Immunological Study Of Toxoplasma Gondii Rhomboid Protease 4 And Cathepsin C1

BackgroundToxoplasma gondii(T.gondii)is an obligate intracellular protozoan parasite with an extremely broad host range inclusive of almost all warm-blooded animals,including human[1].Toxoplasmosis is a zoonotic parasitic disease.Toxoplasma infection can lead to serious consequences in immunosuppressed patients(e.g.HIV infection and malignant tumor radiotherapy).T.gondii infection in pregnant women can be transmitted to the fetus via the placenta,leading to miscarriage,stillbirth,fetal malformation.Toxoplasmosis also poses a serious risk to animal husbandry and human health.T.gondii expresses several proteases,including serine and cysteine proteases,which play an important role in host invasion,intracellular survival and T.gondii differentiation.Rhomboid proteases belong to the serine protease family.T.gondii expresses 5 nonmitochondrial rhomboid proteases,TgROM1 to TgROM5.ROM proteins plays an essential role in T.gondii invasion.TgROM1 is expressed in tachyzoites and bradyzoites.TgROM2 and TgROM3 are expressed in sporozoites.TgROM4 which is expressed in tachyzoites and bradyzoites is localized to the plasma membrane of T.gondii and uniformly distributed on the parasite surface.When T.gondii effectively invades host cells,TgROM4 plays an important role in regulating microneme protein 2(MIC2),MIC3,MIC6,and apical membrane antigen 1(AMA1).TgROM5 is mainly expressed in tachyzoites and involved in the enzymolysis of MIC6 and MIC12.Cathepsins belong to cysteine proteases which play key roles in host-parasite interactions.Five cathepsin proteins are encoded by the T.gondii genome;1 cathepsin B(TgCPB),1 cathepsin L(TgCPL),and 3 cathepsin C(TgCPC1,2,and 3)proteases.Cathepsin C proteases are exopeptidases that are required for T.gondii tachyzoite growth and differentiation.Inhibition of these proteases leads to significant attenuation of infection by T.gondii.TgCPC1 and TgCPC2 are expressed in tachyzoites,and have been reported to occupy dense granules and the parasitophorous vacuole(PV)of tachyzoites.The PV is formed as the parasite invades the host cell.At least one of TgCPC1 or TgCPC2 is required for host cell invasion and growth.And TgCPC1 is expressed at high levels in tachyzoites.However,the immunogenicity of TgCPC1 has not been reported.TgCPC3 has not been detected in the tachyzoites of T.gondii.There are three major infectious stages in the T.gondii life cycle:tachyzoites,cysts,and oocysts.Current drugs are effective for the treatment of acute toxoplasmosis which tachyzoites cause,but they cause many side effects,especially in the fetus,and have no effect on cysts.Therefore,a safe and effective vaccine is important for the prevention and control of toxoplasmosis.Toxoplasma gondii vaccines include whole-worm vaccines,parasite-specific component vaccines and nucleic acid vaccines.DNA vaccines that induce a persistent and strongly protective immune response are a promising option.These important protein on T.gondii which involved in their activities are potential targets for DNA vaccine candidates.Studies have shown that surface antigen 1(SAG1)is a classic and excellent DNA vaccine candidate for T.gondii;the TgROM1 DNA vaccine,TgCPB DNA vaccine and TgCPL DNA vaccine have a protective role in the host against T.gondii infection.Bioinformatics is an interdisciplinary subject involving many fields.It has been widely used to analyze the structure and function of proteins and to predict epitopes.The prediction of epitopes plays an important role in the research of T.gondii vaccine.However,the bioinformatics analysis of TgROM4 and TgCPC1 have not been reported.There are many factors that influence the immune response of vaccines.Immunization strategy is an important factor in determining the effectiveness of immune response,which has gained increasing importance in vaccine research.A prime-boost vaccination strategy induced a more efficient humoral and cellular immune response.Adjuvant can obviously enhance the immune response of the vaccine.Aluminum salt adjuvant and fresse adjuvant are classic adjuvants.a-GalCer is a powerful agonist of natural killer T cell,which can effectively stimulate the natural killer T cells.Studies have shown that a-GalCer can enhance the immune effect of several vaccines.In this study,we predicted the physical and chemical properties,the structures,and antigen epitopes of T.gondii proteins ROM4 and CPC1 by bioinformatics analysis.Then DNA vaccine of T.gondii were constructed based on these proteins.Different immunization strategies were carried out and adjuvant was used to enhance the immune effect,which was evaluated by animal experiments.PurposePredicting the basic characteristics of ROM4 and CPC1 by bioinformatics analysis;predicting the potential T cell and B cell epitopes and providing the theoretical basis for the subsequent construction of the DNA vaccine of T.gondii.Based on these,the ROM4 DNA vaccine was constructed,and the prime-boost immunization strategy was carried out to enhance the immune effect,which was evaluated by animal experiments;the CPC1 DNA vaccine was constructed,combined the adjuvant alpha-galcer to promote the immune response,and then evaluated by animal experiments.This study lays a theoretical foundation for the selection of DNA vaccine targeting T.gondii and the optimization of immunization strategy.Methods1.Bioinformatics analysis:(1)The basic physicochemical properties,phosphorylation sites,acylation sites,transmembrane structure and subcellular location of ROM4 and CPC1 proteins were predict by ProtParam?NetPhos 2.0 Server?CSS-Palm Online Service?TMHMM Server v.2.0 and PSORT II Prediction respectively.(2)DNASTAR was used to predict the secondary Structures and potential B cell epitope of ROM4 and CPC1.(3)IEDB online service was used to predict potential T cell epitope of ROM4 and CPC1.(4)The potential epitope of ROM4 and CPC1 protein was compared with the epitope of SAG1,which is a classic candidate protein for DNA vaccine of T.gondii.2.Construction and verification of DNA vaccines:(1)Based on the bioinformatics analysis of ROM4 and CPC1 protein,we constructed the DNA vaccines.The entire coding sequence of the T.gondii ROM4 and CPC1 gene were amplified using PCR from T.gondii cDNA with synthetic primers.(2)PCR products ROM4 and CPC1 gene fragments were subcloned into the eukaryotic expression plasmid pEGFP-C1 by restriction enzyme digestion and connection to form the recombinant plasmids pEGFP-ROM4(pROM4)and pEGFP-CPC1(pCPC1).Double-enzyme cutting experiments and sequencing analysis were performed to verify the construction of the recombinant plasmids.(3)The recombinant plasmids pROM4 and pCPC1 were propagated in Escherichia coli DH5a,then transfection into HEK 293-T cells for protein expression.Western Blot was performed to verify whether the recombinant plasmids can successfully express the purpose proteins in eukaryotic cells.After successful verification,the immunological effect of the above DNA vaccines with different regimens were assessed on BALB/c mice.3.The evaluation of TgROM4 vaccine.A good antigenic peptides(YALLGALIPYCVEYWKSIPR)on ROM4 protein was selected and synthesized according to the bioinformatics analysis.The DNA vaccines prime and peptide boost immunization strategy was carried out to immune mice.BALB/c mice were randomly divided into 5 groups,including 3 immune groups(peptide immune group,pROM4 immune group and pROM4/peptide immune group)and 2 negative control groups(PBS group and pEGFP-C1 group).The mice were then intramuscular immunized four times,and for the the pROM4/peptide group,mice were injected with pROM4 the first two times and with polipeptide the last two times.After immunization,serum IgG,IgG2a and IgG1 levels were determined using ELISA.The levels of cytokines IFN-y,IL-2,IL-4,IL-10 and IL-12 in the cultured splenocytes were detected.The survival time of mice was evaluated after challenge with tachyzoites of T.gondii RH strain.Additionally,the number of cysts in the brain was determined after intragastric challenge with cysts of T.gondii PRU strain.4.The evaluation of TgCPC1 vaccine.BALB/c mice were randomly divided into 5 groups,including 2 immune groups(pCPC1 immune group and pCPC1/a-GalCer immune group)and 3 negative control groups(PBS group,pEGFP-C1 group,and?-GalCer group).The mice were intramuscular immunized three times,and for the the pCPC1/?-GalCer immune group,a-GalCer was only injected at the final immunization.After immunization,the level of IgG,IgG2a and IgG1 in the serum and the level of cytokines in the cultured splenocytes were detected using ELISA.The survival time of mice was evaluated after challenge with tachyzoites of T.gondii RH strain.Results1.The basic physicochemical properties,phosphorylation sites,acylation sites,subcellular localization,and transmembrane structure of ROM4 and CPC1 proteins were obtained by bioinformatics analysis.The secondary structure of these two proteins were predicted.We predicted their epitopes,and found that similar to SAG1,ROM4 and CPC1 had multiple potential excellent B-cells and T-cell epitopes.This provides a theoretical basis for subsequent experiments.2.Based on the above theoretical analysis,we successfully constructed the DNA vaccine pEGFP-ROM4(pROM4)and pEGFP-CPC1(pCPC1)by PCR,enzyme cutting and connection,and successfully verified.Then we used different strategies to immunize mice and assess their immune response.(1)The evaluation of TgROM4 vaccine.Mice vaccinated with different immunization regimens(peptide,pROM4 and pROM4/peptide groups)elicited specific humoral and cellular responses,with high levels of IgG,IgG2a,and IFN-y(Compared with the negative control group,P<0.05),and pROM4/peptide group showed the highest level.However the levels of IgG1,IL-4,and IL-10 in the serum have no significant difference(P>0.05)between the five groups.The immunized mice(peptide,pROM4 and pROM4/peptide groups)prolonged survival times after challenge with tachyzoites,and reduced numbers of brain cysts after infection compared with negative controls.Especially the pROM4/peptide group had the longest survival time and the least brain cysts.(2)The evaluation of TgCPC1 vaccine.Compared with the negative control groups(PBS group,pEGFP-C1 group,and a-GalCer group),the immune groups(pCPC1 immune group and pCPC1/a-GalCer immune group)had high levels of IgG,IgG2a,IL-2,and IFN-y in the serum,and pCPC1/a-GaICer group was the highest.The level of IL-4 in the a-GalCer group was slightly increased,while there was no significant difference between the immune groups and PBS group.The level of IgG1 and IL-10 had no significant difference between the five groups.The immunized mice prolonged survival times after challenge with tachyzoites,and the pCPC 1/a-GalCer group had the longest survival time.ConclusionWe predicted ROM4 and CPC1 protein of T.gondii through bioinformatics for the first time,and found that they are good antigen of B cells and T cells in theory,and have the structure foundation for T.gondii DNA vaccines.Our animal experiments showed that both TgROM4 and TgCPC1 were excellent candidates for DNA vaccine of T.gondii.And for the first time,we applied the DNA vaccines prime and peptide boost immunization strategy to enhance the immune effect of ROM4 DNA vaccine.Also for the first time,we used adjuvants a-GalCer to enhance the immune effect of new T.gondii DNA vaccine targeting CPC1.These results indicate that the DNA vaccines prime and peptide boost immunization strategy and adjuvant combination with DNA vaccine are the promising methods to improve the immunological efficacy of T.gondii DNA vaccine.