Oral Immunization Of Mice With Attenuated Salmonella Encoding Trichinella Spiralis Calreticulin And Serine Protease 1.1 Confers Protective Immunity In BALB/c Mice

Trichinellosis is a serious zoonotic parasitic disease,which is widely distributed all over the world.The clinical manifestations of Trichinellosis are fever,eyelid and facial edema,muscle pain,etc.It is important to study the protein related to immune escape of Trichinella spiralis for the prevention and treatment of Trichinellosis and the screening of anti-Trichinellosis vaccine.Calreticulin（CRT）is a highly conserved protein that is present in all cells except erythrocytes in higher organisms and plays an important role in the maintenance of intracellular Ca²⁺homeostasis.It was found that the calreticulin of Schistosoma japonicum,hookworm and other parasites had good immunogenicity and antigenicity,and the calreticulin secreted by some parasites could inhibit the activation of the classical complement pathway,thus,calreticulin is a potential candidate for vaccine because it helps the parasite evade the immune attack of the host.In this study,we selected Trichinella spiralis calreticulin（Ts CRT;Gen Bank:KRT34215.1）,cloned and expressed the recombinant protein r Ts CRT,and analyzed its basic biological characteristics,protein expression level and localization in the worm.Our previous studies showed that Trichinella spiralis serine protease play an important role in the invasion,development and immune escape of Trichinella spiralis.In this experiment,the fusion gene Ts CRT+Ts SP1.1 was constructed from Trichinella spiralis calreticulin gene（Ts CRT）and serine proteinase gene（Ts SP1.1）.The fusion gene and the two protein genes were linked to eukaryotic plasmid pc DNA3.1（+）,respectively,the attenuated Salmonella typhimuriumΔcya SL1344 was selected as a vector to deliver DNA vaccine into mice,the humoral and cellular immune responses of mice and the protective effect of mice infected with Trichinella spiralis were studied.Materials and Methods1.Trichinella species,plasmids,strains and laboratory animalsTrichinella spiralis（T1）was used to preserve the species of Kunming mice in our laboratory,p MD19-T was used as clone vector,p QE80L and pc DNA3.1（+）were used as expression vector,BL21 and Rosetta were used for cryopreservation in our laboratory,the attenuated Salmonella typhimuriumΔcya SL1344 was donated by the office of animal epidemic diseases and public safety,College of Animal Science and technology,Henan University of Science and Technology,and the experimental animals were 4-6 weeks old female BABL/c mice,purchased from Henan Experimental Animal Center.2.Bioinformatics analysis of Ts CRTAccording to the Ts CRT amino acid sequence on NCBI,the isoelectric point,molecular weight,signal peptide and transmembrane region of Ts CRT protein were analyzed online using Prot Param and Interpro tools,and the software of Bio XM and MEGA 7.0 was used to compare Trichinella spiralis calreticulin Ts CRT with those of different Trichinella spiralis and other species,and the phylogenetic tree was constructed.3.Biological function identification of Ts CRTAccording to the NCBI sequence,specific primers were designed to amplify Ts CRT gene.The recombinant r Ts CRT protein was cloned and expressed.The mice were immunized with the recombinant protein to obtain anti-r Ts CRT serum,q PCR,Western blot and IFA were used to study the antigenicity of r Ts CRT protein,the transcription and expression of natural Ts CRT protein at different developmental stages of T.spiralis,and the localization of r Ts CRT protein on the surface of T.spiralis.4.Construction of recombinant plasmid and electroporation to attenuated S.typhimuriumTs SP1.1（ACA28930.1）has been cloned and expressed in our laboratory,and anti-r Ts SP1.1 serum was prepared.In this experiment,the domain of Ts SP1.1 was selected to design the fusion gene Ts CRT+Ts SP1.1.Ts CRT+Ts SP1.1,Ts CRT,and Ts SP1.1 were ligated with eukaryotic plasmid pc DNA3.1（+）,respectively.The plasmids were transferred to the electroporation cup,mixed well and electroporated.5.Transcription and expression detection of recombinant plasmids in vitroThe recombinant plasmid and empty plasmid were transfected into 293T cells with Liposome 2000 respectively.After transfection,RNA was extracted from the cells and reverse transcribed into c DNA,specific primers were added to RT-PCR to analyze in vitro transcription.The expression of the protein in vitro can be detected by IFA fluorescence,or by extracting the whole protein of the transfected 293T cells,Western blot was then used for analysis.6.Transcription and expression detection of recombinant plasmids in vivoAfter 2 weeks of oral immunization with DNA vaccines,one mouse in each group was selected and the spleen and mesenteric lymph nodes（MLN）were removed,RNA was extracted from the spleen and MLN of each group,and the spleen and MLN of each group were paraffin-embedded and sectioned,and the transcription of each group of genes in vivo was detected by RT-PCR,IFA fluorescence was used to detect the expression of protein in vivo.7.Vaccination schedules of oral DNA vaccine and sample collectionTwo hundred female BALB/c mice were randomly divided into pc DNA3.1-Ts CRT+Ts SP1.1 group,pc DNA3.1-Ts CRT group,pc DNA3.1-Ts SP1.1 group,pc DNA3.1 group and PBS group.Mice were immunized orally with 1×10⁸ CFU recombinant bacteria.The vaccination was boosted three times at a 2-week interval.At each immune node,the serum levels of anti-Trichinella Ig G,Ig G1,Ig G2a and Ig A were detected;intestinal fluid was collected to detect the levels of histamine,total s Ig A and specific s Ig A in intestinal fluid;spleen,Peyer’s patches and lymph node cell culture supernatants were detected Levels of IFN-γand IL-4.8.Protective effect induced by DNA vaccineTwo weeks after the last immunization,300 Trichinella spiralis were orally administrated to each mouse.At one week after infection,10 mice in each group were killed and the adult worms were recovered,the small intestine was collected and stained with HE and PAS to observe the pathological changes.Intestinal RNA was extracted from the small intestine of each group,and the transcription level of Muc2 was detected by q PCR.At the fifth week after infection,10 mice in each group were killed,the muscle larvae were collected and masseter muscles were collected to observe and compare the number of larvae recovered from different groups and the pathological changes of infected muscles.Two weeks after the last immunization,the serum of mice in each group was collected for antibody-mediated cytotoxicity test（ADCC）against NBL.9.Statistical AnalysisSPSS 21.0 was used to analyze the data.The main statistical methods were one-way Anova and independent t-test,P<0.05 was considered to be statistically significant.Results1.Bioinformatics analysis of Ts CRTThe full-length Ts CRT gene of Trichinella spiralis is 1221 bp,encoding 406 amino acids,with a molecular weight of 47.204 k Da and an isoelectric point p I of 4.65.The protein has a signal peptide and no transmembrane region.The results of sequence alignment and phylogenetic tree show that the sequence of calreticulin Ts CRT is relatively conserved among different species of Trichinella spiralis,and the sequence identity is more than 95%,furthermore,Ts CRT of Trichinella spiralis is closely related to the evolution of the cystic Trichinella spiralis.2.Bioinformatics analysis of Ts CRTWestern blot results showed that r Ts CRT protein could be recognized by infection serum and anti-r Ts CRT serum,which indicated that r Ts CRT protein had good antigenicity The q PCR and Western blot results showed that natural Ts CRT had transcription and expression at different developmental stages of T.spiralis,natural Ts CRT exists mainly on the surface of the parasite and in the embryo.3.Construction of recombinant plasmids,transcription and expression in vitro and in vivoThe results of RT-PCR showed that pc DNA3.1-Ts CRT+Ts SP1.1,pc DNA3.1-Ts CRT,pc DNA3.1-Ts SP1.1,PCDNA3.1 were successfully transferred into attenuated S.typhimurium by electroporation.RT-PCR results showed that the target gene could be amplified successfully with the RNA of the experimental group,but not with the RNA of the control group.Western blot showed that anti-r Ts CRT serum could recognize Ts CRT+Ts SP1.1 and Ts CRT protein,while anti-r Ts SP1.1 serum could recognize Ts CRT+Ts SP1.1 and Ts SP1.1protein,IFA results showed that Ts CRT+Ts SP1.1 and Ts CRT+Ts SP1.1 cells could be recognized by anti-r Ts CRT serum,cells in the Ts CRT+Ts SP1.1 group and Ts SP1.1 group could be recognized by anti-r Ts SP1.1 serum.In vivo protein expression detection:IFA results showed that spleen and MLN sections of Ts CRT+Ts SP1.1 group and Ts CRT group could be recognized by anti-r Ts CRT serum,Ts CRT+Ts SP1.1 and Ts SP1.1 groups were recognized by anti-r Ts SP1.1 serum.4.The immune response induced by DNA vaccineThe levels of Ig G,Ig G1,Ig G2a and Ig A in serum of mice from the second week after immunization were significantly higher than those in the control group(F_{Ig G}=466.492,F_{Ig G1}=535.091,F_{Ig G2a}=1291.021,F_{Ig A}=1138.892,P<0.001),the level of Ig G1 was significantly higher than that of Ig G2a(T_2W=3.070,T_4W=22.574,T_6W=20.969,P<0.001),indicating that oral vaccine induced a Th1/Th2 mixed immune response dominated by Th2 type immunity.Total and specific s Ig A in the intestinal fluid of mice were similarly higher than those in controls(F_{total s Ig A}=482.124,F_{s Ig A}=688.697,P<0.001)and remained at higher levels at week 11 of immunization;However,intestinal histamine levels decreased significantly at week 11 compared with week 6（T=42.983,P<0.001）.The cytokine IL-4and IFN-γlevels similarly increased from week 2 and persisted until Week 11(11w:F_IFN-γ=336.391,F_IL-4=1378.752,P<0.001),the results indicated that mice immunized with Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1 could induce mixed Th1/Th2 immune response.5.Protective effect of DNA vaccineThe adult worm reduction rates of Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1 group were53.4,44.28 and 42.46%,compared with PBS group（F=88.442,P<0.001）.The fecundity of female larvae in Ts CRT+Ts SP1.1 was significantly lower than that in Ts CRT and Ts SP1.1（F=28.819,P<0.001）.The amount of recovered ML in the Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1 groups was 46.05,35.43 and 29.29%less than that in the PBS group,respectively（F=29.072,P<0.001）.The results of HE and PAS staining of intestinal sections showed that the intestinal sections of mice in experimental group were infected with muscle larvae one week later,the intestinal inflammation was mild and the villi were relatively normal,and the width of intestinal villi in the three groups was significantly lower than that in the pc DNA3.1 and PBS control groups（F=11.605,P<0.001）.In addition,the number of goblet cells in the experimental group was significantly lower than that in the pc DNA3.1 and PBS groups（F=13.864,P<0.001）.q PCR results of intestinal mucin Muc2 showed that the transcription levels of mucin Muc2 in the three immune groups were significantly lower than those in the pc DNA3.1 and PBS groups（F=51.730,P<0.001）.The results of HE staining of muscle sections of mice showed that the number of Trichinella spiralis cyst larvae in the three immune groups was significantly lower than that in the PBS group（F=15.835,P<0.01）,and in addition,the number of Trichinella spiralis cyst larvae in the three immune groups was significantly lower than that in the PBS group（F=15.835,P<0.01）,Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1 group also significantly reduced the number of inflammatory infiltrating cells around the capsule（F=79.709,P<0.001）.The number of inflammatory cells in Ts CRT+Ts SP1.1 group was significantly lower than that in Ts CRT and Ts SP1.1 group（F=5.459,P<0.05）.ADCC induced 86.33,80.33 and 79.67%cytotoxicity in the experimental group at 72 h,respectively,which was significantly higher than that in the serum of pc DNA3.1 and PBS groups（F=431.355,P<0.001）.Cytotoxicity was dose-dependent with specific antibodies against Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1(r _{Ts CRT+Ts SP1.1}=0.890,r _{Ts CRT}=0.897,r _{Ts SP1.1}=0.900,P<0.05),in addition,the cytotoxicity increased with the prolongation of culture time(F_24h=5.81,F_48h=68.113,F_72h=431.355,P<0.001).Conclusions1.Expression and purification of Trichinella calreticulin r Ts CRT.Ts CRT is transcribed andexpressed in different stages of Trichinella spiralis（ML,IIL,AW）,mainly located in thecortex and embryo.2.The fusion gene Ts CRT+Ts SP1.1 was designed and combined with Ts CRT and Ts SP1.1to construct DNA vaccine.S.typhimurium was used as vector to deliver DNA vaccine.3.Ts CRT+Ts SP1.1,Ts CRT and Ts SP1.1 DNA vaccines induced a systemic Th1/Th2 mixedimmune response and a local immune response in the intestinal mucosa after oralimmunization of mice,and produced significant immune protection against Trichinellaspiralis challenge infection,Ts CRT+Ts SP1.1 combined immunization effect is betterthan a single DNA vaccine.