Immunoprotection Of Optimized Helicobacter Pylori OipA Oral DNA Vaccine In C57BL/6 Mice

Background:Helicobacter pylori (H. pylori) is a spiral-shaped, microaerophilic,gram-negative bacterium that colonizes the human gastric andduodenal mucosa, It is well known that H. pylori is recognized as the etiological agent of gastric diseases such as chronic atrophic gastritis and peptic ulcers, H. pylori infection is also associated with gastric mucosa-associated lymphoid tissue lymphoma and gastric cance. Current therapies, based on a combination of three or four different antibiotics together with a proton-pump inhibitor,but faces problems such as increasing antibiotic resistance, side effects,recurrence, re-infection and high cost etc. Anti- H. pylori vaccines would be an effective method in prophylactic and therapeutic treatment of H. pylori infection. oipA mainly exists in triple-positive strains and oipA mRNA was relatively higher expression in coccoid shape H. pylori Therefore oipA DNA vaccine may be used for vaccine development against both spiral and coccoid H. pylori. DNA vaccines named as"Third-generation of Vaccines"are a novel vaccination technology which proven to enhanced cellular and humoral immune responses simultaneously. To enhance the immunogenicity of DNA vaccine is the most important,so we optimized H.pylori oipA gene by codon optimization,inserting the Kozak sequence,engineering CpG motifs to investegate whether optimization of oipA gene could enhance the immunogenicityof oipA DNA vaccine and develop strain of vaccine in Attenuated Salmonella typhimurium SL7207 harboring recombinant plasmid pVAX1-oipA and pVAX1-optioipA,then study its protection against H.pylori in C57BL/6 mice.To explore a feasibility of prophylactic and therapeutic effect of Hp DNA vaccine. which lays the foundation for further developing oral Hp DNA vaccine . Methods:1. Construction of recombinant Salmonella carrying oipA gene DNA vaccines①oipA gene was modified by codon optimization,inserting the Kozak sequence and engineering CpG motifs.The optioipA gene and oipA gene was inserted into the eukaryotic expressing vector pVAX1 respectively to develop recombinant plasmid pVAX1-optioipA and pVAX1-oipA.②The AGS cells were transfected by pVAX1-oipA,pVAX1-optioipA and pVAX1 respectively,then the OipA protein expression was confirmed by western blotting,and analyzed the expression level of OipA protein simultaneously.③pVAX1-oipA,pVAX1-optioipA and pVAX1 were converted to LB5000 for methylation and then themodified eukaryoticvector was electrotransfered to final host SL7207 respectively,moreover,the stability of recombinant strains were confirmed.2. protective immunizationSixty special pathogen free (SPF) C57BL/6 mice were randomly divided into 5 group①P BS(10);②S L7207(10);③SL7207/pVAX1(10);④S L7207/pVAX1-oipA(15)⑤S L7207/pVAX1-optioipA(15); each mouse was immunized by oral inoculation at 0,2 -week. Blood were collected from mice angulus oculi at 2 week after the last immunization,Sera specific anti-OipA IgG and subtype IgG1,IgG2a were measured by indirect ELISA. Four weeks after last immunization,mice were challenged with Hp SS1 at a dose of 5×108CFU at 0,2,4-d. Mice were sacrificed 4 weeks after last challenged, rapid urease test, quantitative culture of H. pylori and histology examination of gastric mucosa were performed. Splenocytes were cocultured with H. pylori whole cell sonicate(Hp WCS) for 3 days and the suspension were collected for measurement IFN-γand IL-4 by ELISA.Results:1. Construction of recombinant Salmonella carrying oipA gene DNA vaccinesPCR identification, enzyme digestion and sequencing revealed that the recombinant plasmid pVAX1-oipA, pVAX1-optioipA were constructed successfully;The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed a corresponding production,moreover,the expression level of pVAX1-optioipA was higher than pVAX1-oipA;PCR identification,enzyme digestion revealed that the recombinantstrains SL7207/pVAX1-oipA,SL7207/pVAX1-optioipA were constructed successfully. the stability of recombinant strains were confirmed.2. protective immunization2.1 Two weeks after last immunization, immunized mice had been induced anti-OipA IgG and subtype IgG1,IgG2a antibodies;and the levels of anti-OipA IgG and subtype IgG1,IgG2a antibodies in pVAX1-optioipA group was obviously higher than pVAX1- oipA group(P< 0.01).2.2 Compared with control group,significantly higher levels of IFN-γand IL-4 were detected in mice splenocytes immunized with pVAX1-oipA and pVAX1-optioipA(P< 0.01), moreover,the level of IFN-γand IL-4 of pVAX1-optioipA was higher than pVAX1-oipA(P<0.01). To optimize oipA gene would enhanced Th1 and Th2 response.2.3 C57BL/6 mice achieved specific immunoprotection by oral inoculation with vaccine strain.The protection rate in pVAX1-oipA group and pVAX1-optioipA was 40% and 73.33% respectively. H.pylori colonization density were significantly decreased(p<0.01), compared with SL7207/pVAX1-oipA, the Hp colonization density were significantly decreased and the degree of gastric mucosa inflammation were also decreased in SL7207/ pVAX1-oipA group( p<0.01 ).Conclusions:1.pVAX1-oipA and pVAX1-optioipA eukaryotic expression vectors were constructed successfully,The AGS cells transfected with pVAX1-oipA and pVAX1-optioipA had expressed a corresponding production,moreover,the expression level of pVAX1- optioipA was higher than pVAX1-oipA, so optimization of H. pylori oipA gene could improve protein expression .2.Attenuated Salmonella typhimurium SL7207 containing pVAX1-oipA,pVAX1- optioipA were successfully constructed .It can protect C57BL/6 mice against H pylori infection and to optimize oipA gene would enhance the protection efficiency of H. pylori DNA vaccine. 3. Oral inoculation with oipA DNA vaccine would facilitated a mixed Th1 and Th2 response,to optimize oipA gene would enhance both cellular and humoral immune responses in C57BL/6 mice.