Study Of Recombinant Lactococcus Lactis As Mucosal Delivery Vector

Lactococcus lactis (L.lactis) is a gram-positive bacterium that is used for foodand fermentation industry and generally regarded as safe (GRAS). L.lactis is used asan ideal mucosal delivery vector, which has the advantages of weak antigens, withoutextracellular enzyme activity and without colonization in gastrointestinal tract.Although L.lactis has many achievements in expressing bacterial or virus antigengenes, upon L.lactis with the application of preventing highly pathogenic avianinfluenza H5N1virus has not been developed yet.Highly pathogenic avian influenza H5N1virus has posed a great threat to healthof humans and animals, while mutation of hemagglutinin (HA) gene of virus genomemay easily lead to antigen drift or antigen shift, and then result in extremelydifficulties and challenges in preventing the infection of avian influenza virus. HAprotein, as a protective antigen of influenza virus, has been developed for thepreparations of avian influenza vaccines.In this thesis, we focused on the HA or HA1gene of highly pathogenic avianinfluenza virus H5N1subtype with nisin-controlled expression (NICE) system,constructing different expresson types of recombinant L.lactis and evaluating immuneeffects after combined with adjuvant or coated by enteric capsule. The objective ofthis thesis is to establish recombinant L.lactis-based technology platform of vaccinedelivery, provide a feasible idea for preventing and controlling the infection of highlypathogenic avian influenza virus and expand further research to other viral antigengenes.In this thesis, there are following main innovations.(1) Based on nisin-controlled expression system, Three different expressiontypes of recombinant L.lactis were successfully constructed in the first time.(2) HA or HA1protein was correctly located and expressed in the different partsof L.lactis. (3) Surface displayed and secretory recombinant L.lactis provided100%and80%immune protection efficiency, respectively. After combined withadjuvant.(4) Surface displayed and secretory recombinant L.lactis provided100%immune protection efficiency, respectively. After coated by enteric capsule.(5) Based on Surface displayed and secretory recombinant L.lactis, deliverytechnology platform for avian influenza vaccines was successfullyconstructed.Three different expression types of recombinant L.lactis by conventionalmethods of molecular biology, they were non-secretory recombinant L.lactis,L.lactis(pNZ8150-HA), secretory recombinant L.lactis, L.lactis(pNZ8110-HA), andsurface displayed recombinant L.lactis, L.lactis (pNZ8110-pgsA-HA1), respectively.Briefly, we used plasmid pGEM-HA as template and designed Sca I and Hind III sitesin forward and reverse primers, respectively. PCR product and expression plasmidpNZ8150without secretion signal were digested by Sca I/Hind III, respectively.After ligation, the product was named as pNZ8150-HA.Similarly, we used expression plasmid pNZ8110with secretion signal ssUSP andplasmid pGEM-HA as framework and template, respectively. Nae I and Hind III sitesin forward and reverse primers were designed for PCR reaction. PCR product andexpression plasmid pNZ8110were digested by Nae I/Hind III, respectively. Afterligation, the product was named as pNZ8110-HA.Firstly, we used Bacillus subtilis genome as template, Spe I site and linker-1(5’tcctcctggggatcc3’) were designed for PCR reaction in forward and reverse primers,respectively. PCR product was pgsA gene. Secondly, HA1gene was obtained by PCRreaction using plasmid pGEM-HA as template, linker-2(5’ggatccccaggagga3’) andHind III were designed. Finally, because of the complementary of linker-1andlinker-2, pgsA gene and HA1gene were fused by bridge PCR reaction. The PCRproduct (pgsA-HA1gene) and expression plasmid pNZ8110were digestedrespectively. After ligation, the product was named as pNZ8110-pgsA-HA1.pNZ8110, pNZ8150-HA, pNZ8110-HA and pNZ8110-pgsA-HA1weretransferred to competent L.lactis NZ9000by electroporation, respectively. Screened positive clones were named as L.lactis(pNZ8110), L.lactis(pNZ8150-HA),L.lactis(pNZ8110-HA) and L.lactis(pNZ8110-pgsA-HA1), respectively.L.lactis(pNZ8110) was used as a negative control.Recombinant L.lactis was induced for expression by nisinA, the optimalinducible expression conditions were that the final concentration of nisinA was1ng/ml, and induction time was three hours.To test heterologous preoteins expression in different parts of recombinantL.lactis, after nisinA induction, supernatants and lysates of L.lactis(pNZ8110)、L.lactis(pNZ8150-HA)、L.lactis(pNZ8110-HA) and L.lactis(pNZ8110-pgsA-HA1)were analyzed by western blot, respectively. Western blot analysis indicated thatL.lactis(pNZ8110) had not HA-specific protein band in the supernatant and lysate,this result revealed L.lactis(pNZ8110) did not produce HA protein (about64kDa).L.lactis(pNZ8150-HA) had not specific protein band in the supernatant, but detectedin the lysate, this indicated L.lactis(pNZ8150-HA) was a non-secretion recombinantL.lactis. L.lactis(pNZ8110-HA) had HA-specific protein band (about64kDa), thisshowed L.lactis(pNZ8110-HA) was a secretion recombinant L.lactis. AlthroughL.lactis(pNZ8110-pgsA-HA1) did not produce specific protein band in thesupernatant, a specific protein band(molecular weight: about82kDa) was detected inthe lysate, further experiment was needed to confirm thatL.lactis(pNZ8110-pgsA-HA1) was a surface displayed recombinant L.lactis. Aftersurface of recombinant L.lactis was treated by polyclonal anti-HA serum, the relativefluorescence intensity was analyzed by flow cytometry and immunofluorescenceassay. Results revealed L.lactis(pNZ8110-pgsA-HA1) was positive, and indicated itexisted specific antigen protein on its surface. By contrast, L.lactis(pNZ8110)、L.lactis(pNZ8150-HA) and L.lactis(pNZ8110-HA) were negative. So constructions ofthree different recombinant L.lactis were correctly confirmed by western blot, flowcytometry and immunofluorescence analysis. Expression of heterologous protein wascorrectly located at the different parts of L.lactis. In addition, concentration ofheterologous protein was assayed by Brodfford method, results showed the finalconcentrations of L.lactis(pNZ8150-HA), L.lactis(pNZ8110-HA) andL.lactis(pNZ8110-pgsA-HA1) were1.4813mg/ml,5.7144mg/ml and5.2288mg/ml, respectively。To explore the optimal immunization dose and the impact of adjuvant onimmune efficiency, we adopted immunization strategy that immunization time wasday1～3, day14～16and day28～30天(total times: nine). Immunization dosewas50ul,100ul,150ul and200ul，respectively. Concentration of recombinantL.lactis was1011CFU/ml. when recombinant L.lactis was combined with adjuvant,cholera toxin B subunit (CTB) was used as a adjuvant, its dose was1mg every time.The same doses of PBS, L.lactis(pNZ8110), PBS+CTB and L.lactis(pNZ8110)＋CTBwere uses as controls. Each dose group was10mice,To obtain the optimal immunization dose, HA-specific serum IgG and fecal IgAantibodies were analyzed by ELISA. Compared with IgG and fecal IgA antibodieslevels of four different dose groups, the optimal combination dose was150ulrecombinant L.lactis and1mg CTB every time. Among the optimal conditions,immune effect of L.lactis(pNZ8110-pgsA-HA1)＋CTB group was the best,L.lactis(pNZ8110-HA)＋CTB group was the better and L.lactis(pNZ8150-HA)＋CTB group was the worst. Immune effect was investigated by ELISpot assay,hemagglutination inhibition and virus challenge experiments, after mice were orallyadministrated with the optimal combination dose. Mice were immunized withL.lactis(pNZ8110-pgsA-HA1)＋CTB or L.lactis(pNZ8110-HA)＋CTB, and not onlyproduced high levels of humoral immune and mucosal immune responses, but alsoproduced cellular immune response. The mice of L.lactis(pNZ8110-pgsA-HA1)＋CTB group obtained complete protection,100%survival, the survival ofL.lactis(pNZ8110-HA)＋CTB group was80%, after mice were treated with H5N1virus lethal challenge. These results demonstrated two vaccines developed includingsecretory and surface displayed recombinant L.lactis combined with adjuvant CTBthat provided effective immune protection against H5N1virus challenge.Although the use of mucosal adjuvant CTB could improve immune efficiencyof recombinant L.lactis, especially secretory and surface displayed recombinantL.lactis, the higher dose (150ul, concentration of recombinant L.lactis was1011CFU/ml), more immune times and the potential danger of CTB prompted us tosearch for another more safe, effective and feasible method. We tried to hope that the less immunization dose obtained the better immune effect. According to the immunecharacteristics of recombinant L.lactis, recombinant L.lactis immunized by oraladministration was mostly degraded by stomach acid, less recombinant L.lactisentered into small intestine and much less ones were uptaken by villi M cells. In viewof this condition, we designed an experiment that recombinant L.lactis coated byenteric capsule, mice immunized with this enteric capsule could avoid the aciddegradation, as well as targeting intestinal site, and improve immune efficiency ofrecombinant L.lactis.To briefly narrate, we named L.lactis(pNZ8110), L.lactis(pNZ8150-HA),L.lactis(pNZ8110-HA) and L.lactis(pNZ8110-pgsA-HA1) as L1, L2, L3and L4,respectively. After L.lactis was coated by enteric capsule, they were accordinglynamed as capsule-L1, capsule-L2, capsule-L3and capsule-L4, respectively. Detailedimmunization strategy was that immunization time was0,2,4,6weeks (every otherweek, total times: four) enteric capsule coating10μl recombinant L.lactis that itsconcentration was1011CFU/ml, immunization dose was1capsule/time. To highlightthe advantage of enteric capsule, the same doses of PBS、L1、L2、L3and L4wereused as a reference, respectively, in which PBS、L1and capsule-L1as negativecontrols. Each dose group was10mice.HA-specific serum IgG and fecal IgA were detected by ELISA and secretorylevels of IFN-γ were analyzed by ELISpot assay. Results showed the antibody levelsand secretory levels of IFN-γ of capsule-L3group and capsule-L4group weresignificantly higher than that of other immune groups. These results indicated miceimmunized by capsule-L3or capsule-L4not only produced humoral immuneresponse, mucosal response, but also induced cellular immune response.Microneutralization assay of serum from other aspects supported the immune effectof capsule-L3and capsule-L4. Mice treated by capsule-L3or capsule-L4showedcomplete protection (100%survival), while the survivals of other groups were lessthan or equal40%, after H5N1virus lethal challenge. So two developed vaccines(capsule-L3and capsule-L4) could provide a strong immune protection and provide anew and feasible idea for controlling the infection of highly pathogenic avianinfluenza H5N1virus. In summary, we successfully constructed three different expression types ofrecombinant L.lactis. HA or HA1antigen expression was accurately located at thedifferent part of recombinant L.lactis after nisinA inducing. Secretion recombinantL.lactis and surface displayed recombinant L.lactis were combined with mucosaladjuvant CTB showed the best immune effect, respectively. These results showedL.lactis(pNZ8110-HA) and L.lactis(pNZ8110-pgsA-HA1) developed for controllingthe infection of highly pathogenic avian influenza H5N1virus. In addition, afterenteric capsule coating L.lactis(pNZ8110-HA) and L.lactis(pNZ8110-pgsA-HA1),respectively, mice immunization with capsule-L3or capsule-L4obtained completeprotection. These results revealed two developed vaccines (capsule-L3andcapsule-L4) showed greater potential for preventing highly pathogenic avianinfluenza H5N1virus.