Construction Of An Anti-helicobacter Pylori Vaccine Using A Food-grade Lactic Acid Bacterium As The Vector And Evaluation Of Its Immune Efficacy

Helicobacter pylori infection is one of the most important risk factors for the development and progression of gastritis,peptic ulcer and gastric cancer.Effective vaccination will be effective to prevent and control population-based Hp infection.Hp neutrophil-activation protein A(NapA)and adhesion A(HpaA)play indispensable roles in the immune response against Hp infection,which have been become two attractive potential alternative antigens in Hp vaccine research.In the studies for expressing and devilering heterologous proteins,L.lactis have been the one of the most frequently used vaccine delivery vehicles.AimThis work was aimed to construct an engineered recombinant strain expressing Hp hpaA and napA gene respectively by using food-grade expression system L.lactis NZ3900/pNZ8149.The recombinant strains were given to mice via an intragastric route,the immunoprotective efficiency and the underlying mechanism would be evaluated and investigated,thus laying crucial foundation for the development of Hp oral vaccines.Methods1.The engineered strain L.lactis NZ3900/pNZ8149-hpaA and NZ3900/pNZ8149-napA were constructrd by gene recombinant technology,the immunoreactivity of HpaA and NapA protein were identified by SDS-PAGE and Western blotting after induced by nisin.2.SPF grade,BALB/c mice were separated into HpaA group,NapA group,HpaA+NapA group,8149 group,GM17 group and baseline group randomly according to the weight,mice in the first five groups were given the dosage of 200 ?L NZ3900/pNZ8149-hpaA(1×1010 CFUs bacteria),NZ3900/pNZ8149-napA(1×1010 CFUs bacteria),the mixture of two bacteria(containing 1×1010 CFUs bacteria respectively),NZ3900/pNZ8149(1×1010 CFUs bacteria)and GM17 liquid medium via an intragastric route once a week and continued for six weeks.Half amount mice from the above intervention groups were executed seven days after the last vaccination,blood sample and intestinal feces were collected.ELISA was applied to assess specific IgG and SIgA antibodies;Thl,Th2 and Th17 type cytokines in spleen cell cultures were measured by ELISA.The baseline group was not given any intervention.3.Fourteen days after the last vaccination,the mice were orally challenged with 2×109 CFUs Hp bacteria four times at intervals of two days and the basline group was given without any intervention.Seven days after the last challenge infection,the mice in all the groups were sacrificed,urease reaction and qPCR were used to determined the Hp colonization in the stomachs.4.Recombiant strains were continue cultured for 12 h,thus the growth curves could be drawn.Besides,the ratio of recombinant plasmid retaining heterologous gene was determined in the 50th generation bacteria without antibiotic pressure.Additionly,plate count method was utilized to count the bacterial colonies in medium corresponding to different OD600,thus the relationship curves of OD600 and CFU/mL were obtained by Matlab software.Results1.The recombinant L.lactis strain NZ3900/pNZ8149-hpaA and NZ3900/pNZ8149-napA were successfully constructed,which were verified by restriction enzyme digestion,PCR,and gene sequencing.SDS-PAGE analysis showed that HpaA was expressed as a 27 kDa protein and constituted about 33.3%of the whole bacteria cell lysate proteins after induced by nisin,similarly,NapA was expressed as a 15 kDa protein and the percentage in whole bacteria cell lysate proteins was 18.6%.Western blotting analysis demonstrated that HpaA and NapA could produce immunze response with anti-Hp serum.2.Assessment the production of specific antibodies:The level of antigen specific IgG and SIgA antibodies in HpaA group,NapA group and HpaA+NapA group were increased compared to 8149 group and GM17 group(P<0.05).The level of anti-HpaA IgG antibody in HpaA+NapA group was increased compared to HpaA group(P<0.05),however,this significant statistical difference could not observed when compared to NapA group as for anti-NapA IgG antibody(P>0.05).The level of anti-HpaA SIgA antibody in HpaA+NapA group was lower than HpaA group and the level of anti-NapA SIgA antibody in HpaA+NapA group was lower than NapA group(P<0.05).3.Measurement of the cytokines:(1)The levels of IL-17,IL-23 and IFN-? in groups immunized with antigens were higher than 8149 group and GM17 group,NapA group was higher than HpaA group and HpaA+NapA group for the level of IFN-y(P<0.05).(2)IL-2:HpaA+NapA group was higher than 8149 group and GM17 group and HpaA group was higher than GM17 group(P<0.05).(3)IL-12:HpaA group and NapA group were higher than 8149 group and GM17 group and HpaA+NapA group was higher than GM17 group(P<0.05).(4)IL-4:HpaA+NapA group was lower than 8149 group and GM17 group and HpaA group and NapA group were lower than 8149 group(P<0.05).(5)IL-10:HpaA group,NpaA group and HpaA+NapA group were lower than 8149 group(P<0.05).4.Determination of Hp colonization in the stomachs:the results of urease reaction showed that OD550 in HpaA group and HpaA+NapA group were lower than 8149 group and GM17 group and NapA group was only lower than GM17 group(P<0.05).The results of qPCR demonstrsted that HpaA group,NapA group and HpaA+NapA group were lower than 8149 group and HpaA+NapA group was lower than HpaA group(P<0.05).However,this significant statistical difference could not observed in HpaA+NapA group and NapA group(P>0.05).5.The results showed that hpaA and napA gene were detectable in all the examined colonies of the 50th engineered L.lactis strain,genetic positive rates were 100%.After the measurement of OD600 and bacterial colonies in the different phage of cultivation,the growth curves and the relationship curves between and OD600 and CFU/mL of recombinant strains were successfully obtained.Conculsion1.The recombinant L.lactis strain expressing Hp HpaA and NapA were successfully construced taking advantage of food-grade L.lactis expression system via gene combinant technology,which named NZ3900/pNZ8149-hpaA and NZ3900/pNZ8149-napA.Furthermore,the HpaA and NapA proteins induced by recombinant strain had favourable immunoreactivity.2.The two recombinant strains orally immunized alone or combined,an obvious increase for antigen-specific serum IgG and feces SIgA antibodies were determined.Besides,Thl and Th17 type immunze resposes could be observed;Additonly,Hp colonization in the stomaches were decreased in the antigen vaccination groups.Furthermore,the immunoprotective efficiency of recombinant strain expressing HpaA could be enhanced by the recombinant strain expressing NapA.3.The above recombinant strains had obvious immunoprotective efficacy and favourable genetic stability,this study have potential application in Hp oral vaccine researches.