| Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important food borne pathogen capable of causing nonbloody or bloody diarrhea, hemorrhagic colitis (HC) and potentially fatal hemolytic uremic syndrome (HUS). A very low infectious dose (100 to 200 organisms) for EHEC O157:H7 infection has been estimated from outbreak investigations. This is different from most of other pathogens which are commonly over 1,000,000 organisms for infection.One of the important virulence trait of EHEC O157:H7 is the capacity to produce attaching and effacing (A/E) lesions on enterocyte. This property is encoded by a 43kb pathogenicity island (PAI) termed the locus of enterocyte effacement (LEE). The genes of LEE are organized into five polycistronic operons, including LEE1, LEE2, LEE3, LEE4 and tir/eae. LEE1 contains ler (LEE-encoded regulator) gene. The product of ler transcriptionally activates LEE1, LEE2, LEE3, LEE4 and tir/eae. The ler gene product is a central up-regulator of the virulence genes of LEE.Another important virulence factor of EHEC O157:H7 is Shiga-toxin (Stx), encoded by a prophage integrated into the chromosome of O157:H7. This potent cytotoxin is the factor that leads to death and many other symptoms in patients infected with EHEC O157:H7. Stxs produced by O157:H7 include Stx1 and Stx2. The holotoxin of Stx composed of a single enzymatically active A subunit noncovalently associated with a pentamer of B subunits. The A subunit is a N-glycosidase that cleaves a specific adenine residue on 28S rRNA in 60S ribosomal subunits. The pentamer of B subunits mediates holotoxin binding to receptors on eukaryotic cells.Due to the treatment of infection with EHEC O157:H7 is difficult because antibiotics do not change the cause of enteritis and may increase the incidence of HUS, the best method to prevent O157:H7 infection is developing a vaccine strategy. To control outbreaks caused by EHEC O157:H7 and reduce mortality due to HUS, a safe and effective vaccine is required. Many efforts have been made by several research groups to develop vaccine. But there is no any vaccine could be used in clinic. An ideal broad-spectrum O157:H7 vaccine probably should be a live attenuated vaccine originated from EHEC O157:H7.In order to obtain an attenuated vaccine candidate, a ler deletion mutant of O157:H7 was constructed by use of suicide vector pCVD442. Meanwhile, due to potential instability of the prophage carrying the stx gene, the prophage was cured with serial passages of bacteria and confirmed by PCR and DNA sequencing. We constructed a ler/stx deletion mutant of EHEC O157:H7, termed as F25, in 2002.Shiga toxins are important virulence factors and have strong immunogenicity. Many studies had showed that Stxs have good protective efficacy. F25 lacking the prophages encoding Shiga toxins, can not produce Shiga toxins and has not the immunogenicity of Stx. Two kinds of mutant were constructed by site-directed mutagenesis with PCR in the active center and membrane spanning region of the A subunit of Stx1 and Stx2.Site-directed mutagenesis was carried out with PCR. The DNA fragments including the stx1 or stx2 mutant genes were ligated to pUC18 to construct plasmids to produce mutant Stxs. E. coli DH5αwas used as the host strain for the wild-type and mutant Stxs production. Mutant Stx1 with glutamic acid at position 167 was replaced by aspartic acid (E167D), arginine at position 170 was replaced by glycine (R170L), alanine at position 231 was replaced by aspartic acid (A231D) and glycine at position 234 was replaced by glutamic acid (G234E). Mutant Stx2 with glutamic acid at position 166 was replaced by aspartic acid (E166D), alanine at position 219 was replaced by aspartic acid (A219D) and glycine at position 232 was replaced by glutamic acid (G232E). Vero cell cytotoxicity of the mutant Stx1 and Stx2 was assayed by comparison with the wildtype toxins. Neither decrease of viability nor change of cell morphology was observed for the cell inoculated with mutant toxins.The recombinant plasmid encoding stx1 and stx2 mutant genes was transformed into F25 to construct the live attenuated vaccine candidate, termed as F105.The cultural supernatants of EHEC O157:H7 EDL933, F25 and F105 were inoculated into vero cell cultures, respectively. The results showed that strain EDL933 has potent cytotoxicity to vero cell, the cytotoxicity of F25 was inactivated and the cytocoxicity of F105 was reduced 10-4 folds compared with EDL933.To assess the pathogenicity of vaccine candidates F25 and F105, we established a mice model for EHEC O157:H7 infection. Three groups of weaned mice (weight 10-12 gram) were intragastrically inoculated with a dose of 4×109 CFU wild type strain EDL933, and vaccine candidates F25 and F105, respectively. And all groups of mice were simultaneously intraperitoneal with mitomycin. The function of mitomycin is to increase the sensitivity of mice to O157:H7. Mice were observed daily for clinical signs of disease including weight change. Mice inoculated with F25 and F105 were gained weight normally and experienced no clinical signs. In contrast, mice inoculated with EDL933 exhibited weight loss and all dead in four days. This result demonstrated that these two vaccine candidate strains F25 and F105 were safety to mice.There are three groups (A to C) of mice (weight about 20 gram). Groups A and B of mice were intragastrically immunized with a dose of 5×109 CFU of vaccine candidate strains F25 and F105, respectively, and the group C of mice as control. Fourteen days after immunization, all groups of mice were intragastrically inoculated with wild type O157:H7 EDL933 strain. Samples of fecal were collected from all groups of mice. These fecal samples were tested by PCR everyday after challenged with EDL933. The results showed that wild type EDL933 strain could be detected from fecal sample of group C at the 13th day after challenged by EDL933. In contrast, wild type EDL933 strain could not be detected from fecal samples of groups A and B at the 6th day after challenged by EDL933. The results showed that strains F25 and F105 could be used as vaccine to reduce the colonization of EHEC O157:H7 in the intestine of animal.The adult female mice (weight about 28 gram) were divided into three groups, A to C, and group A as control. Groups B and C were intragastrically immunized twice by vaccine candidate strains F25 and F105, respectively. For all immunizations, the oral does was 1010 CFU. The 7th day after the first immunization, the female mice were mated. The 14th day after the first immunization, groups B and C mice were boosted with the same does. The suckling mice born by pregnant mice were orally challenged with O157:H7 EDL933 at 7 days of age. The results showed that 73.2% of the sucking mice born by Group B mice (immunized by F25) were survival and 83.0% of the sucking mice born by Group C mice (immunized by F105) were survival. In contrast, 16.8% of the suckling mice born by control group mice (non-vaccinated) were survival.Mice were (weight about 20 gram) divided three groups, A to C, and group A as control. Groups B and C were intraperitoneally immunized twice by vaccine candidate strains F25 and F105, respectively. For all immunizations, the does is 2×107 CFU. The 14th day after the first immunization, groups B and C were boosted with the same does. The 21st day after the first immunization, all groups of the mice were intraperitoneally challenged with a does of 2×107 CFU wild type EHEC O157:H7 EDL933. The results showed that 11/20 of the Group B mice (immunized by F25) were survival and 14/20 of the Group C mice (immunized by F105) were survival. In contrast, 0/20 of the control group mice (non-vaccinated) were survival.The results demonstrated that F25 (O157 ler/stx deletion mutant) and F105 (F25 carrying recombinant plasmid encoding mutant Stx1 and Stx2) lost the cytotoxicity to vero cell and were safety to mice. F25 and F105 could protect mice from the challenge of wild type strain of O157:H7. This study showed that F25 and F105 could be used as attenuated vaccine candidates against EHEC O157:H7.
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