Salmonella Typhimurium Flagellin Enhance Special Antibodies Response Research

1. Research BackgroundAs one kind of peritrichate, the flagella of Salmonella typhimurium, which is composed ofa basal body, hook, motor, and filament, plays a major role in its motility and specialantigenicity[1, 2] . The special antigen, H antigen, is not only the systematical standards, butalso a potent inducer of innate immunity. Flagellin is a highly conserved bacterial proteinwhich is the most important part of the flagella and elicits TLR5 dependent inflammatoryresponses in both animals and plants [3, 4] and plays an significant role in bacterialpathogenicity by facilitating motility and adhesion to host mucosal tissues [5-8]. Recentstudies have shown that flagellin is associated with strong adaptive immune responses,mainly Th2-type, with the production of mucosal [9-12] and systemic IgA.Flagellin caninduce the antigen-special response which is combined with flagellin through the innateimmunology induced by it. Accordingly they have the profound significance as adjuvants,and recombinant flagellin in a soluble or fused form has been reported to play an importantrole as adjuvant for novel vaccines against epitope-based influenza vaccines [13], Yersiniapestis [8, 14], Plasmodium falciparum [15] , Tetanus toxoid [10], West Nile virus (WNV) [16]and inactivated influenza virus (A/PR/8/34, HIN1 subtype) [17] . The safety of flagellin usedas adjuvant is without doublts, because there are no seriously adverse reactions during allof the experiments.In view of the established role of the innate immune response in the development ofadaptive immunity, the strong innate response to flagellin may be the reason why flagellinpresents as an effective adjuvant. The innate immue is the firstline defense againstmicrobial pathogen. It can recognize and discriminate the different pathogen and as theinitiator of the events that are essential for the developent of an adaptive immune response,in which antigen presenting cells play the important role. Pattern recognition receptorexpressed on antigen presenting cells can recognize the corresponding pathogen associatedmolecular pattern expressed on pathogen [18-20]. Flagellin is a proteinic pathogen associatedmolecular pattern which can be recognized by pattern recognition receptor, Toll-likereceptor 5 mainly [21-23]. The direct binding of flagellin to the TLR5 expressed on cells suchas endothelial and epithelial cells, monocytes, macrophages, and immature dendritic cellsinitiates the MyD88 depended signal passway, products proinflammatory cytokines such as TNF-α,IL-6 and IL-12, and up-regulate some costimulatory molecule, CD80, CD86, MHCet al [24], then stimulate cells maturation, enhance the ability of presenting antigen, andmigrate to secondary lymphoid sites . Accordingly Th cells are attracted, stimulated,differentiated to another typical cell and finally initiate the adaptive immunity. Meanwhile,the flagellin within the cytosol of macrophages is detected through the Nod-like receptot(NLR) IPAF and NAIP5. IPAF is the mainly cytosol receptor, however, NAIP5 justenhance the detection of the flagellin with IPAF.Failing to enhance the immunogenicities of antigens to obtaina protective and sustainedresponse in humans is, so far, still a formidable problem with which many vaccines todayhave confronted including the blood-stage antigen apical membrane antigen 1 (AMA1)which is the leading malarial vaccine candidate of Plasmodium falciparum. [25,26] Despite ofthe conjugation of Alhydrogel or Montanide ISA720, the special antibody in human bodyto AMA1 still remained a low level. The addition of the TLR9 agonist CpG ODN to theAMA1/Alhydrogel formulation greatly increased the functional antibody responses inhuman, and the GIA can averagely reach 90%, in which 96% is the highest. However,along with the side effect associated with CpG ODN in an adult phase III trail, thepotential safety hazard became the biggest obstacle to the application of CpG ODN.This research focused on the immunopotention of flagellin which is conjugated withOVA/Alhydrogel formulation instead of CpG ODN. All the results has provided theevidence that the Salmonella flagellin is a potent immunopotentiator of specific immunityin mice immunized with ovalbumin .2. ObjectivesThis study tries to research the ability of flagellin as a immunopotentiator among BALB/cmice, through comparing the special antibodies in the serum during a randomizedcontrolled trial.3. Methods3.1 Analyzing the quantity of antibody to OVA from immunized BALB/c mice, includingIgG/IgG2a/IgG2b/IgG3, by EILSA. 3.2 Analyzing the concentration of IL-4 and IFN-γin supernatant of splenocytes whichwere isolated from immunized mice sacrificed after 14 days post-boost and stimulated byOVA and PMA.3.3 Contract TLR5-specific bioactivity assay: transfect the RAW264.7 cell strain withplasmid pUNO-hTLR5 expressing the human TLR5 through the TurboFect agent.4. Results4.1 The vaccine formulations of OVA+Alhydrogel+Flagellin administered intramuscularlyinduced significantly higher antibody titers comparing to OVA+Alhydrogel, meanwhile nostatistically significant difference in serum IgG titers were detected between mice injectedwith flagellin and CpG ODN. Moreover, the period of higer antibody titers is longer in thegroup co-administered with flagellin than those with CpG ODN. In the whole experiment,the immunological enhancement of flagellin is comparable to CpG ODN's.4.2 The flagellin coadminisered intramuscularly induced higher IgG2a and IgG2b ratio ofspecial antibody, and produced higer IFN-γand less IL-4 than the other group. Based onthe above results detected with the doses tested, we found that flagellin enhanced both theTh1 and Th2 responses, Th1-biased. On the other hand the increased IgG2b levels activatecomplement via the classical pathway. It has been suggested that the classical pathway isthe most effective immune defense against microorganisms, and flagellin may thereforehave some advantages as an adjuvant for enhancing the humoral immunity againstinfection.4.3 When Vaccine formulations were administered subcutaneously and intraperitoneally,the group coadministered with flagellin induced significantly higher special antibody titersthan the group injected only with Alhydrogel and significantly lower than the ones withCpG ODN.4.4 The immune response generanted in mice with each vaccine formulationadministratered subcutaneously and intraperitoneally is the same as the results of groupsimmunized intramuscularly. Howerve, there is no higer ratio of IgG2b in groupsimmunized subcutaneously, It supposed that the administrer's way is not suitable foractivating complement system.4.5 Get the RAW264.7 cell line which expresses TLR5 consistently. 5. ConclusionsFlagellin is a promising candidate immunopotentiator which can induce the comparableimmunological enhancement as CpG ODN does. Co-administration of flagellin with OVAand Alhydrogel induces enhanced humoral and cellular immune responses, and induces themix Th1/Th2 humoral respons, Th1 mainly.