Identification Of Inhibitory Molecules Against Goat Interleukin-4 And Interferon-γ From Haemonchus Contortus

Haemonchosis can cause anaemia,malnutrition,ascites and even death of samll ruminants,resulting in the loss of productivity and great economic impact.The Haemonchus contortus excretory and secretory,protein(HcESPs)showed satisfied immunogenicity and could regulate the host immune response.Previous studies indicated that parasitic helminth had evasion mechanism to subvert or suppress host immunity in order to ensure their survival.IL-4 and IFN-y could regulate T cells differentiation.Researches suggested that Haemonchus contortus(H.contortus)could induce the specific Th2 immune response.Therefore,the studies on the antagonistic molecules of IL-4 and IFN-y in HcESPs were helpful to reveal the immunoregulating mechanism and to further understand the relationship between host cytokines and parasite infection.Besides,these cytokine inhibitors were considered the candidate vaccine antigen for the treatment and prevention of Haemonchosis.Furthermore,the identification of the binding areas and sites of inhibitors and cytokines could reveal further mechanism of immune evasion.1 Identification of the inhibitory mo.lecules against goat interleukin-4 derived from Haemonchus contortusIn this study,three proteins,Glutathione S-transferase domain containing protein(HcGST),Transthyretin domain containing protein(HcTTR)and Calponin actin-binding domain containing protein(HcCab),were respectively identified to bind to goat IL-4 by co-immunoprecipitation(co-IP)assays along with Yeast two-hybrid screening.Meanwhile,cells proliferation analysis showed that recombinant HcTTR(rHcTTR)could block the IL-4 induced proliferation of peripheral blood mononuclear cells(PBMCs)of goat,while recombinant HcGST and recombinant HeCab did not.In addition,the rHcTTR could also downregulate the transcriptions of candidate genes in IL-4 engaged JAK/STAT pathway.These results indicated that HcTTR was a novel antagonist against goat IL-4 from H contortus excretion and secretion proteins(HcESPs)and would improve our understanding of the relationship between host cytokines and parasite infection.2 Identification of the binding domain of Haemonchus contortus Transthyretin domain containing protein and interleukin-4In this study,the protein structure of HcTTR was divided into three parts:N-terminal TTR(N-TTR),C-terminal TTR(C-TTR)and the containing domain 52(TTR52).Yeast two hybrid tests were performed to analyze the interaction between these domains and goat IL-4.The results showed that TTR52 domain could bind to IL-4,while N-TTR and C-TTR did not.GST-PULLDOWN analysis also showed that TTR52 could combine with IL-4 recognized by His monoclonal antibody.Therefore,TTR52 was the binding domain of goat cytokine IL-4.This result provided a theoretical basis for further understanding about the mechanism of antagonism.3 Studies on the potential roles of HcTTR in protecting local crossbred goats against Haemonchus contortus infectionIn our previous study,it was reported that HcTTR was a novel antagonist of the goat cytokine interleukin-4(IL-4),and was involved in the regulation of host immune responses,implying that it might be applied for therapeutic strategies or vaccine development against the infection of H.contortus.Thus,the protective capacities of recombinant HcTTR(rHcTTR)against H.contortus infections via active and passive immunization trials were examined.For the passive protection trials,H.contortus infected goats were intravenously immunized twice with 5 mg of total IgG containing anti-rHcTTR goat polyclonal antibodies.The results showed that the significant rates of reduction in egg shedding and worm burden was 58.12%and 64.61%、respectively,as compared with the positive control group.For the active protection trials,local goats were vaccinated twice with 500 μg of rHcTTR to generate antigen-specific circulating antibodies,resulting in 63.7%reduction in eggs shedding and 66.4%reduction in worm burdens after H.contortus challenge.In both passive and active trials,the immunized goats displayed higher mucosal IgA levels and less anaemic compared to the challenged positive controls.Pen trials indicated that HcTTR generated partial immune protective effects against H.contortus challenge and it could be a promising vaccine candidate for development of effective strategy to control H.contortus.4 Identification of inhibitory molecules against goat interferon-y derived from Haemonchus contortusTo identify the antagonist of goat IFN-y from HcESPs.In this study,co-IP and LC/MS-MS analysis were used to screen IFN-y binding proteins in excretory secretory proteins.Yeast two hybrid assays were used to further identify the binding proteins.The results showed that Haemonchus contortus Glutathione S-transferase domain protein(HcGST)and Enolase domain protein(HcENO)could bind to IFN-y.The preliminary study of these two binding proteins indicated that recombinant HcGST(rHcGST)could inhibit the nitric oxide(NO)secretion from monocytes induced by IFN-y,while recombinant HcENO did not.These findings further revealed the relationship between host and parasite and could help us better understand how the host immune responses were regulated by parasite.5 Studies on the potential roles of Y75B8A.8(Hc8)in protecting local crossbred goats against Haemonchus contortus infectionHaemonchus contortus Y75B8A.8(Hc8)belonging to Haemonchus comortus excretory and secretory proteins(HcESPs)was investigated as a functional inhibitor of goat interleukin-2(IL-2).In this research,the recombinant Hc8(rHc8)and the goat polyclonal antibodies containing total IgG anti-rHc8 were performed to evaluate the protective capacities of Hc8 against H.contortus infections via active and passive immunization trials.In active and passive immunization trials,local crossbred female goats with ages of 9-12 months-old were randomly divided into three groups,five in each group,respectively.Parasitological examinations(egg shedding and worm burdens).the IgA levels in MH samples and hemoglobin levels were detected.Goats were intravenously immunized with 5 mg of total IgG containing anti-rHc8 goat polyclonal antibodies for twice consistently brought about reductions of 38.01%and 46.35%in egg shedding and worm burdens in passive trial,respectively,when compared with the challenge control group.Excitedly,the active immunization twice with 500 μg of this rHc8 protein resulted in the generation of high levels of antigenspecific circulating antibodies,induced a 69.6%reduction in egg shedding and a 54.9%reduction in abomasal worm burden when co-administered with Freund’s adjuvant to local crossbred female goats.Immunized groups had higher mucosal homogenate IgA and hemoglobin levels than challenge controls in both passive and active immunization trials.The results indicated that Hc8 could provide partial protection and implied for its role in application for therapeutic strategies against the infection of H.contortus.