Immune Protective Effects Of Haemonchus Contortus Dendritic Cells Stimulating Protein Hepatocellular Carcinoma-Associated Antigen 59 On Goats

Hepatocellular carcinoma-associated antigen 59(HCA59),Cyclization recombination enzyme-actin like-6 protein(CREA6),Aromatic amino acid β-eliminating lyase(Aromatic amino acid beta-eliminating lyase threonine aldolase domain containing protein,TA-1),SCP-like extracellular domain containing protein(SCP),and Globin domain containing protein(GB),which are important components of Haemonchus contortus excretion and secretion antigen(HcESPs).The former four proteins have been shown to bind to goat PBMCs in vitro.The Haemonchus contortus GB protein has been shown to bind to goat IL-2.Dendritic cells(DCs)are the only multifunctional cells that can initiate primary immune responses and regulate adaptive and innate immunity through antigen uptake and processing,stimulating lymphocyte and cytokine release.Biodegradable and biocompatible PLGA and Chitosan nano-adjuvants can enhance the body’s immunity by prolonging the release of antigen,prolonging the time of antigen recognition by immune cells,promoting the uptake of antigen by DCs,and regulating the pattern of antigen presentation.Haemonchus contortus is an important digestive nematode,which is mainly parasitic in the abdomen of ruminants.The excretory secretion of Haemonchus contortus contains a variety of proteins and is pleiotropic.On one hand,it appears to help immune protection of the host and immune escape against the worm,on the other hand,HcESP can also mediate immunosuppression.In this research,the above five recombinant proteins were selected from HcESP to verify their effects on mononuclear dendritic cells,then HCA59 was selected owing to its strong stimulation on dendritic cells.Afterwards,HCA59-PLGA and HCA59-Chitosan nano vaccines were constructed to verify the immune response induced in mice.The immunoprotective effect of HCA59-PLGA nanovaccine on goats was eventually verified.1 The identification of antigens which can affect the functoins of DCs in goatsThe goat peripheral blood mononuclear cells(PBMCs)were isolated from healthy goat anticoagulant obtained by venipuncture.Monocytes(attached cells)were obtained from the PBMCs cultured for one night.The traditional GM-CSF/IL-4 combined stimulation method was used as the positive control group,and rHCA59,rCREA6,rTA-1,rSCP and rGB were used for the experimental group to stimulate the monocytes for 7 days.The obtained cells were observed and identified by optical microscopy and flow cytometry.The transcription of key genes in the WNT pathway related to md-DCs maturation and differentiation were observed by RT-PCR.The results showed that under light microscopy,cells obtained from the experimental group had typical dendritic cell morphology,similar as the positive group.Flow cytometry results demonstrated that the percentage of CD80 of rHCA59,rCREA6,rTA-1,rSCP and rGB group were significantly higher than that of monocyte control group,while the the five experimental groups had a relatively lower expression of CD 14,CD 11c and CD 172a when compared with those in monocytes.RT-PCR results showed that rHCA59,rCREA6,rTA-1,rSCP and rGB could activate the transcription of key genes in WNT pathway.The above results indicate that rHCA59,rCREA6,rTA-1,rSCP and rGB could promote the proliferation,differentiation and maturation of md-DCs.Above all,rHCA59 was identified to have the most strong stimulation functions on the proliferation,differentiation and maturation of md-DCs.2 Preparation of rHCA59-PLGA and rHCA59-Chitosan nanoparticlesPolymeric nanoparticles were prepared under aseptic condition according to the double emulsion(w/o/w)solvent evaporation method.The working concentration of PVA was explored before starting the preparation of nanoparticles.Three PVA concentration gradients were designed,which were 1%,4%,and 6%,respectively.At 6%PVA,a high encapsulation efficiency of the final PLGA particles was got(89%).After scanning electron microscopy,the obtained nanoparticles not only had a large number of particles,but also had a relatively small particle size and were generally around 180-402nm at 6%PVA.The rHCA59-Chitosan nanoparticles antigen formulation was prepared using the ionic gelation method.After scanning electron microscopy,the obtained nanoparticles not only had a large number of particles,but also had a small particle size and were generally around 200-500nm.3 Effects of different nanoparticles adjuvants on the immunogenicity of rHCA59 in miceThe rHCA59-PLGA,rHCA59-Chitosan,rHCA59-PLGA-Chitosan(rHCA59-PLGA and rHCA59-Chitosan particles mixture)and rHCA59-CFA(complete Freund’s adjuvant)vaccines were injected into the mice respectively on day 0,and the blood was collected on the 14th day.The sera from blood were collected for the detection of the levels of cytokines and antibodies by ELISA.The spleens of mice were obtained and spleen lymphocytes and dendritic cells were separated.The proliferation of lymphocytes was observed by CCK-8 test kit.T cell typing and dendritic cell phenotype changes were detected by flow cytometry.The results showed that under the rHCA59-PLGA-Chitosan or rHCA59-PLGA antigen presentation system,mice lymphocytes were proliferated and high levels of special humoral immunity(IgM,IgG1,IgG2a)and cell-mediated immune responses(IFN-γ,IL-4 and IL-17)were also caused.They also resulted in the increase of CD3+CD4+T and CD3+CD8+T cells,enhancing the percentage of CD11C+CD83+ and CD11C+CD86+ on dendritic cells.The above results indicated that in the future studies,rHCA59 might be a good candidate molecule for the preparation of a single-dose nanovaccine against the infection of Haemonchus contortus.4 Immune protection of goats caused by rHCA59-PLGA nanovaccineThe experimental group goats were subcutaneously immunized with rHCA59-PLGA vaccine on day 0,boosted two weeks later,and challenged Haemonchus contortus L3(8000)together with the positive control group two weeks after booster immunization.On the 63rd day,the goats were slaughtered and the amount of Haemonchus contortus in the abdomen was examined.Goat sera were taken on days 0,14,28,43,54 and 63,respectively,and serum levels of cytokines and antibodies were measured by ELISA.Goat feces were taken on the 50th,52nd,54th,56th,58th,60th and 62th day of the experiment,and EPG(number of eggs per gram of feces)was detected by saturated saline floating method.The results indicated that under the rHCA59-PLGA antigen presentation system,goats produced higher level of humoral immunity(IgA,IgE,IgG)and cell-mediated immune responses(IL-9,TGF-β,IL-4,IL-17).The average egg reduction rate was 44.1%.The total reduction rate of H.contortus was 54.6%,the reduction rate of female H.contortus was 52.8%,and the reduction rate of male H.contortus was 58.6.%.The above results indicated that the rHCA59-PLGA vaccine could induce the goats to produce partial protection against the infection of Haemonchus contortus.