To Construct And Evaluate The Effects Of Enterohemorrhagic Escherichia Coli O157:H7 Vaccine Candidates In Mice

ObjectsEnterohemorrhagic Escherichia coli 0157:H7(0157:H7)is a zoonotic enteric pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome(HUS)in humans.EHEC 0157:H7 infection lack of effective prevention and treatment.The effective vaccine is the best way to prevent the infection.In this study,we constructed the novel bivalent antigen EspA-Tir-M as a candidate subunit vaccine and inserting EspA-Tir-M as into L.acidophilus as a live vector candidate vaccine and evaluate the protection effect of vaccines.MethodsFirst,we amplify the fusion genes EspA-Tir-M by overlap extension PCR and connect the fusion genes with the plasmid pET28a(+)to construct the Escherichia coli prokaryotic expression system.The expression of the protein EspA-Tir-M was induced and protein was purified and concentrated.Then the prepared purified protein was administered by intranasal and subcutaneous routes in mice.After immunization,we collected the the serum and fecal samples to detect the levels of IgG,SIgA,and the cytokines such as IFN-y,IL-4 and IL-10.Assessment of the protective effect of immunization against EHEC 0157:H7 challenge was conducted after the last immunization.The protective effects of intranasal immunization were compared to subcutaneous immunization.In addition,we developed a candidate EHEC 0157:H7 vaccine by inserting the bivalent antigen EspA-Tir-M into plasmid pMG36e to construct the L.acidophilus expression system.The recombinant L.acidophilus was identified by multiple PCR and gene sequencing.The protein expressed by the recombinant L.acidophilus was detected by SDS-PAGE and Western Blot.Finally,we evaluated the specific immune responses elicited in mice and protection against EHEC 0157:H7 challenge both in vitro and in vivo.Inhibition of EHEC 0157:H7 by recombinant L.acidophilus by the colonized LoVo cells was evaluated using the competition and exclusion assays.The ability of recombinant L.acidophilus inhibits EHEC 0157:H7-induced attaching and effacing lesions was tested by fluorescence actin staining.Oral immunization with recombinant L.acidophilus induced the specific antibodies and cytokines were detected in mice.Furthermore,the assessment of the protective effect of immunization against EHEC 0157:H7 challenge was conducted.ResultsWe construct the Escherichia coli prokaryotic expression system to express the fusion protein EspA-Tir-M successfully.The protein EspA-Tir-M was soluble mainly and the purity was 90%.Immune responses to the fusion protein administered by intranasal and subcutaneous routes were compared in mice.Results showed higher levels of specific mucosal and systemic antibody responses induced by intranasal as compared to the subcutaneous immunization.Intranasal immunization enhanced the concentration of interleukin-4,interleukin-10,and interferon-?,while subcutaneous immunization enhanced only the latter two.In addition,intranasal immunization protected against EHEC 0157:H7 colonization and infection in mice at a rate of 88.9%.Histopathological analysis revealed that vaccination reduced colon damage,especially when administered intranasally.In contrast,subcutaneous immunization elicited a weak immune response and exhibited a low protection rate.In addition,we first report about using the L.acidophilus expressing EspA-Tir-M antigen successfully.The recombinant L.acidophilus expressed EspA-Tir-M fusion protein was mainly a secreted protein.It can exclude EHEC 0157:H7 from LoVo cells at rates of nearly 94%and 60%in exclusion and competition assays,respectively.Additionally,recombinant L.acidophilus inhibited the induction of attaching and effacing(A/E)lesions by EHEC 0157:H7 cells in vitro.Oral immunization recombinant L.acidophilus with also enhanced interferon-? and interleukin-4 and-10 production.The recombinant L.acidophilus protected against EHEC 0157:H7 colonization and infection in mice at a rate of 77.8%.Histopathological analysis revealed that orally administered reduced or inhibited A/E lesions and toxin-induced systemic injury.ConclusionThis is the first report that we construct the novel EspA-Tir-M fusion protein as subunit vaccine and the novel oral recombinant L.acidophilus expressing EspA-Tir-M as live vector vaccine successfully.Both of them were effective to induce immune responses and protected against EHEC 0157:H7 colonization and infection in mice.They are the promising candidate vaccines against EHEC 0157:H7 infection.