Immunogenicity Of Novel Fusion Proteins Immunized Against Enterohemorrhagic Escherichia Coli O157:H7 Infection In Mice Models

Since enterohemorrhagic E.coli(EHEC)O157:H7 first identified as a human pathogen in 1982,different countries including China had reported different scale of outbreaks and sporadic cases.Human infection is assoiated with a wide range of clinical illness,including asymptomatic shedding,and about 5%-10%develops to bloody diarrhoea,haemorragic colitis,haemolytic uraemic syndrome,and even death.EHEC O157:H7 is found regularly in the faeces of healthy cattle,and is transmitted to humans through contaminated food,water,and direct contact with infected people or animals. Treatment of EHEC O157:H7 infection with antimicrobial agents is controversial,and management of patients is only supportive,because antimicrobial agents may increase the risk of HUS.During the past decade,EHEC O157:H7 has evolved from a clinical novelty to a global public-health concern.Based on the reasons above,researches on the EHEC O157:H7 vaccine were considered essential,for the usage of vaccine may be a simple,economical,and fast management of EHEC O157:H7 infection.Pathogenesis of EHEC O157:H7 are adherence factors,toxins,hemolysin and so on. These molecules and their segments are the available targets of protective antigens.Intimin is a key protein that mediates the attaching and effacing(A/E) lesion.In this study we chose the immunoprotective segment of Intimin to immunize BALB/c mice, and obtain the serum to analyze the immunogenicity and immunoreactivity of this truncated Intimin(Int281).Then test its ability to decrease EHEC O157:H7 adhesion onto HEp-2 cells and BALB/c mice.We also chose the immunoprotective B subunits of Shiga toxins to provide a wide scope protection of EHEC O157:H7 infecton and its severe sequela.We fuse the two B subunits of Shiga toxins named 2S to induce anti-toxin response,and further fuse 2S with Int281 named SSI to induce both anti-toxin and anti-adhesion responses.Part 1.Prokaryotic expression and immunogenic analysis of Int281EHEC strains induce a characteristic attaching and effacing(A/E) lesion on epithelial cells.A/E lesion formation requires Intimin,an outer membrane adhesion protein.The cell binding activity of Intimin is localized at the C-terminal 281 amino acids of the polypeptide.Intimin of EHEC O157:H7 is distinct from other serotypes,and it is typeγand their differences are concentrated in the C terminal.So we chose the immunogenic C-terminal Int281 as a vaccine candidate to study,we cloned the int281 gene from EHEC O157:H7 EDL933 by PCR.The product of PCR was inserted into pMD18-T cloning vector.The gene of int281 in cloning plasmid was sequenced and cut down by Nde I and Not I endonucleases.Then it was recombined with the same cut pET-22b(+). The expression plasmid certified by sequencing was transformed into E.coli BL21 (DE3),and induced by IPTG to express the recombinant Int281.This protein was detected by SDS-PAGE,and its immunoreactivity was validated by western blot.The BALB/c mice were immunized with this antigen.Titers of mice serum total IgG against Int281 protein were detected by ELISA.The proteins were expressed as inclusion bodies, which were denatured with8 mol/L urea,renatured by several grades of urea,and dialyzed into PB solusion.The expression rate of total bacteria proteins is 25%.The renatured proteins were purified by Ni²⁺ affinity chromatograghy and the final purity is above 95%.The result of western blot suggested that purified Int281 proteins could bind with antibodies of lysed O157:H7,which indicated that it had the same or similar conformation as the native proteins.In indirect ELISA,the serum IgG specific for Int281 reached the peak point at 1:10⁶ ten days after the third immunization.Three months later, the antibodies kept almost the same level.These studies showed that purified Int281 has an excellent immunogenicity that deserves further study on evaluating its protection against intestinal colonization by EHEC O157:H7.Part 2.Immunogenicity of a novel Stx2B-Stx1B fusion protein in a mice model of EHEC O157:H7 infectionPrevious works done by our laboratory had proven the B subunits of Stxs are nontoxic immunogens,and chicken polyclonal antibodies anti-Stx1 B and anti-Stx2 B can inhibit toxicities to HeLa cells.Passive immunization of mice with chicken polyclonal anti-Stx1B and anti-Stx2B antibodies protected mice from the effects of crude toxins challenge,separately,and not cross-reacted.Considering all the above,we fused the two B subunits to form a new fusion protein named 2S to provide a wide scope of protection against EHEC O157:H7 infection.We introduced a soft linker between Stx2B and Stx1B,and expressed it in E.coli.The production is rather high,the purification procedure is very simple,which based on an anion exchange chromatography method.The purified 2S was confirmed by ELISA using either Stx1B-specific or Stx2B-specific antibodies,and results showed that the two components in 2S have antigenicity the same as the two mono-subunits.Since EHEC infection causes systemic immune responses,we intend to induce humoral reactivity by the adjuvant CFA used in first vaccination and IFA in boosting through the i.p.pathway. 2S reserves antigenic epitopes of the two immunogenic components of the toxins,even enhanced their immunigenicities in mice.It induces Th2 responses in mice humoral reaction,and develops high level neutralizing antibodies for protecting mice from lethal dose challenge of EHEC O157:H7.We consider this novel fusion protein provides broad spectrum protection against heterologous Shiga toxins,and a vaccine candidate against EHEC O157:H7 infection.Part 3.Immunogenicity of Stx2B-Stx1B-Int281 fusion protein in a mice model of EHEC O157:H7 infectionThe pET-22b(+)-ssi plasmid constructed by us was the same as the design.And recombinant SSI expressed higly in the E.coil BL21(DE3).The molecular weight was confirmed by SDS-PAGE and Immunoblotting.The iateralso certificated that this fusion protein could be recognized by chicken anti-EHEC O157:H7 bacteria proteins polyantibodies,and proved that this recombinant protein was immunoreactive.The antigenic epitopes confirmation by ELISA showed us the fusion protein could display the epitopes of each component besides a little difference.That might due to the middle position and confirmation of Stx1B that might make it some difficult to expose.The SSI and each Single protein were injected into BALB/c mice,and we collected sera from each immunized group.The anti-Stx2B and anti-Int281 ELISA titers were almost at the same level as the single proteins,but low anti-Stx1B IgG titers.The SSI immunized mice sera also compared with Int immunized group in the anti-adherence test. The results suggested that the former performed better than the latter in this anti-EHEC O157:H7 adhesion to HEp-2 cells and it could provide full protection against at least 10⁹ CFU of the bacteria in vitro.The pathology analysis suggested that mice immunized by SSI could provide effective protection against EHEC O157:H7.The cecum and colon from both SSI and Int281 immunized group showed no obvious changes,while the organs from model group had significant inflammation.SSI might provide protection against kidney failure syndrome,such as blood tendency,increased permeability of glomerular and renal tubular.All of the above results proved SSI was effective in preventing or cure EHEC O157:H7 infection and its complication,and worth to further study as a vaccine candidate...