Preliminary Study Of Flagellin FliC Mutant As HPV L2 Peptide Antigen Vector

Currently, two HPV vaccines have been commercially developed based on HPV major capsid protein L1, but their protection are mainly for HPV 16 and HPV 18 infection and associated lesions, and can only prevent 70% of Cervical cancer worldwide, with weak cross-protection or no cross-protection for other HPV types.According to the HPV detetion rate of cervical cancer specimens worldwide, HPV 18 is second only to HPV 16. Patients with HPV18-associated carcinomas have a relative risk of death 2.4 times greater than that of patients with HPV16-associated carcinomas. The minor capsid protein L2 of HPV contains a plurality of cross-neutralizing epitopes, which are mainly located in L2N (aa.13-154) region.Therefore, this area is an ideal object of research on L2. L2 can only induce a low titer of neutralizing antibody, Thus enhancing the immunogenicity of L2 is the key point to develop a novel HPV L2 vaccine.Flagellin can induce inflammatory response and DC (Dendritic cell, DC) maturation through TLR5 signaling pathway, and can enhance the immunogenicity of antigen which is mixed or expressed with flagellin as fusion protein, but the adjuvant activity of flagellin existed in fusion proteins is higher than that of flagellin mixed with antigen. The immunogenicity of HA1 of influenza virus H1N1 will be enchanced when it is inserted to replace the hypervariable region of flagellin to form a fusion protein, but it may also lead to some inflammatory response in some people including muscle pain, chill and headache when immunizing with this fusion protein. According to the previous studies, Deletion of D2 and D3 domains can reduce far fewer systemic inflammatory responses and abrogate detectable inflammatory side effects in mouse.According to the HPV detetion rate of cervical cancer specimens worldwide, HPV 18 is second only to HPV 16. Patients with HPV18-associated carcinomas have a relative risk of death 2.4 times greater than that of patients with HPV16-associated carcinomas. In order to find a protein carrier which possesses adjuvant activity and safety to apply in L2 vaccine, we choose the Salmonella typhimurium flagellin FliC as protein carrier. We deleted different regions of hypervariable region (ND2+D3+CD2a+CD2b) which can induce flagellin-specific antibody, and retained sites (ND1 and CD1) for TLR5 recognition to construct different FliC mutants; then we selected the HPV 18 L2 N polypeptide as an antigen, which is inserted into flagellin mutants’deleted area to construct the fusion genes. The fusion genes were then cloned into pET22b vector. After prokaryotic expression, purification and immunization of mice with flagellin fusion proteins, we further to measure the flagellin-specific ELISA binding antibody and neutralizing antibody against HPV18.The results were as followed:1. In our research, we constructed three fliC mutants,whose D3, D3+CD2a or D2+D3 was deleted. fliC mutants and fliC were cloned into pET22b vector by Nde Ⅰ and Hind Ⅲ restriction sites, then indentified by sequencing, thus, we obtained pET22b-fliC expression vector.18 L2N gene was inserted to replace the deleted region.18 L2N gene was cloned to replace the deleted region directed by Nhe Ⅰ and Afl Ⅱ restriction sites. Then, constructed pET22b-fliC△D 3/L8 L2N, pET22b:fliC△D3CD2a/18 L2N and pET22b-fliC△D 2D 3/18 L2N vectors were indentified by Nhe Ⅰ and Afl Ⅱ digestion.2. The constructed plasmids and pET 42a-18 L2N were transformed into BL21(DE3). Proteins were expressed in BL21(DE3) induced by 0.5 mM IPTG at 30℃ for 4 h.18 L2N and FliC mutant/18 L2N fusion proteins were all highly expressed as inclusion body in E.coil with 6×Histag at the C-terminal. They were all purified by washing the inclusion body and Ni-Sepharose affinity chromatography. The denatured proteins were renatured by dialysis. Endotoxin was removed by Q-Sepharose HP anion exchange chromatography. Residual endotoxin was quantified by LAL test, which indicated all the samples were below the 50 EU/mg. By SDS-PAGE and Western blot analysis, the purity of all the proteins were higher than 90% and were existed as monomers by Native-PAGE. FliC was expressed as soluble protein in E.coil and purified by Ni-Sepharose affinity chromatography. By linear elution and optimizing the concentration of imidazole in binding buffer, we got highly purified FliC.3. Mice were immunized three times at interval of two weeks with equimolar 18 L2N and FliC mutant/18 L2N fusion proteins. Data from ELISA coated with FliC indicated that The FliC specific IgG titer is low in the serum of mice in all groups two weeks after the first immunization without significant difference among all the groups. Two weeks After the third immunization, the titers of FliC△D3/18 L2N group were significantly higher than that of FliC△D 3C D 2a/18 L2N and FliC △D2D3/18 L2N group. There is no significant difference between FliC△D 3CD 2a/18 L2N and FliC △ D2D3/18 L2N group in the FliC specific IgG titer.4. Within the first week after the first immunization, the daily body weight change of each mouse was measured at the same time. Only the body weight of FliC△ D 3/18 L2N immunized mice decreased in the first day after immunization and until the fourth day, FliC△ D 3/18 L2N im munized mice achieved a similar level of weight change as the other three groups of mice.5. Two weeks after the third immunization, the neutralizing antibody titers against HPV 18 were measured by HPV pseudovirus neutralization assay. The titer of HPV 18 neutralizing antibody of FliC△D 2D 3/18 L2N group and FliE △D 3/18 L2N group were higher than that of other groups, but only significant when compared with that of 18 L2N group. The titers of FliC △D 3C D 2a/18 L2N group were relatively low without significant difference when compared with that of 18 L2N group.In summary, this study demonstrated that FHC△D2D3 can enhance the production of neutralizing antibody induced by 18 L2N and the FliC-specific ELISA binding antibody can be reduced by deleting the hypervariable region, implying the intramolecular adjuvant potential of FliC AD2D3 in HPV L2 vaccine.