| lipopolysaccharide (LPS), which is a component of Gram negative(G~-) bacteria cell wall, has been considered as a very important factor starting the pathologic cascade leading to multi-organ failure and death. Respecting of the high disease incidence and disservice of endotoxemia, more and more researches about how to prevent and cure the disease caused by LPS have been interested in. Up to now, there is no ideal antagonists against LPS and lipid A for clinical prophylaxis and therapy. There were some evidences that the pre-presence of antibodies against LPS are protective and the amount of antibody against core glycolipid of LPS in vivo is correlated with the survival in sepsis patients, but the protection of the antibody is limited . And the protection against LPS by passive application of anti-LPS antibody was indicated to be time phase limited and hard to master.Therefore, to develop a vaccine which can elicit secondary immune response and to provide broad spectrum prevention and protection against LPS is very necessary. There are two major difficulties in preparing effective LPS vaccine with broad spectrum protection: (1) LPS is a kind of Thymus-independent antigen (TI antigen), which can not elicit secondary antibody response and high affinity antibodies with strong opsonization; (2) there are too many kinds of specificities of strains and serotypes in Gram negative bacteria with LPS.Our objective is to screen the peptide mimotopes of the conservative epitopes on LPS, it will change the properties of lipopolysaccharide epitope into peptide epitope, change the T cell independent antigen (TI antigen) into T cell dependent antigen (TDantigen), so that it will be able to induce effective secondary immune response and immunological memory.In the previously research, we synthesized three peptides mimetics of S.typhi-LPS, mice immunized with these peptides produced anti-LPS antibodies, but the titer of these antibodies was too low to provide effective protection against attack by bacteria. Based on those research, mimotopes to conservative structure of LPS would be screened from phage display cyclic7-peptide library by using purified polyclonal anti-S.typhi LPS antibodies with cross protection against G" bacteria infection, and one of the peptides mimicry to LPS epitope was synthesized and named as 13L. Four peptide mimics included 13L and 13a^ 13K 12w were conjugated to Blue Carrier (BC) separately as vaccine candidates to immunize the mice, the antigenicity and immunogenicity of these peptides were identified by serials of experiment in vivo and in vitro. We also would observe whether these peptide-vaccine can induce a effective protection against bacterium challenge and inhibit LPS induced septic shock or not.This research can be divided into the following three parts.Part I Screening and identification of the LPS peptide mimotopes from phage displayed peptide librariesThe LPS mimotopes were screened from c7c phage display peptide library by using polyclonal antibody against E.coli LPS 2630 (L2630) purified with affinity chromatograph and the antigenicity of selected clones was identified by ELISA. After 3 rounds of biopanning, 12 out of 20 phage clones were identified as positive clones which could bind to polyclonal anti-LPS 2630 antibody, and 7 of these 12 phage clones showed binding with polyclonal anti-S.Typhi LPS 7261 antibody. The deduced amino acid sequence analysis showed 7 of these clones had the conservative sequence: X-DXXL-XX or X-XDXXL-X (being apply for patent). These results suggested that the peptides displayed on phage clones can mimic to cross-reactive epitopes of LPS 2630, and 7 of these phage clones may mimic to common epitopes of LPS 2630 and LPS 7261.Part II The synthesis and identification of peptide mimics of LPSIn this part, four peptide mimics of LPS 13a(SACPSWASFWCGG) > 13b(SACFQFYPAACGG)> 12w(GPPQWFFSQPQL)and BL(SACQEXXXXGCGG)(being apply for patent) were redesigned and synthesized by adding SA and GG on the two sides of origin sequence for stability of the conformation. The antigenicity and immunogenicity of peptide mimics of LPS were identified.Four peptide mimics were conjugated with BSA as carrier, separately. The binding of 13L-BSA with polyclonal rabbit an&S.typhi antibody, polyclonal rabbit ani-bacillus E.coli antibody and 1B6 (monoclonal antibody for S.typhi) was the best among these four peptide mimics. The specific inhibition test indicated that four' peptide mimics could inhibit the binding of anti-LPS polyclonal Ab with different kind of LPS in ELISA, and the inhibition of 13L was best too. These results indicated that four synthetic peptides could be mimetic to epitopes of LPS.The titer and binding specificity of anti-LPS antibody from Balb/c mice immunized with peptide-BC (Blue carrier) conjugate. The antisera from mice were detected for binding with different LPS including S.Typhi LPS 7261, E.Coli LPS 2630, J5LPS and Re595 Lipid A. The results showed that antibodies against different LPS were produced in all of mice immunized with four kinds of peptide-BC conjugate separately, and the titer of antibody increased with the times of immunization, and the secondary immune response to LPS from different species was observed. The antisera from immunized mice could bind with S.Typhi LPS 7261, E.Coli LPS 2630. These results suggested that these four synthetic peptide mimics successfully simulated the conservative or cross-reactive epitope of LPS from different species, and the characteristics of TI (thymus independent) antigen for LPS has been changed to be of TD(thymus dependent) antigen. The feasibility of the epitope mimicry method used for making polysaccharide antigen peptide mimics as vaccine was also proved.Based on immunization with four kinds of peptide mimics separately , the mixture of combinatory peptide mimics was tested for eliciting production of antibodies. The results showed that immunization with mixture of peptide mimics could elicit better antibody production than using with single peptide mimics.We also tried to use inactivated S.Typhi to boost immunization with peptide mimics, which means the epitope with different carrier. It might be surprisingly that antibody titer of mice immunized with peptide mimics increased obviously by boosting with inactivated S.Typhi.In addition, the antisera from mice and rabbit immunized with 13L-BC conjugate were found to bind with LipidA, LTA and PGN of Gram+ bacterial.Part III Peptide mimics as experimental vaccine elicited protective immunity against bacterial challenge and endotoxic shockIn order to characterize four peptide mimics of LPS as candidate vaccine , the animal model with bacteria challenge and endotoxic shock induce by injection of LPS were established. The survival time of mice attacked with LD90 S.typhi 0.2 mL of 0.5x107/mL ip and blood pressure of mice injected with 200 ug/mouse LPS were observe.Peptide mimics was conjugated to Blue Carrier (BC) as experimental vaccine, BALB/c mice were immunized with peptide-BC conjugate emulsion using Freud's adjuvant. Specific antibodies against S.typhi-LPS 7261 and E.c |
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