| Haemonchus contortus is a hematophagous gastrointestinal parasite affecting small ruminants.Parasitism by H.contortus is a great concern of sheep and goat producers around the globe and accounts for billions of dollars in economic loss worldwide.To control the infestation of this worm,the unchecked use of anthelmintics is sometimes suboptimal in terms of safety and effectiveness.H.contortus continuously released its secretions comprising excretory/secretory proteins(Hc ESPs)in its host.These Hc ESPs interact with the host immune system and the biological molecular pathways that underlie the immunomodulatory effects.However,different breeds of sheep and goats show differences in their natural resistance to H.contortus.The immune response of sheep and goats exhibited depends on the age,breed,sex,physiological status,climate,and housing conditions.This study evaluated the immunogenic maturation of goat monocyte-derived dendritic cells(DCs)and the upregulation of toll-like receptors induced by five different H.contortus antigens.A mice model was also used to determine the efficacy of these antigens with the adjuvant activity of bio-polymeric nanoparticles(PLGA,Chitosan).Finally,the goats received nano-vaccine in separate and mixed antigens formulations to determine the efficacy of these preparations in protecting goats against H.contortus infections.In conclusion,the preparation containing two H.contortus antigens(r Hc ARF1 and r HCA59)encapsulated into PLGA nanoparticles is safe for host animals and exerts immunoprophylactic effects against H.contortus infection.1.The comparison of five antigens of H contortus to activate DC in-vitroThe nematode H.contortus is a significant pathogenic species in the worldwide veterinarian sector and is a major constraint on small ruminant health and production in tropical and subtropical areas.Previously,it was found that several proteins of H.contortus were involved in the stimulation of host immune responses.Monocytes play a significant role in the inflammatory response,which is crucial to activating innate responses against pathogens.Besides,monocytes can also differentiate into DCs under inflammatory situations.However,the information about selecting superlative antigens with immunogenic efficacies on host monocyte-derived DCs(md-DCs)is lacking.In the current study,the stimulatory effects of five H.contortus recombinant proteins,including elongation factor-1α(EF-1α),arginine kinase(AK),excretory/secretory-15(ES-15),excretory/secretory-24(ES-24),and ADP-ribosylation factor 1(ARF1),on the maturation of goat md-DCs were reported.These recombinant proteins were purified separately in the E.coli expression system and incubated with isolated goat peripheral blood mononuclear cells(PBMC).Immunofluorescence assay(IFA)results confirmed the binding of these molecules to the md-DCs compared to control groups.As for flow cytometry analysis,recombinant proteins stimulated md-DCs maturation via the up-regulated expression of the costimulatory molecules CD80 and MHC-II.Quantitative RT-PCR data showed a significant increase in the expression of specific genes of the WNT and toll-like receptor(TLR)signaling pathways.The result of ELISA indicated higher levels of IL-10,IL-12,IFN-γ,and TNF-αsecretions by md-DCs compared to negative(p ET-32a His-Tag)and blank(PBS)control groups.The data provides valuable support in selecting potential antigens for future studies on the immunomodulation of the host against H.contortus infections.2.The DCs-stimulating antigens of H.contortus(ES-15,ES-24,and ARF1)encapsulated in bio-polymeric nanoparticles enhance immune responses in mice modelH.contortus cause a worldwide disease of small ruminants,and there are very few descriptions of recombinant antigens being protective of combating the worm infestation.The aim of this study was to evaluate immune responses induced by H.contortus recombinant ES-15(r Hc ES-15),ES-24(r Hc ES-24),and ARF1(r Hc ARF1)antigens incorporated with bio-polymeric nanoparticles(NP)in mice model.Briefly,three antigens were encapsulated in poly(lactic-co-glycolic acid)(PLGA)and chitosan(CS)NP first and then injected subcutaneously into ICR mice at 0 days.On day 14,mice were humanly killed,and their blood and spleens were isolated for further experiments.Scanning electron microscopy(SEM)was carried out to characterize the antigen-loaded NP.The mice’s immune responses were gauged in terms of allergic reactions,lymphocyte proliferation,serum-specific antibody productions(Ig G1,Ig G2a,and Ig M),and cytokine expressions(IL-4,IL-10,IL-17,IFN-γ,and TGF-β)through ELISA.Flow cytometry was performed to determine the proliferation of CD3e~+CD4~+and CD3e~+CD8a~+T cells and phenotypical changes of splenic CD11c~+CD83~+and CD11c~+CD86~+DCs.The results demonstrated that recombinant r Hc ES-15,r Hc ES-24,and r Hc ARF1 antigens were successfully incorporated in polymeric NPs.They activated the humoral immune responses characterized by increased specific antibodies,high levels of cytokines,and potent lymphocyte proliferation.The mice immunized with the antigens-loaded NP formulations showed higher percentages of splenic DCs and T cells than control groups.Among three antigens,r Hc ARF1 triggered a more robust immune response than r Hc ES-15 and r Hc ES-24 in experimental animals.These findings suggest that r Hc ARF1 encapsulated in NPs conferred a better immunity to the infection of this nematode,providing the foundation for further development of a commercial vaccine against H.contortus.3.Vaccination of r Hc ARF1 encapsulated in PLGA nanoparticles elicits immune protection against H.contortusH.contortus,presenting a worldwide distribution,is one of the significant pathogenic gastrointestinal nematodes of sheep and goats.The eggs of this nematode contaminate the pasture grazed by animals,particularly by small ruminants.After getting entrance into the abomasum,it feeds on blood of the host animals.Affected animals usually present dehydration,weakness,production loss,and anemia.Young lambs show a high fetal rate after the worm infestation.The preparation containing r Hc ARF1 antigen encapsulated in PLGA nanomaterial(r Hc ARF1-PLGA)was tested to protect against H.contortus infection in goats.Fifteen goats were randomly allocated into three groups.On days 0 and 14,the goats of the experimental group were immunized with the preparation.After 14 days of the second immunization,goats in experimental and positive control groups were challenged with 8000infective larvae(L3).Positive and negative controls were unvaccinated,while the negative control was not challenged with L3.The results demonstrated that after immunization with the r Hc ARF1-PLGA preparation,the experimental group produced high levels of antigen-specific serum Ig G,Ig A,and Ig E antibodies.In addition,elevated levels of IL-4,IL-9,IL-17,and TGF-βwere observed in the experimental group compared to the control groups.After the L3 challenge,the mean eggs per gram(EPG)of the experimental group was reduced compared to the positive control group(*P<0.05).There was a significant difference in abomasal worm burdens between the experimental group(74.2±17.13)and the positive control group(163.6±88.57;***P<0.001).This study concluded that the use of r Hc ARF1nanovaccine showed strong protection in goats against H.contortus infection.4.Protective responses induced by the cocktails encompassing r HCA59 and r Hc ARF1incorporated in biodegradable PLGA nanoparticles to control H.contortus infectionThe concept of therapeutic vaccines is based on the activation of the immune system against pathogenic microorganisms.Herein,we employed r HCA59 and r Hc ARF1 antigens.The two proteins were encapsulated into PLGA nanoparticles and were then used to immunize the host animal against H.contortus infection.Briefly,fifteen goats were divided randomly into three groups(Groups 1 to 3).We immunized goats in Group 1 with antigens-loaded NPs(r HCA59+r Hc ARF1-PLGA)on days 0 and 14.Group 2 was an unvaccinated positive control challenged with 8000 H.contortus L3.Likewise,Group 3(negative control group)was unvaccinated and not challenged with L3.Eight thousand infective L3 were challenged in goats in Group 1 after 14 days of second immunization.The results of ELISA showed high levels of specific serum Ig G,Ig A,and Ig E productions and significantly higher production of IL-4,IL-9,IL-17,and TGF-βafter vaccination.In the meantime,the fecal eggs and the abomasal worm burdens were reduced by 69.4%and 63.2%,respectively in r HCA59+r Hc ARF1 group.Moreover,goats in Group 1 that received NP-based vaccines showed high levels of neutrophils,lymphocytes,and decreased hemoglobin levels after the L3 challenge.The study suggested that r HCA59+r Hc ARF1-PLGA preparation conferred partial protection and could be a good candidate for developing effective nanovaccines against H.contortus infection. |
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