Construction Of Nanovaccines And Its Immunoprotections Against Toxoplasma Gondii In Mice And Chickens

As a vital obligate intracellular parasitic protozoan,Toxoplasma gondii(T.gondii)is the causative agent of toxoplasmosis.Usually,T.gondii can be controlled by immunocompetent hosts.Nevertheless,individuals with inhibited immunity may develop severe clinical symptoms such as encephalitis,ophthalmia,and even death.As a serious disease widely distributed in the world,T.gondii could infect many types of animals,including sheep,goats,pigs,chickens,cattles,horses,etc.As the intermediate hosts of T.gondii,these susceptible animals generated great potential risks in human toxoplasmosis.T.gondii-infections in pigs,goats,and sheep could lead to acute symptoms,even abortions and stillbirths of newborn animals.As an important source of high-quality protein in China,chicken is also considered as the important intermediate host for toxoplasmosis.Although the free-range chickens showed clinical symptoms,it could be the potential sources of human and carnivore toxoplasmosis.At present,common drugs against toxoplasmosis are restricted to combinations of pyrimethamine and sulfadiazine.However,this treatment may contribute to numerous side effects,drug residues and no effect on chronic infection.In recent years,important progresses have been made in the development of vaccines against toxoplasmosis,especially in live-attenuated vaccines,DNA vaccines,recombinant protein vaccines and live vector-based vaccines.Although above vaccines alleviated T.gondii infection,none of these were able to effectively eliminate the formation of cysts and provide enough protection.Therefore,it is imperative to develop a safe and efficient vaccine for T.gondii.To decrease immune dosage and avoid the degradation,many researches have focused on effective delivery system.Currently,many synthetic delivery systems have been proved to be effective in improving the immune responses.Approved by the food and drug administration(FDA),poly(lactic-co-glycolic acid)(PLGA)have been widely used in vaccine and drug delivery system,and characterizes by easy biodegradability,nontoxicity,and good biocompatibility.Due to being biocompatible,biodegradable,and relatively safe,chitosan(CS)is also widely applied in partial food and biology medicine.Herein,we made a new attempt to identify T.gondii antigens with good immunogenicity,the antigens were then entrapped in PLGA or chitosan nanospheres to synthesize the nano-subunit vaccine or the nano-DNA vaccine against T.gondii.Our researches contribute a lot to the immunogenic improvements of subunit or DNA vaccine,and developments of nanomaterial vaccines against T.gondii.1 Identification of potential vaccine antigens against toxoplasmosisProkaryotic expression system and affinity chromatography method were conducted to obtain five recombinant T.gondii proteins.Five purified T.gondii proteins,together with 3recombinant T.gondii proteins preserved in our laboratory,were respectively immunized at the zero and seven day of the trial with BALB/c mice to evaluate the immunoprotection.The titers of antibodies generated by immunized vaccines were detected by ELISA method,and the concentration of cytokine IFN-γ,IL-4,IL-10,and IL-17 were illustrated by double antibody sandwich ELISA,the MHC molecule expressions in murine spleen lymphocytes were analyzed by flow cytometry.A lethal dose of T.gondii RH strain were challenged intraperitoneally to construct the survival curve seven days after the first immunization.Obtained by affinity chromatography,five recombinant proteins,named rTgH2A1,rTgPSA1,rTgSIR2,rTgRPP2,and rTgNADH,showed a clear single target band in SDS-PAGE analysis with the molecule weights were 37.5 k Da,44.7 k Da,58.0 k Da,29.7 k Da,and 78.8 k Da respectively.According to the results of western blot analysis,five recombinant proteins could be detected by sera from T.gondii-infected rats,and the native proteins could also be detected by sera from recombinant proteins immunized rats.Such results indicated good reactionogenicities of obtained recombinant proteins.In in vivo trials,eight recombinant proteins could induce a high-titer antibody response.Except for rTgPSA1 and rTge IF-5A,other six recombinant proteins could induce higher Ig G2a than Ig G1,eliciting a Th1 biased immunity.The secretions of IFN-γ,IL-4,and IL-17 in sera were regulated,while the secretions of IL-10 remained unchanged.Evaluated by flow cytometry analysis,rTgRPP2,rTgRab18,rTgRab43,and rTge IF-5A could induce higher expression of MHC-I molecules,indicating the endogenous antigen presentation was activated.Analyzed by flow cytometry,rTgH2A1,rTgPSA1,rTgSIR2,rTgRPP2,rTgRab18,rTgRab43,and rTge IF-5A could generate higher expression of MHC-II molecules,indicating exogenous antigen presentation was activated.Immunization with recombinant proteins,except for rTgNADH,conferred significant protection,with prolonged survival time in animals.Collectively,the results of this research indicated that rTgH2A1,rTgPSA1,rTgSIR2,rTgRPP2,rTgRab18,rTgRab43,and rTge IF-5A could provide partial immune protection against acute toxoplasmosis.2 Synthesis of nano subunit and DNA vaccine against T.gondiiTo synthesize the nano subunit vaccine against T.gondii,the PLGA and chitosan nano-materials were obtained to entrap rTgH2A1 and rTgPSA1.In the current research,two PLGA nanospheres and two chitosan nanospheres were successfully synthesized,named rTgH2A1-PLGA,rTgPSA1-PLGA,rTgH2A1-CS,and rTgPSA1-CS respectively.Based on the scanning electron microscope(SEM)imagine,the mean diameters of prepared nanospheres were 98 nm,104 nm,102 nm,and 100 nm respectively.The encapsulation efficiency(EE)of rTgH2A1-PLGA,rTgPSA1-PLGA,rTgH2A1-CS,and rTgPSA1-CS nanospheres were74.8%,68.4%,67.4%,and 67.3%,whereas the loading capacity(LC)of synthesized nanospheres were 4.3%,3.7%,1.2%,and 1.3%respectively.Furthermore,we aslso constructed the DNA vaccine,p VAX1-TgRPP2 and p VAX1-TgSIR2,and the well-constructed plasmids were transfected into HEK 293-T cells to detect its expressions.Exanimated by laser confocal microscope,the two constructed plasmids could be efficiently expressed in eukaryotic cells.To synthesize the nano DNA vaccine against T.gondii,the PLGA and chitosan nano-materials were obtained to entrap p VAX1-TgRPP2 and p VAX1-TgSIR2.In the current research,two PLGA nanospheres and two chitosan nanospheres were successfully synthesized,named p VAX1-TgRPP2/PLGA,p VAX1-TgSIR2/PLGA,p VAX1-TgRPP2/CS,and p VAX1-TgSIR2/CS respectively.Based on the SEM imagine,the mean diameters of prepared nanospheres were 97.30 nm,91.51 nm,102.02 nm,and 106.21 nm respectively.The EE of p VAX1-TgRPP2/PLGA,p VAX1-TgSIR2/PLGA,p VAX1-TgRPP2/CS,and p VAX1-TgSIR2/CS nanospheres were 54.30%,75.62%,48.35%,and54.71%,whereas the LC of synthesized nanospheres were 3.07%,1.28%,0.85%,and 3.47%respectively.3 Immune protection of nano subunit vaccine against T.gondiiBALB/c mice and Hy-line chickens were divided equally,and each animal was received an injection containing rTgH2A1-PLGA,rTgH2A1-CS,rTgPSA1-PLGA,rTgPSA1-CS,rTgH2A1-IF,or rTgPSA1-IF.The titers of antibodies generated by immunized nanospheres and emulsions were detected by ELISA method,the concentration of cytokine IFN-γ,IL-4,IL-10,and IL-17 were illustrated by double antibody sandwich ELISA,and the optimal administration route was determined by challenging experiments.To evaluate the CD4~+and CD8~+T lymphocytes in chicken spleen,the flow cytometry analysis was conducted.Fourteen days after the boost immunization,a lethal dose of T.gondii RH strain were challenged intraperitoneally to construct the survival curve in mice,and 10~7 of T.gondii RH strain were challenged intraperitoneally to illustrate the parasite burdens and the growth efficiency in chickens fourteen days after the secomd immunization.All these results proposed that four nanospheres as well as two emulsions could elicit significantly humoral and Th1-biased cellular immunity,and modulating the secretions of IFN-γ,IL-4,and IL-17 in the sera of mice.The challenging trials showed that both rTgH2A1-PLGA and rTgPSA1-CS nanospheres could significantly prolonged the survival time of mice,while the two synthesized nanospheres could also elicit high levels of antibodies,modulate the secretion of cytokines,promote the generation of CD4~+and CD8~+T lymphocytes,enhance the growth efficiency,and decrease the parasite burdens in chickens.Furthermore,as substitutes for each other,PLGA and chitosan nanospheres as well as its emulsions showed a remarkable advantage through the intramuscular route in mass vaccination.4 Immune protection of nano DNA vaccine against T.gondiiBALB/c mice were first equally divided into serval groups,and each animal was received an injection containing p VAX1-TgRPP2/PLGA,p VAX1-TgRPP2/CS,p VAX1-TgSIR2/PLGA,and p VAX1-TgSIR2/CS nanospheres.The titers of antibodies generated by immunized nanospheres and emulsions were detected by ELISA method,the concentration of cytokine IFN-γ,IL-4,IL-10,and IL-17 were illustrated by double antibody sandwich ELISA,and the maturity of dendritic cells(DCs)and the expression of MHC molecules were determined by flow cytometry.By using cell counting kit-8 reagent,the proliferation of murine splenic lymphocytes was investigated,and the flow cytometry was also used to determine the proportion of CD4~+and CD8~+T lymphocytes in murine splenic lymphocytes.Fourteen days after the secod immunization,a lethal dose of T.gondii RH strain were challenged intraperitoneally to evaluate the parasite burdens in animals.Accompanied by the generation of antibodies and cytokines,animals immunized with nanospheres could generate significantly humoral and Th1-biased cellular immunity.Furthermore,the synthesized nanospheres could promote the maturity of DCs and expression of MHC molecules,accelerate the proliferation of T lymphocytes,improve the proportion of CD4~+and CD8~+T lymphocytes.Induced by p VAX1-TgRPP2/PLGA,p VAX1-TgRPP2/CS,p VAX1-TgRPP2/PLGA,and p VAX1-TgSIR2/CS noospheres,obviously enhanced immunity and the significantly reduced parasite burdens were observed in T.gondii-infection model.According to the immunoprotections on mice,p VAX1-TgRPP2/PLGA and p VAX1-TgSIR2/PLGA nanospheres were considered better.To evaluate the immunoprotections on chickens,p VAX1-TgRPP2/PLGA and p VAX1-TgSIR2/PLGA nanospheres were synthesized again and the animal trials were conducted.At day 0 and 14,synthesized nanospheres were immunized with chickens,and fourteen days after the second immunization,the immune protection was analyzed by the in vivo trials.The results showed that p VAX1-TgRPP2/PLGA and p VAX1-TgSIR2/PLGA nanospheres could both trigger significant high levels of Ig Y,and could modulate the cytokines in chickens.Meanwhile,two tested nanospheres could significantly enhance the generation of CD4~+and CD8~+T lymphocytes,promote the growth efficiency,and decrease the parasite burdens in chickens.To sum up,delivered by PLGA or chitosan,p VAX1-TgRPP2 and p VAX1-TgSIR2 might be the promising vaccines against acute toxoplasmosis.Furthermore,p VAX1-TgRPP2/PLGA and p VAX1-TgSIR2/PLGA nanospheres also showed a remarkable advantage in generating effective immunoprotections in chickens through the intramuscular route.