Construction Of Trichinella Spiralis Galectin Oral Vaccine And Its Induced Protective Immunity

Trichinellosis is a severe,globally distributed food-borne zoonotic disease,human Trichinella infection is primarily caused by eating raw or undercooked meat containing the encapsulated Trichinella larvae.The process of human infection can be divided into two stages,including the intestinal stage and the muscle stage.The main clinical manifestations are diarrhea and abdominal pain as well as fever,myalgia,myocarditis,anaphylaxis（intestinal phase）,facial edema and encephalitis（muscle phase）.The intestinal mucosal epithelium is the first natural barrier against the invasion of T.spiralis,and it is also the preferential interaction site between the host and Trichinella spiralis.Whether T.spiralis invades intestinal epithelial cells（IECs）is the key to infecting the host.Conventional Trichinella vaccines have been developed using subcutaneous injection,but this route of immunization fails to elicit strong mucosal responses and adaptive immunity.In addition,the results of previous experiments showed that the reduction rate of worms after immunization was not high,and the ideal immune protection effect could not be achieved.Lactic acid bacteria（LAB）,as a class of recognized safety grade（GRAS）microorganisms,have become the preferred bacteria for vaccine carrier research because they can express and secrete exogenous antigenic proteins and colonize local intestinal mucosa.Galectins are characterized by a unique carbon recognized domain（CRD）sequence motif binding toβ-galactoside,and constitute an evolutionary conserved family.Several galectins or galectin-like proteins have been identified in some parasites and are presumed to play roles in the invasion process and immune regulation.Trichinella spiralis galectin（Tsgal Gen Bank accession XM＿003381608.1）biological characteristics and functions were identified,and the results indicated that Tsgal might interact with IECs and participate in the invasion of host intestinal epithelium by T.spiralis in the early stage of infection.In this study,recombinant Lactobacillus plantarum NC8-p SIP409-pgs A’-Tsgal was constructed to explore its immune protection effect on Trichinella spiralis.The results showed that oral vaccine immunization of mice could induce Th1/Th2 mixed immune responses and intestinal mucosa-specific immune responses.Oral immunization of mice showed significant protection against Trichinella infection and reduced intestinal and muscle inflammatory responses,laying the foundation for the development of new vaccines against Trichinella.Materials and methods1.Trichinella species,experimental animals,plasmids and strainsTrichinella spiralis（T1）was used to preserve Kunming mice in our laboratory;The experimental animals were BALB/c mice purchased from Henan Provincial Laboratory Animal Center;The cloned strain was Escherichia coli DH5α,and the expression strain was Lactobacillus plantarum NC8;the cloning vector was p MD19-T,and the expression vector was p SIP409-pgs A’.2.Construction of recombinant Lactobacillus plantarum NC8The p QE-80L-Tsgal plasmid stored in the laboratory was used as the template to amplify the target gene,and then connected with the expression vector（p SIP409-pgs A’）to construct the recombinant expression plasmid p SIP409-pgs A’-Tsgal.Then electrotransferred into L.plantarum NC8 to construct recombinant L.plantarum NC8-p SIP409-pgs A’-Tsgal.SDS-PAGE,Western blot and IFT were used to verify the expression of Tsgal protein in the constructed recombinant bacteria and whether it could be successfully expressed on the surface of the recombinant bacteria.3.Biological characteristics of recombinant Lactobacillus plantarum NC8The growth curve of the recombinant L.plantarum NC8 was determined to verify whether the gene insertion affected the growth of L.plantarum NC8.In vitro simulated gastric acid environment to verify the acid resistance of recombinant L.plantarum.4.Protective immunity of recombinant Lactobacillus plantarum NC8 against Trichinella spiralis225 BALB/c mice were randomly divided into five groups:recombinant L.plantarum NC8+α-lactose group,α-lactose group,recombinant L.plantarum NC8 group,NC8 empty bacteria group and PBS group.The mice were orally immunized,and the test samples were collected according to the time points of the immunization process.ELISA was used to detect the levels of Ig G,Ig G1,Ig G2a,Ig A in the serum and the levels of s Ig A,histamine in the intestine of the mice after immunization and infection.Cytokine content（IFN-γand IL-4）in spleen,mesenteric lymph nodes（MLN）,Peyer’s patches,and intestinal lamina propria（ILP）.Two weeks after the last immunization,the mice were challenged with Trichinella spiralis.The AW and ML were counted at 7 dpi and 35 dpi,and their histopathological changes were observed.To analyze the immune protection effect of recombinant L.plantarum NC8 vaccine on mice.5.Statistical analysisAll the data were analyzed by SPSS 26.0 software and the results were shown as mean±standard deviation（SD）.One-way ANOVA or the Student’s t-test was used to analyze the differences among various groups.P<0.05 was defined as statistical significance.Results1.Construction of recombinant Lactobacillus plantarum NC8The sequencing results showed that the Tsgal gene had 99.87%similarity with the Tsgal gene sequence provided by Gen Bank.SDS-PAGE results showed that the recombinant L.plantarum NC8 had a protein band at the molecular weight of 30.4 k Da;Western blot results showed that rTsgal could be recognized by infected serum and anti-rTsgal serum;IFT results showed that green fluorescence could be observed on the surface of bacteria using anti-rTsgal serum and infection serum as primary antibodies,but normal serum could not.All the above experiments confirmed that the recombinant L.plantarum NC8-p SIP409-pgs A’-Tsgal was successfully constructed.2.Biological characteristics of recombinant Lactobacillus plantarum NC8The growth curve showed that the insertion of the recombinant plasmid did not affect the growth of L.plantarum NC8.The experimental results of simulating the gastric acid environment in vitro showed that the recombinant L.plantarum still has a certain viability in the extremely acidic environment,which can ensure that the bacteria enter the digestive tract of mice through the gastric environment,laying the foundation for the subsequent immune experiments.3.Protective immunity of recombinant Lactobacillus plantarum NC8 against Trichinella spiralisFive groups of mice were immunized according to groups,and samples were collected according to the time points of the immunization process and the corresponding indicators were detected.The results of serum antibody levels showed that from the second week of the first immunization,the levels of Ig G,Ig G1,Ig G2a and Ig A in the recombinant L.plantarum NC8 group and the recombinant L.plantarum NC8+α-lactose group were significantly higher than NC8 group,α-lactose group and PBS control group(F_{Ig G}=220.810,F_{Ig G1}=138.256,F_{Ig G2a}=57.905,F_{Ig A}=5147.182,P<0.0001).Ig G1 levels were significantly higher than Ig G2a levels at 4 and 6 weeks after oral immunization with recombinant L.plantarum NC8(t_4W=14.923,P_4W<0.0001;t_6W=3.580,P_6W<0.005);The levels of different types of s Ig A in the intestinal fluid of mice in different groups were detected.The results showed that after 3 times of immunization,the levels of total s Ig A and specific s Ig A in the immunized mice in the two groups of recombinant L.plantarum NC8 group were significantly higher than other groups(F_Total=308.853,F_Special=164.843,P<0.0001).Moreover,oral immunization of mice with recombinant L.plantarum NC8 or NC8 empty bacteria can induce intestinal mucosal immune responses and histamine secretion.The cytokine results showed that the levels of IFN-γand IL-4 produced by the spleen,MLN,Peyer’s patches and ILP cells of mice immunized with recombinant L.plantarum NC8 gradually increased.Trichinella challenge infection was performed after immunization,and AW was collected at 7 dpi.Compared to the PBS group,the mice vaccinated with recombinant L.plantarum NC8,recombinant L.plantarum NC8+lactose,lactose and empty NC8 exhibited 57.28,63.92,32.61 and 10.73%（F=177.029,P<0.0001）.Moreover,immunization of mice with recombinant L.plantarum NC8,recombinant L.plantarum NC8+lactose,lactose and empty NC8 produced a 53.30,58.77,31.28,and 21.17%reduction of muscle larval burdens at 35 dpi（F=49.222,P<0.05）.Histopathological changes of intestine and muscles in immunized mice showed that vaccination of mice with recombinant L.plantarum NC8 significantly hindered larval invasion,ameliorated intestinal inflammation and relieved the inflammatory reaction of the muscle tissues.Conclusion（1）A Trichinella galectin oral vaccine,recombinant Lactobacillus plantarum NC8-p SIP409-pgs A’-Tsgal,was successfully constructed using L.plantarum.（2）The recombinant L.plantarum NC8 can survive and express rTsgal in an acidic environment.（3）Mice immunized with rTsgal oral vaccine can induce Th1/Th2 mixed immune response and intestinal mucosa specific immune response.（4）Oral immunization of mice with rTsgal vaccine showed significant protection against Trichinella infection and alleviated intestinal and muscle inflammatory responses.