Protective efficacy of multiepitope vaccines constructed from common antigens of Eimeria species in chickens.

Clinical avian coccidiosis is typically caused by coinfection with several Eimeria species. Recombinant protein and DNA vaccines have shown promise in controlling coccidiosis. On this basis, DNA vaccines that encode multiple epitopes from different Eimeria species may provide broad protection against coinfections. In this study, we designed a fusion gene fragment, 14EGT, that contained concentrated T-cell epitopes from four common antigens of Eimeria species (14-3-3, elongation factor 2, glyceraldehyde-3-phosphate dehydrogenase, and transhydrogenase). The multiepitope DNA vaccine pVAX1-14EGT and recombinant protein vaccine pET-32a-14EGT (r14EGT) were then created based on the 14EGT fragment. Subsequently, cellular and humoral immune responses were measured in vaccinated chickens. Vaccination-challenge trials were also conducted, where the birds were vaccinated with the 14EGT preparations and later exposed to single or multiple Eimeria species to evaluate the protective efficacy of the vaccines. According to the results, vaccination with 14EGT preparations effectively increased the proportions of CD4+ and CD8+ T cells and the levels of Th1 and Th2 hallmark cytokines. The levels of serum IgG antibodies were also significantly increased. Animal vaccination trials revealed alleviated enteric lesions, weight loss, and oocyst output compared to those of the control groups. The preparations were found to be moderately effective against single Eimeria species, with the anticoccidial index (ACI) ranging from 160 to 180. However, after challenge with multiple Eimeria species, the protection provided by the 14EGT preparations was not satisfactory, with ACI values of 142.18 and 146.41. Collectively, the results suggest that a multiepitope vaccine that encodes the T-cell epitopes of common antigens derived from Eimeria parasites could be a potential and effective strategy to control avian coccidiosis.

Haemonchus contortus HcL6 promoted the Th9 immune response in goat PBMCs by activating the STAT6/PU.1/NF-κB pathway.

Th9 cells play a crucial role in parasite immunity. The development of Th9 cells is facilitated by several cytokines. Key transcription factors, such as STAT6, STAT5, and PU.1, are known to enhance IL-9 expression during the Th9 immune response. NF-κB-mediated transduction pathways participate in the induction of IL-9. In a previous study, we unveiled a unique ribosomal protein derived from Haemonchus contortus excretory-secretory proteins (HcESPs) that interact with host Th9 cells. In the present study, the effects of the Haemonchus contortus ribosomal protein L6 domain DE-containing protein (HcL6) on IL-9 secretion, Th9 differentiation, and IL-9 transcription were assessed by employing ELISA, flow cytometry, and qPCR methodologies. The observations revealed the transcriptional upregulation of several key genes within the Th9 immune response pathway. Moreover, silencing STAT6, PU.1, and NF-κB was found to attenuate the Th9 immune response. In this study, we unveiled the Th9 immune response-inducing capabilities of HcL6 and elucidated some of its underlying mechanisms. These findings suggest that HcL6 is an immunostimulatory antigen capable of inducing the Th9 immune response. These insights could prove instrumental in identifying potential candidate antigens for the development of immunoprophylactic strategies against H. contortus infections.

Hepatocellular carcinoma-associated antigen 59 and ADP-ribosylation factor 1 with poly (lactic-co-glycolic acid): A promising candidate as nanovaccine against haemonchosis.

Haemonchus contortus (H. contortus) ADP-ribosylation factor 1 (Hc-ARF1) and Hepatocellular carcinoma-associated antigen 59 (Hc-HCA59) are recognized to largely regulate the immune responses of host cells. However, studies about the protective efficacy of the two molecules are poorly unknown. In this research, combinations of recombinant Hc-HCA59 (rHc-HCA59) and Hc-ARF1 (rHc-ARF1) proteins were amalgamated with poly (lactic-co-glycolic acid) (PLGA) nanoparticles adjuvant in order to investigate their protection potential against H. contortus in goats. The results demonstrated that the levels of IgG, IgA, IgE, and IL-4 were noticeably enhanced in the rHc-HCA59 and rHc-ARF1 (rHc-HCA59+rHc-ARF1) group before H. contortus third-stage larvae (L3) challenge. After the L3 challenge, the levels of IL-17, IL-9, and TGF-ß were considerably upregulated in the rHc-HCA59+rHc-ARF1 group. In the meantime, the abomasal worm burdens and the fecal eggs were reduced by 63.2% and 69.4% respectively in the rHc-HCA59+rHc-ARF1 group. According to the studies, PLGA nanoparticles immobilized with rHc-HCA59 and rHc-ARF1 proteins conferred partial protection and were expected to be a potential candidate for developing nano vaccines to combat goat haemonchosis.

Identification of excretory and secretory proteins from Haemonchus contortus inducing a Th9 immune response in goats.

Th9 cells have been shown to play crucial roles in anti-parasite immunity, pathogenic microbe infection, and allergy. Previous studies have demonstrated that Haemonchus contortus excretory and secretory proteins (HcESPs) induce the proliferation of Th9 cells and alter the transcriptional level of IL-9 as well as its related pathways in the Th9 immune response after infection. However, the exact molecule(s) in HcESPs inducing the Th9 immune response is not yet known. In this study, flow cytometry, co-immunoprecipitation (Co-IP) and shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) were used, and a total of 218 proteins from HcESPs that might interact with goat Th9 cells were identified. By in vitro culture of Th9 cells with HcESPs, 40 binding proteins were identified. In vivo, 38, 47, 42 and 142 binding proteins were identified at 7, 15, 35 and 50 days post-infection (dpi), respectively. Furthermore, 2 of the 218 HcESPs, named DNA/RNA helicase domain containing protein (HcDR) and GATA transcription factor (HcGATA), were confirmed to induce the proliferation of Th9 cells and promote the expression of IL-9 when incubated with goat peripheral blood mononuclear cells (PBMCs). This study represents a proteomics-guided investigation of the interactions between Th9 cells and HcESPs. It provides a new way to explore immunostimulatory antigens among HcESPs and identifies candidates for immune-mediated prevention of H. contortus infection.

A multiepitope vaccine encoding four Eimeria epitopes with PLGA nanospheres: a novel vaccine candidate against coccidiosis in laying chickens.

With a worldwide distribution, Eimeria spp. could result in serious economic losses to the poultry industry. Due to drug resistance and residues, there are no ideal drugs and vaccines against Eimeria spp. in food animals. In the current study, a bioinformatics approach was employed to design a multiepitope antigen, named NSLC protein, encoding antigenic epitopes of E. necatrix NA4, E. tenella SAG1, E. acervulina LDH, and E. maxima CDPK. Thereafter, the protective immunity of NSLC protein along with five adjuvants and two nanospheres in laying chickens was evaluated. Based on the humoral immunity, cellular immunity, oocyst burden, and the coefficient of growth, the optimum adjuvant was evaluated. Furthermore, the optimum immune route and dosage were also investigated according to the oocyst burden and coefficient of growth. Accompanied by promoted secretion of antibodies and enhanced CD4+ and CD8+ T lymphocyte proportions, NSLC proteins entrapped in PLGA nanospheres were more effective in stimulating protective immunity than other adjuvants or nanospheres, indicating that PLGA nanospheres were the optimum adjuvant for NSLC protein. In addition, a significantly inhibited oocyst burden and growth coefficient promotion were also observed in animals vaccinated with NSLC proteins entrapped in PLGA nanospheres, indicating that the optimum adjuvant for NSLC proteins was PLGA nanospheres. The results also suggested that the intramucosal route with PLGA nanospheres containing 300 µg of NSLC protein was the most efficient approach to induce protective immunity against the four Eimeria species. Collectively, PLGA nanospheres loaded with NSLC antigens are potential vaccine candidates against avian coccidiosis.

Recombinant dynein light intermediate chain of Haemonchus contortus affects the functions of goat immune cells in vitro.

Haemonchus contortus dynein light intermediate chain (HcLIC), an essential excretory/secretory protein of Haemonchus contortus, has been shown to have antigenic features. Neverthless, understanding of its immunomodulatory roles on host immune cells remains limited. Herein, HcLIC gene was amplified by polymerase chain reaction (PCR) and cloned in prokaryotic expression vector pET32a. The protein was expressed by IPTG and purified by affinity chromatography using HisTrap™ FF column. The localization of HcLIC in adult H. contortus woms was detected by immunohistochemical analysis. Immunofluorescence assay (IFA) was carried out to test the binding ability of rHcLIC to goat peripheral blood mononuclear cells (PBMCs). Furthermore, the effects of HcLIC on cell migration and cell apoptosis were evaluated when goat PBMCs were co-incubated with rHcLIC protein. The results revealed that rHcLIC was expressed in the cuticle tissues of adult H. contortus. IFA confirmed the binding of HcLIC on the surface of goat PBMCs. Moreover, functional analysis revealed that the interaction between rHcLIC and host immune cells significantly suppressed cell migration, suggesting that parasite might lessen the production of cytokines and chemokines that signal the migration of host immune cells towards infection site. Moreover, rHcLIC treatment improved cell apoptosis efficiency which might lower the immune cells quantity and thereby downregulate host immunity, enabling parasite survival within host. These results suggested that decrease trend of migration along with induction of apoptosis might be an immunosuppressive strategy of H. contortus. Overall, these findings add to our understanding of HcLIC, and the mechanisms involved in H. contortus immune escape during host-parasite interaction.

Em14-3-3 delivered by PLGA and chitosan nanoparticles conferred improved protection in chicken against Eimeria maxima.

Eimeria maxima (E. maxima) are an intracellular apicomplexan protozoan that causes intestinal coccidiosis in chickens. The purpose of this research was to develop a novel delivery approach for recombinant E. maxima (rEm) 14-3-3 antigen to elicit enhanced immunogenic protection using poly (D, L-lactide-co-glycolide) (PLGA) and chitosan (CS) nanoparticles (NPs) against E. maxima challenge. The morphologies of prepared antigen-loaded NPs (PLGA/CS-rEm14-3-3 NPs) were visualized by a scanning electron microscope. The rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs-immunized chicken-induced changes of serum cytokines, IgY-antibody level, and T-lymphocyte subsets and protective efficacies against E. maxima challenge were evaluated. The results revealed that encapsulated rEm14-3-3 in PLGA and CS NPs presented spherical morphology with a smooth surface. The chickens immunized with only rEm14-3-3 and PLGA/CS-rEm14-3-3 NPs elicited a significant (p<0.05) higher level of IFN-γ cytokine, stimulated the proportions of CD4+/CD3+, CD8+/CD3+ T-cells, and provoked sera IgY-antibody immune response compared to control groups (PBS, pET-32a, PLGA, and CS). Whereas, PLGA-rEm14-3-3 NP-immunized chicken provoked a higher level of IFN- γ production and IgY-antibody response rather than CS-rEm14-3-3 and bare antigen, relatively. The animal experiment results ratified that PLGA-rEm14-3-3 NP-immunized chicken significantly alleviated the relative body weight gain (%), decreased lesion score, and enhanced oocyst decrease ratio compared to CS-rEm14-3-3 NPs and only rEm14-3-3. The anti-coccidial index of the chicken vaccinated with the PLGA-rEm14-3-3 NPs was (180.1) higher than that of the Cs-rEm14-3-3 NPs (167.4) and bare antigen (165.9). Collectively, our statistics approved that PLGA NPs might be an efficient antigen carrier system (Em14-3-3) to act as a nanosubunit vaccine that can improve protective efficacies in chicken against E. maxima challenge.

Histone deacetylase SIR2 in Toxoplasma gondii modulates functions of murine macrophages in vitro and protects mice against acute toxoplasmosis in vivo.

Silent information regulator 2 (SIR2) in histone deacetylase (HDAC) is particularly conserved and widely expressed in all eukaryotic cells. HDAC is a crucial post-translational modification protein regulating gene expression. In the present study, a Toxoplasma gondii (T. gondii) silent information regulator 2 (TgSIR2) gene in HDAC was cloned and the modulation effects of recombinant TgSIR2 (rTgSIR2) on murine Ana-1 macrophages were characterized in vitro. The results indicated that rTgSIR2 had a good capacity to eliminate T. gondii by promoting proliferation, apoptosis, and phagocytosis, and modulating the secretion of nitric oxide (NO), pro-inflammatory cytokines, and anti-inflammatory cytokines. In in vivo experiments, animals were immunized with recombinant TgSIR2, followed by a lethal dose of T. gondii RH strain challenge 14 days after the second immunization. As compared to the blank and control group, the animals immunized with rTgSIR2 could generate specific humoral responses, as demonstrated by the significantly high titers of total IgG and subclasses IgG1 and IgG2a. Significant increases of IFN-γ, IL-4, and IL-10 were seen, while no significant changes were detected in IL-17. The percentage of CD4+ and CD8+ T lymphocytes in animals immunized with rTgSIR2 significantly increased. A significantly long survival time was also observed in animals vaccinated with rTgSIR2 14 days after the last immunization. All these results clearly indicate that rTgSIR2 played an essential role in modulating host macrophages and offered the potential to develop a therapeutic strategy against T. gondii.

Characteristics of Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase of the parasitic nematode Haemonchus contortus and its modulatory functions on goat PBMCs in vitro.

Biotin lipoyl attachment and 2-oxoacid dehydrogenase acyltransferase (BLAODA), as an essential excretion of Haemonchus contortus (HcESPs), was identified to have antigenic functions. T helper-9 (Th9) cells secrete interleukin (IL)-9, a signature cytokine associated with tumour immunology, allergy and autoimmunity. Nonetheless, the understanding of modulatory functions of BLAODA on Th9 and other immune cells is limited. In this study, the BLAODA gene was cloned, and the recombinant (r) protein of BLAODA (rHcBLAODA) was expressed and immunoblotting was performed. The results revealed that HcBLAODA gene was successfully cloned and rHcBLAODA protein was expressed. The localization of rHcBLAODA was confirmed on the surface of gut sections from adult H. contortus. The rHcBLAODA protein capability to react precisely with anti-H. contortus antibodies were confirmed by immunoblotting and immunofluorescence assay (IFA). Further functional analysis showed that interaction of rHcBLAODA with host cells significantly enhanced Th9 cells generation, IL-9 expression, nitric oxide production and cell apoptosis while suppressing the cells proliferation and cells migration depending on the concentration. Overall, these findings suggest that rHcBLAODA protein could modulate the host immune response by inducing Th9 cells to secrete IL-9 cytokine in vitro.

Rhomboid protein 2 of Eimeria maxima provided partial protection against infection by homologous species.

Rhomboid-like proteases (ROMs) are considered as new candidate antigens for developing new-generation vaccines due to their important role involved in the invasion of apicomplexan protozoa. In prior works, we obtained a ROM2 sequence of Eimeria maxima (EmROM2). This study was conducted to evaluate the immunogenicity and protective efficacy of EmROM2 recombinant protein (rEmROM2) and EmROM2 DNA (pVAX1-EmROM2) against infection by Eimeria maxima (E. maxima). Firstly, Western blot assay was conducted to analyze the immunogenicity of rEmROM2. The result showed that rEmROM2 was recognized by chicken anti-E. maxima serum. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay revealed apparent transcription and expression of EmROM2 at the injection site. qRT-PCR (quantitative real-time PCR), flow cytometry and indirect ELISA indicated that vaccination with rEmROM2 or EmROM2 DNA significantly upregulated the transcription level of cytokines (IFN-γ, IL-2, IL-4, IL-10, IL-17, TGF-ß and TNF SF15), the proportion of CD8+ and CD4+ T lymphocytes and serum IgG antibody response. Ultimately, a vaccination-challenge trial was performed to evaluate the protective efficacy of rEmROM2 and pVAX1-EmROM2 against E. maxima. The result revealed that vaccination with rEmROM2 or pVAX1-EmROM2 significantly alleviated enteric lesions, weight loss, and reduced oocyst output caused by challenge infection of E. maxima, and provided anticoccidial index (ACI) of more than 160, indicating partial protection against E. maxima. In summary, vaccination with rEmROM2 or pVAX1-EmROM2 activated notable humoral and cell-mediated immunity and provided partial protection against E. maxima. These results demonstrated that EmROM2 protein and DNA are promising vaccine candidates against E. maxima infection.

Protection studies of an excretory-secretory protein HcABHD against Haemonchus contortus infection.

Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/ß-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory-secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.

The excretory-secretory antigen HcADRM1 to generate protective immunity against Haemonchus contortus.

The prevention, treatment and control of Haemonchus contortus have been increasingly problematic due to its widespread occurrence and anthelmintic resistance. There are very few descriptions of recombinant antigens being protective for H. contortus, despite the success of various native antigen preparations, including Barbervax. We recently identified an H. contortus excretory­secretory antigen, H. contortus adhesion-regulating molecule 1 (HcADRM1), that served as an immunomodulator to impair host T-cell functions. Given the prophylactic potential of HcADRM1 protein as a vaccine candidate, we hereby assessed the efficacies of HcADRM1 preparations against H. contortus infection. Parasitological and immunological parameters were evaluated throughout all time points of the trials, including fecal egg counts (FEC), abomasal worm burdens, complete blood counts, cytokine production profiles and antibody responses. Active vaccination with recombinant HcADRM1 (rHcADRM1) protein induced protective immunity in inoculated goats, resulting in reductions of 48.9 and 58.6% in cumulative FEC and worm burdens. Simultaneously, passive administration of anti-HcADRM1 antibodies generated encouraging levels of protection with 46.7 and 56.2% reductions in cumulative FEC and worm burdens in challenged goats. In addition, HcADRM1 preparations-immunized goats showed significant differences in mucosal and serum antigen-specific immunoglobulin G (IgG) levels, total mucosal IgA levels, haemoglobin values and circulating interferon-γ, interleukin (IL)-4 and IL-17A production compared to control goats in both trials. The preliminary data of these laboratory trials validated the immunoprophylactic effects of rHcADRM1 protein. It can be pursued as a potential vaccine antigen to develop an effective recombinant subunit vaccine against H. contortus under field conditions.

Actin-depolymerizing factor from Eimeria tenella promotes immunogenic function of chicken dendritic cells.

Dendritic cells play a crucial role in inducing antigen-specific immunity to pathogens. During host-parasite interaction, host immune response to the parasite molecules is considered essential for recognizing novel antigens for control strategies. Therefore, in the present study, chicken dendritic cells (DCs) (ChDCs), derived from spleens were used to evaluate their capacity to proliferate and differentiate autologous T lymphocytes in response to actin-depolymerizing factor from Eimeria tenella (EtADF). Immunoblot analysis showed that recombinant EtADF protein (rEtADF) was able to interact with rat anti-rEtADF antibodies. The immunofluorescence test confirmed rEtADF binding on ChDCs surface. Flow cytometric analysis revealed that phenotypes for MHCII, CD1.1, CD11c, CD80, and CD86 were increased in ChDCs after rEtADF treatment. qRT-PCR results indicated that ChDCs triggered TLR signaling in response to rEtADF, and suppressed Wnt signaling. Transcript levels of CD83, CCL5, and CCR7 in ChDCs were improved following rEtADF treatment. In addition, rEtADF promoted DC-directed T cell proliferation and differentiation of naïve T cells into CD3+/CD4+ T cells in DC/T cell co-incubation system. Cytokine analysis of rEtADF-pulsed ChDCs showed increased levels of IL-12 and IFN-γ, while IL-10 and TGF-ß remained unchanged. Moreover, rEtADF-treated ChDCs enhanced production of IFN-γ when incubated with T cells, and IL-4 secretion remained unchanged. Our findings indicted that rEtADF could facilitate the polarization of Th1 immune cells by triggering both host DCs and T cells. Our findings provide useful insights into future work aimed at anticoccidial vaccine strategies.

Recombinant ubiquitin-conjugating enzyme of Eimeria maxima induces immunogenic maturation in chicken splenic-derived dendritic cells and drives Th1 polarization in-vitro.

Dendritic cells (DCs) are key linkages between innate immunity and acquired immunity. The antigens that promote the functions of DCs might be the effective candidates of novel vaccine. In this research, the ability of ubiquitin-conjugating enzyme (UCE), a recognized common antigens among chicken Eimeria species, to stimulate DCs of chickens were evaluated. We cloned UCE gene from Eimeria maxima (EmUCE), and its protein expression was confirmed by SDS-PAGE and western-blot. Immunofluorescence assay confirmed the binding of rEmUCE on the surface of chicken splenic-derived DCs (ChSP-DCs). Flow cytometric analysis showed that rEmUCE-treated ChSP-DCs increased MHCII, CD1.1, CD11c, CD80, and CD86 phenotypes. qRT-PCR indicated that transcript levels of maturation markers CCL5, CCR7, and CD83 in ChSP-DCs were upregulated in response to rEmUCE. Following rEmUCE treatment, chSP-DCs activated TLR signaling and inhibited Wnt signaling. Moreover, rEmUCE promoted DC-mediated T-cell proliferation in DC/T-cell co-incubation. Interestingly, CD3+/CD4+ T-cells were significantly enhanced when rEmUCE-treated chSP-DCs were co-incubated with T-cells. Cytokine secretion pattern of rEmUCE-stimulated ChSP-DCs revealed that the production of IL-12 and IFN-γ was increased whereas IL-10 and TGF-ß were unchanged. Likewise, the co-incubation of ChSP-DCs with T-cells indicated increased production of IFN-γ but not IL-4. Collectively, rEmUCE could polarize DCs to immunogenic phenotype and shift the immune cells towards Th1 response. Our observations provide valuable insight for future research aimed at vaccine development against avian coccidiosis.

Recombinant elongation factor 1 alpha of Haemonchus contortus affects the functions of goat PBMCs.

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF-1α) was cloned and expressed. The influences of recombinant HcEF-1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF-1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF-1α was bound on surface of PBMCs. Moreover, the productions of IL-4, TGF-ß1, IFN-γ and IL-17 of cells were significantly modulated by the incubation with rHcEF-1α. The production of interleukin IL-10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF-1α was increased significantly, whereas MHC-I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF-1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host-parasite relationship.

Proteomic analysis revealed T cell hyporesponsiveness induced by Haemonchus contortus excretory and secretory proteins.

Haemonchus contortus has evolved highly integrated and sophisticated mechanisms to promote coexistence with hosts. The excretory-secretory (ES) products generated by this parasite contribute to the regulation of the host immune response to facilitate immune evasion and induce chronicity, but the proteins responsible for this process and the exact cellular mechanisms have yet to be defined. In this study, we identified 114 H. contortus ES proteins (HcESPs) interacting with host T cells and 15 T cell binding receptors via co-immunoprecipitation and shotgun liquid chromatography-tandem mass spectrometry analysis. Based on bioinformatics analysis, we demonstrated that HcESPs could inhibit T cell viability, induce cell apoptosis, suppress T cell proliferation and cause cell cycle arrest. Furthermore, the stimulation of HcESPs exerted critical control effects on T cell cytokine production profiles, predominantly promoting the secretion of interleukin (IL)-10, IL-17A and transforming growth factor-ß1 and inhibiting IL-2, IL-4 and interferon-γ production. Collectively, these findings may provide insights into the interaction between ES proteins and key host effector cells, enhancing our understanding of the molecular mechanism underlying parasite immune evasion and providing new clues for novel vaccine development.

Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina modulates the functions of chicken dendritic cells to boost Th1 type immune response and stimulates autologous CD4+ T cells differentiation in-vitro.

Dendritic cells (DCs) play a pivotal role to amplify antigen-specific immune responses. Antigens that sensitize T cells via antigen-presentation by DCs could enhance the capacity of host immunity to fight infections. In this study, we tested the immunogenic profiles of chicken DCs towards Glyceraldehyde-3-phosphate dehydrogenase from Eimeria acervulina (EaGAPDH). Immunoblot analysis showed that recombinant EaGAPDH (rEaGAPDH) protein was successfully recognized by rat sera generated against rEaGAPDH. Interaction and internalisation of rEaGAPDH by chicken splenic-derived DCs (chSPDCs) was confirmed by immunofluorescence analysis. Flow cytometry revealed that chSPDCs upregulated MHCII, CD1.1, CD11c, CD80, and CD86 cell-surface markers. Moreover, mRNA expressions of DC maturation biomarkers (CCL5, CCR7, and CD83) and TLR signalling genes (TLR15 and MyD88) were also upregulated whereas those of Wnt signalling were non-significant compared to negative controls. rEaGAPDH treatment induced IL-12 and IFN-γ secretion in chSPDCs but had no effect on IL-10 and TGF-ß. Likewise, DC-T cell co-culture promoted IFN-γ secretion and the level of IL-4 was unaffected. Proliferation of T cells and their differentiation into CD3+/CD4+ T cells were triggered in chSPDCs-T cells co-culture system. Taken together, rEaGAPDH could promote Th1 polarization by activating both host DCs and T cells and sheds new light on the role of this important molecule which might contribute to the development of new DCs-based immunotherapeutic strategies against coccidiosis.

EtMIC3 and its receptors BAG1 and ENDOUL are essential for site-specific invasion of Eimeria tenella in chickens.

Avian coccidian parasites exhibit a high degree of site specificity in different Eimeria species. Although the underlying mechanism is unclear, an increasing body of evidence suggests that site specificity is due to the interaction between microneme proteins (MICs) and their receptors on the surface of target host cells. In this study, the binding ability of E. tenella MICs (EtMICs) to different intestinal tissue was observed by immunofluorescence to identify the key surface molecule on the parasite responsible for the site specificity. Subsequently, we identified the corresponding host-cell receptors by yeast two-hybrid screening and glutathione-S-transferase pull-down experiments and the distribution of these receptors was observed by immunofluorescence in chicken intestinal tissues. Finally, we evaluated the efficacy of receptor antiserum against the infection of E. tenella in chickens. The results showed that EtMIC3 could only bind to the caecum while EtMIC1, EtMIC2, and EtAMA1 did not bind to any other intestinal tissues. Anti-serum to EtMIC3 was able to block the invasion of sporozoites with a blocking rate of 66.3%. The receptors for EtMIC3 were BCL2-associated athanogene 1 (BAG1) and Endonuclease polyU-specific-like (ENDOUL), which were mainly distributed in the caecum. BAG1 and ENDOUL receptor antiserum reduced weight loss and oocyst output following E. tenella infection, showing partial inhibition of E. tenella infection. These data elucidate the mechanism of site specificity for Eimeria infection and reveal a potential therapeutic avenue.

Recombinant cold shock domain containing protein is a potential antigen to detect specific antibody during early and late infections of Haemonchus contortus in goat.

BACKGROUND: Haemonchus contortus (H. contortus) is one of the most important parasites that cause huge economic losses to small ruminant industry worldwide. Effective prognosis and treatment depend upon the early diagnosis of H. contortus infection. To date, no widely-approved methods for the identification of prepatent H. contortus infection are available to identify prepatent H. contortus infection properly. The aim of this study was to evaluate the diagnostic potential of recombinant cold shock H. contortus protein (rHc-CS) during early and late infections of H. contortus in goat. RESULTS: Purified rHc-CS exhibited a clear band, with a molecular weight about 38 kDa. H. contortus eggs were not detected by fecal egg count technique from feces collected at 0 to 14 days post infection (D.P.I). However, eggs were detected at 21, 28 and 35 D.P.I. Hence, results of immunoblotting assay showed specific anti rHc-CS antibody detection in all goat sera collected at early stage (14 D.P.I) and late stage (21-103 D.P.I) of H. contortus infection. Furthermore, no cross reactivity was observed against Trichinella spiralis, Fasciola hepatica and Toxoplasma gondii or uninfected goats. Among several evaluated rHc-CS indirect-ELISA format variables, favorable antigen coating concentration was found 0.28 µg/well at 37 °C 1 h and overnight at 4 °C. Moreover, optimum dilution ratio of serum and rabbit anti-goat IgG was recorded as 1:100 and 1:4000, respectively. The best blocking buffer was 5% Bovine Serum Albumin (BSA) while the best time for blocking, serum incubation and TMB reaction were recorded as 60, 120 and 10 min, respectively. The cut-off value for positive and negative interpretation was determined as 0.352 (OD450). The diagnostic specificity and sensitivity of the rHc-CS, both were recorded as 100%. CONCLUSION: These results validated that rHc-CS is a potential immunodiagnostic antigen to detect the specific antibodies during early and late H. contortus infections in goat.

Y75B8A.8 (HC8) protein of Haemonchus contortus: A functional inhibitor of host IL-2.

Interleukin 2 (IL-2) is an important immune regulatory factor in the immune response of the host. However, little is known about the inhibitor of host IL-2 in Haemonchus contortus infection. In this study, we found that globin domain-containing protein (HCGB) and Protein Y75B8A.8 (HC8) from H contortus excretory and secretory products are two binding proteins of IL-2 in goats. The yeast two-hybrid screening further validated the positive interactions of IL-2 with HCGB and HC8. Meanwhile, we found that HC8 had inhibitory effects on IL-2-induced peripheral blood mononuclear cell (PBMC) proliferation, while HCGB did not. Furthermore, transcriptional analysis revealed that HC8 could block the IL-2-activated signalling pathway. Our results showed that HC8 was a functional inhibitor of goat IL-2.