Preparation Of Muli-epitopes Vaccine And Its Fuction Of Immune Protection Against To GAS Infection

Objective: Group A streptococcus (GAS) is the most common bacterial pathogen of Gram-positive bacteria which can cause serious invasive infection, empoison diseases and autoimmunity diseases. By epidemiology discover in 5 citys of China, there are 50%, even 70%~ 80% in countrysides, of children ages in 8~13 infected [1].In all diseases caused by GAS, the most serious disease is hypesensitivity after upper airway infection, such as poststreptococcal acute glomerulonephritis, and rheumatic fever. The effective vaccine against to GAS is not produced until now, as its proteins on the surface contains epitopes known to cross-react with tissues of human, as well as, GAS possesses multiplicity Serotypes [2].About GAS vaccine research,,the candidates have fucused on the surface M protein for 20 years, which is a primary virulence factor of GAS. Although M protein has well immunogenicity and immune protection, it is restricted as a vaccine candidate because it have multiple serotypes and often causes tissue crossreactions. In recent years, many researchers have driven their attention to other surface protein of GAS as vaccine candidates. Fba, a novel Fibronectin-binding protein expressed on the surface of GAS, was reported by Terao Y, and had been found that it exists in 18 serotypes at least by Southern blot[3]. Our former researches have showed that Fba possessed strong immunogenicity and immune protection against GAS[4,5], thus, Fba protein is considered a vaccine candidate of GAS.A immunodominant epitope Fba96-118 has been identified by monoclonal antibody (named A8) of anti-GAS, and 15 linear epitopes of Fba have been identified by polyclonal antibody of anti-GAS in our before research . According to this, we designed two peptides containing series pitopes, one of them is named P5, has contained 8 epitopes, another is recombinant peptide P5 and specific sequnces from four serotypes of M protein. Two recombinant nucleotide sequence were Cloned and expressed, then animals were immunized with the two proteins,and challenged with GAS to evaluate the protective fuction of vaccine protein subsequently. Flow cytometry, Real-time PCR, ELISA, HE staining and other experimental techniques were used to evaluate cellular and humoral immunity induced by the new synthetic vaccines.Methods:1 Immunodominant epitopes were identified by bioinformatics from the existing linear epitopes of Fba via phages display, The gene of peptide P5 was amplified by PCR, and then expressed in E.coli.2 The type-specific sequences from M protein of four serotypes 1,3,6,18 of GAS were selected via Gene sequence alignment with databases of human tissues. The genes Mb was amplified by PCR, and then expressed in E.coli.3 Recombination gene P5 + Mb were amplified by PCR and expressed in E.coli. The whole Fba gene was amplified by PCR and expressed in E.coli. Peptides P5 + Mb, P5 and protein Fba were analyzed by Western-blot, then purified.4 Balb/c mice were randomly individed into 4 groups, every group is 8, and immunized with P5 + Mb, P5 and Fba protein respectively, PBS as control. Mice were immunized with the same dose at an interval of 10 days, and boosted three times. Blood was obtained from mice inner canthal before next immunization, 10 days after last immunization, mice were challenged by GAS.5 10 days after the last immunization, half of mice in each group were killed, and spleen cells were used to detect CD4+T cell and CD8+T cell by flow cytometry (FCM), mRNA of cytokines IL-4, IL-12, IFN-γby Real-time PCR. The level of IgG was analysed by ELISA.6 Mice were challenged with lethaled dose GAS(M+Fba+) to evaluate the protective rates on other half of the mice after four times immunization. 7 In order to evaluate whether cross-immunity with mice tissues would induce by vaccines immunization, the heart and kidney of the mice were observed on morphology, and pathology with HE staining. At same time, mice, with same weeks of age as experiment mice, were infected with GAS , and 5 days later were killer together as control.Results:1 Three prokaryotic expression plasmids, PET28a/P5+Mb, PET28a/P5, PET28a / Fba,were constructed and expressed successfully. Western blot analysis showed that P5 + Mb, P5 and Fba can significantly bind mAb A8.2 Percentage of CD4 + T cells and CD8+ T cells of splenocytes from mice immunized with P5+Mb were the highest among all the groups, which showed significant difference with PBS control group by Flow cytometry. Other groups had no significant difference with PBS control group.3 Cytokines IL-4 was significant higher in group P5+Mb immunized than other groups, while cytokine IL-12 was significantly higher in group Fba immunized than the control group, and IFN-γwere higher in group P5+Mb and Fba immunized. Between other groups have no significant difference by Real-time PCR.4 Levels of serum IgG of each experimental group gradually increased after mice were immunized with the three peptides or proteins. P5 + Mb group, P5 group and Fba group were 1:407680, 1:101920 and 1:203840, respectively. The titers of IgG among three groups were not significant differences.5 Mice of PBS group all died after 3 days challenged. Protective rates in P5 + Mb group, Fba group and P5 group were 80%, 60% and 20%, separately. Mice immunized with each peptides or proteins were evoked significantly protective immune responses compared with the PBS control.6 The volume of heart and kidney were bigger significantly in GAS infection group than other groups. HE staining showed that heart valve was damaged seriously, renal tubular tissue was destroyed in GAS infection group. P5 + Mb group, P5 group and Fba group were no significant difference with the control group, the immune factors induced by vaccines don't damage heart and kidney tissues.Conclusions:1 Construction and expression peptide P5 + Mb, P5 and protein Fba successfully.2 P5 + Mb, P5 peptides and Fba protein have protective effect on GAS infection, and P5 + Mb peptide showed significantly fuction in immune protection and enhances the efficacy of cellular and humoral immunity.3 P5 + Mb , P5 peptides and Fba protein have no significantly immunological cross-reaction.with tissues of heart and kidney of mice...