti-Em14-3-3 antibody titers of both rEm14-3-3 and pVAX1-14-3-3 immunized groups showed higher levels compared with those of PBS, pET32a (+), and pVAX1 controls (P < 0.05). The IFN-γ and tumor growth factor-ß (TGF-ß) levels showed significant increments in the rEm14-3-3 and pVAX1-14-3-3 immunized groups, when compared with those in the negative controls (P < 0.05). These results demonstrated that Em14-3-3 could be used as a promising antigen candidate for developing vaccines against E. maxima.

Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii 10 kDa excretory-secretory antigen (TgESA10).

Toxoplasma gondii 10 kDa excretory-secretory antigen (TgESA10) is involved in the early stages of host invasion. The aim of this study was to evaluate the immune protective efficacy of a DNA vaccine encoding TgESA10 gene against acute T. gondii infection in mice. The gene sequence encoding TgESA10 was inserted into the eukaryotic expression vector pVAX I, and the efficacy of intramuscular vaccination of BALB/c mice with pVAX-ESA10 was analyzed. Mice immunized with pVAX-ESA10 elicited high titers of total IgG, IgG1, IgG2a, IgA and IgM antibodies, while IgE showed no changes. Analysis of cytokine profiles revealed significant increases of IFN-γ, IL-4 and IL-17, while no significant changes were detected in TGF-ß1. Additionally, we found that pVAX-ESA10 enhanced the activation of CD4(+) and CD8(+) T cells and the expression of MHC-I and MHC-II molecules in spleen in mice. Immunization with pVAX-ESA10 significantly prolonged survival time (14.3 ± 1.7 days) after challenge infection with the virulent T. gondii RH strain, compared with the control groups which died within 8 days. These results suggested that TgESA10 DNA vaccine could trigger strong humoral and cellular responses and induce partial protection against acute toxoplasmosis.

Protective immunity against acute toxoplasmosis in BALB/c mice induced by a DNA vaccine encoding Toxoplasma gondii elongation factor 1-alpha.

BACKGROUND: Toxoplasma gondii can infect almost all warm-blood animals including human beings. The high incidence and severe damage that can be caused by T. gondii infection clearly indicates the need for the development of a vaccine. T. gondii elongation factor 1-alpha (TgEF-1α) plays an important role in pathogenesis and host cell invasion for this parasite. The aim of this study was to evaluate the immune protective efficacy of a DNA vaccine encoding TgEF-1α gene against acute T. gondii infection in mice. METHODS: A DNA vaccine (pVAX-EF-1α) encoding T. gondii EF-1a (TgEF-1α) gene was constructed and its immune response and protective efficacy against lethal challenge in BALB/c mice were evaluated. RESULTS: Mice inoculated with the pVAX-EF-1α vaccine had a high level of specific anti-T. gondii antibodies and produced high levels of IFN-gamma, interleukin (IL)-4, and IL-17. The expression levels of MHC-I and MHC-II molecules as well as the percentages of both CD4(+) and CD8(+) T cells in mice vaccinated with pVAX-EF-1α were significantly increased (p < 0.05), compared with those in all the mice from control groups (blank control, PBS, and pVAXI). Immunization with pVAX-EF-1α significantly (p < 0.05) prolonged mouse survival time to 14.1 ± 1.7 days after challenge infection with the virulent T. gondii RH strain, compared with mice in the control groups which died within 8 days. CONCLUSIONS: DNA vaccination with pVAX-EF-1α triggered strong humoral and cellular responses and induced effective protection in mice against acute T. gondii infection, indicating that TgEF-1α is a promising vaccine candidate against acute toxoplasmosis.