KR100766775B1 - Toxoid vaccine preventing the infectious disease of Enterohamorrhagic Escherichia coli - Google Patents

Abstract

The invention field relates to a hemorrhagic E. coli infection prevention toxoid vaccine, chapter that is causative of a break of the sheet and more particularly of hemorrhagic E. coli infections produce a toxin (Shigatoxins) or the same concept, Vero toxin (verotoxins) hemorrhagic Escherichia coli (Enterohamorrhagic Escherichia coli : EHEC) is isolated from domestic cattle and used as a vaccine strain. Toxoids for preventing hemorrhagic E. coli infections containing the detoxified Shigatoxin as an active ingredient by mass production and inactivation of Shigatoxin secreted from these vaccine strains. It relates to a vaccine.

The toxoid vaccine for preventing hemorrhagic E. coli infection according to the present invention can be used not only to prevent hemorrhagic diarrheal disease of calf and to reduce the infection rate in livestock, but ultimately to prevent human hemorrhagic E. coli infection.

Intestinal hemorrhagic E. coli infection, EHEC, Shigatoxin, Verotoxin, Toxoid vaccine

Description

Toxoid vaccine preventing the infectious disease of Enterohamorrhagic Escherichia coli}

Figure 1 shows the Shiga toxins (Shiga toxins) gene, which is characterized in the domestic bovine hemorrhagic E. coli (EHEC) isolate strain in the present invention. [One; EC377 (stx1; 180 bp), 2; EC611 (stx2; 255bp, ehx A; 534bp), 3; EC381 (stx1 + stx2, ehxA), 4; O157: H7 (stx1 + stx2, ATCC43894)]

The invention field relates to a hemorrhagic E. coli infection prevention toxoid vaccine, chapter that is causative of a break of the sheet and more particularly of hemorrhagic E. coli infections produce a toxin (Shigatoxins) or the same concept, Vero toxin (verotoxins) hemorrhagic Escherichia coli (Enterohamorrhagic Escherichia coli : EHEC) is isolated from domestic cattle and used as a vaccine strain. Toxoids for preventing hemorrhagic E. coli infections containing the detoxified Shigatoxin as an active ingredient by mass production and inactivation of Shigatoxin secreted from these vaccine strains. It relates to a vaccine.

The toxoid vaccine for preventing hemorrhagic E. coli infection according to the present invention can be used to prevent hemorrhagic diarrheal disease of calf and to reduce the infection rate in livestock, and ultimately to prevent human hemorrhagic E. coli infection.

Intestinal hemorrhagic E. coli infections cause hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in humans, and hemorrhagic dysenteria in young calves in cattle and edema disease in weaning pigs. It is known to cause. In the case of calves in animals, hemorrhagic diarrhea is caused by toxins produced by these strains, and there is no therapeutic agent. It is becoming. On the other hand, the fungus that causes edema of weaning pigs is a variant and is known to be independent of human disease.

In addition to the well-known E. coli O157: H7, various serotypes, such as O26 and O111, have been isolated from patients with enterohepatitis E. coli infections. E. coli is widely distributed in livestock such as cattle. For this reason, when E. coli, which produces Shigatoxin of a specific serotype, is used as an antigen of the vaccine, only a limited effect of preventing the serotype can be expected. Therefore, the importance and interest for toxins that all serotypes are commonly produced and are a direct cause of pathogenesis have emerged.

The major toxins produced by hemorrhagic Escherichia coli (EHEC) are found to be Shigatoxin 1 (Stx1) and 2 (Stx2), and are known to produce one or both species of toxins, depending on the species.

As a study for effectively preventing E. coli (EHEC), the development of mutant strains using genetic recombination technology, that is, to produce attenuated strains by mutating genes that produce toxins, is being conducted. It is expected to take. An easier approach is to mass-produce and effectively inactivate the toxin produced by the strain and use it as a prophylactic vaccine antigen.

Not all E. coli strains that have genes that produce Shigatoxin produce toxins in normal culture, and are known to have different forms of toxin production according to isolated breeders, serotypes, and culture conditions. That is, the bacterium can promote toxin production as a means of defense in an environment that is disadvantageous for its survival, such as antibiotic treatment, and EHEC isolated from herbivores such as cows rather than pigs has been reported to have strong toxin production.

Shigatoxin is known to be inactivated by formalin treatment, which is used to kill or detoxify common species. It is estimated that the molecular weight of the toxin is very small, such as Stx1 is 32 molecular weight (MW) and Stx2 is 7.7 MW, but this is not clear, and it is indefinite and safely inactivates and binds various adjuvants to the toxin. Increasing immunity by increasing the research is being conducted.

Therefore, the present inventors isolate the hemorrhagic Escherichia coli (EHEC) prevalent in domestic cattle through the distribution investigation on the causative bacteria in domestic cattle, such as domestic cattle, and selected strains with strong pathogenicity and excellent toxin production ability, and toxin production from these strains By establishing palpation and inactivation techniques, confirming safety and using them as antigens of preventive vaccines, it has been found to be able to prevent calf hemorrhagic diarrheal disease and reduce infection rate in livestock, and ultimately contribute to human hemorrhagic E. coli infection. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a toxoid vaccine for the prevention of enterohemorrhagic E. coli (EHEC) infection.

In order to achieve the above object, the present invention provides an EC377 strain selected from domestic bovine hemorrhagic E. coli isolates.

In addition, the present invention provides an EC611 strain selected from domestic bovine hemorrhagic E. coli isolates.

In addition, the present invention provides an EC381 strain selected from domestic cattle-derived E. coli isolates.

In addition, the present invention provides a method for promoting the production of Shigatoxin, characterized in that the addition of 0.5 ~ 1.0ug / ml mitomycin C in the culture of E. coli strains.

In the present invention, the Shiga toxin is 0.2 to 0.5% glutaaldehyde (glutaraldehyde) for 1 to 3 hours, 0.2 to 1.0% lysine (lysine) added to the Shiga characterized in that the detoxification Toxin inactivation method is provided.

In addition, the present invention provides a toxoid vaccine for preventing hemorrhagic E. coli infections, which comprises at least one of inactivated Shigatoxin cStx1 and cStx2 toxoid antigens as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present invention is a strain selected from the strains of domestic hemorrhagic E. coli isolates with strong pathogenicity and excellent toxin production, EC377 strain (O55, Stx1), Shiga toxin 2 that produces Shiga toxin 1 (Stx1) and serotype O55 (Stx2) were generated and EC611 strains with serotype O6 (O6, Stx2) and both Shigatoxins 1 and 2 were generated and the EC381 strain (O153, Stx1 + Stx2) with serotype O153 was provided.

In addition, the present invention is 0.5 ~ 1.0ug in Brain Heart Infusion broth (BHI) or Tryptic soy broth (TSB), which is a general growth medium used in the culture of intestinal hemorrhagic E. coli strains Addition of / ml mitomycin C (Mitomicin C) provides a production promoting method for mass production of Shigatoxin. As described above, when incubated with the addition of mitomycin C, it was confirmed that an increase in toxin titer of 8 to 128 times occurred compared to general culture (see Tables 5 and 6).

In addition, the present invention is treated with 0.2 ~ 0.5% glutaraldehyde (glutaraldehyde) for 1 to 3 hours in order to effectively detoxify the produced Shigatoxin, 0.2 to 1.0% Lysine (Lysine) is added to the A method of inactivating toxins is provided.

Shigatoxin was confirmed to be a highly toxic substance by confirming virulence through the semi-injection dose in mice (Table 7). Therefore, there is a need to inactivate the strong virulence of Shigatoxin in preparing a vaccine by using the Shigatoxin as a toxoid antigen. The concentration of glutaraldehyde used in the conventional formalin treatment or in the present invention is used. When it is low or not treated with lysine, it is confirmed that inactivation is not made, in the present invention, 0.2 to 0.5% glutaraldehyde (glutaraldehyde) for 1 to 3 hours, 0.2 to 1.0% lysine (Lysine) The inactivation of Shigatoxin was added, thereby establishing an effective inactivation method. In more detail, the concentration of glutaraldehyde should be adjusted according to the amount of toxin to be inactivated, and as shown in Table 8 below, when the sugatoxin titer is 1: 1,140 to 2,125, 0.3% of the article is required. It was found that rutaaldehyde was used to sufficiently inactivate the toxin. The glutaaldehyde for inactivation is preferably used in a concentration of 0.2 ~ 0.5%, when using a concentration of 0.2% or less may cause problems with sufficient inactivation of toxin, and also of glutaraldehyde itself In view of toxicity, it is preferable to use as little as possible. Therefore, it is preferable to inactivate by appropriately adjusting and using it according to the content (titer) of toxin within the range of 0.5% or less. The concentration of lysine depends on the concentration of glutaaldehyde used, and in order to safely and sufficiently neutralize toxic glutaaldehyde components, it is preferable to use a concentration of 1 to 2 times the content of glutaaldehyde. Tables 9-11)

In addition, the present invention for the prevention of intestinal hemorrhagic E. coli infection characterized in that it contains at least one of the above safe and effectively inactivated Shigatoxin 1 (cStx1) and Shigatoxin 2 (cStx2) toxoid antigen as an active ingredient. Toxoid vaccine is provided.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Characteristics of Strains

1) EHEC distribution in livestock

Sixty strains (10.0%) of 15 species, including O26, were isolated from 599 domestic cattle feces (287 cattle, 312 pigs). ), Seven species (3.1%) including cow O6 and two species (4.5%), including pig O8 (see Table 1, 2004).

In addition, the presence of an ehe A gene, which is a pathogenic factor of EHEC, which adheres to the epithelial epithelial cell, and the presence of enterohemolysin ( ehx A, gene related to hemolysis ) are related to the pathogenic strength of EHEC. As reported, Table 1 also shows the test results of the presence of eae A and ehx A genes for each of the Shigatoxin- producing strains characteristic of EHEC. As a result, it was confirmed that the eae A and ehx A gene retention rates of Shigatoxin- producing strains were very low.

2) Shigatoxin Formation Capacity of EHEC Isolates

Shigatoxin production in EHEC isolates (91) was positive for 40 weeks (44.0%), and Hanwoo was 28 (43.1%) out of 43 weeks, 9 (40.9%) out of 22 cows and 26 pigs. Three weeks (11.5%) showed a big difference between livestock species (see Table 2).

Example 2 Vaccine strain selection

In the present invention, EC377 strains (O55, Stx1), EC611 strains (O6, Stx2), and EC381 strains (O153) as a vaccine strain were subjected to investigation of the toxin production and pathogenicity of mice in the EHEC outdoor isolate, which is a popular strain of Shiga toxin production in domestic farms. , Stx1 + Stx2), respectively (see Table 3), and the half-lethal dose (LD ₅₀ ) for mice in the vaccine strain was EC377 4.6 x10 ⁷ cfu, EC611 5.0x 10 ⁷ cfu, and EC381 1.7x10. Calculated at ⁷ cfu (see Table 4).

Example 3 Shigatoxin Formation Promoting Effect

Shigatoxin-producing bacteria, selected vaccine strains, were cultured by adding 0.5 ug / ml mitomycin C to Brain Heart Infusion broth (BHI). It was confirmed that the toxin titer increased by 128 times. This is a 4- to 64-fold increase compared to the antibiotic 0.1% Polymixin B, an antibiotic used to promote Shigatoxin secretion, and 0.1% polymyxin 200 compared to 0.5ug / ml mitomycin C. As a high concentration of antibiotics, there are some disadvantages in terms of cost and safety (see Table 5).

In addition, the concentration of mitomycin was added to 0.05ug / ml, 0.1ug / ml, 0.2ug / ml, 0.5ug / ml, and 1.0ug / ml, respectively, and as a result, it was confirmed that the promoting effect of Shigatoxin production was 1.0ug / ml. There was no synergistic effect in the above addition, and the addition of less than 0.5 ug / ml resulted in a decrease in the effect of promoting the production of Shigatoxin (see Table 6).

Example 4 Verification of Pathogenicity and Inactivation of Shigatoxin

1) Pathogenicity of Shigatoxin in Mice

The LD ₅₀ of Shigatoxin in mice was 1,900 toxin titers and 1,900 and protein content of 334 µg / 0.2 ml for Stx1, whereas Stx2 showed a strong virulence of toxin titer 1: 103 and 21 µg / 0.2 ml of protein. (See Table 7).

2) Inactivation and Safety of Shigatoxin

In order to effectively detoxify the toxin, 0.2 to 0.5% glutaraldehyde was treated for 1 to 3 hours, and 0.2 to 1.0% lysine was added to establish a method for inactivating a shigatoxin. 8). On the other hand, when the formalin treatment method was used, or the glutaaldehyde concentration was low or the lysine was not treated, it was found to cause mortality at the time of mouse inoculation, indicating that inactivation was not sufficiently achieved. It was also confirmed that the aldehydes could be safely used by neutralizing their own toxicity (see Tables 9-11).

As can be seen in Table 10, 0.11% glutaaldehyde (G) treatment caused the death of the mouse was determined that the content is insufficient to inactivate Stx1 and Stx2 of Toxin titers 1: 2,048.

As shown in Table 11, Shiga Toxin 2 (Stx2), which is relatively toxic compared to Shiga Toxin 1 (Stx1), was treated with 0.2% and 0.5% glutaaldehyde (G) as a result. It was found that toxin 2 was inactivated. However, when lysine was not treated, it was confirmed that the mouse died due to the toxicity of glutaaldehyde itself, and it was found that the concentration of lysine (L) corresponding to 1 to 2 times of glutaaldehyde is required for the purpose of neutralizing it. there was.

The control group 2 of Table 11 is a case where the mouse was treated with glutaaldehyde, or glutaraldehyde and lysine. When only glutaaldehyde was treated, the mouse died of its own toxicity, and when treated with lysine. It was confirmed that mouse death did not occur due to the neutralization of glutaaldehyde toxicity by lysine.

Example 5 Preparation of Toxoid Vaccine

Enter B. broth, mitomycin C (0.5 ug / ml), and enter the hemorrhagic E. coli (EHEC) strain that produces Shigatoxin 1 (Stx1) and Shigatoxin 2 (Stx2) 37 Incubate by shaking at 48 ° C. for 48 hours each. The culture solution was centrifuged (8,000 rpm, 30 minutes), the supernatant was collected, concentrated, dialyzed in PBS for 3 days, and the centrifuged supernatant was again filtered through 0.45 μm of crude Shiga toxin (Verotoxin) solution. It is done. Using the BCA Protein Assay Reabent Kit (Pierce Co), the crude Shiga toxins (cStxs) concentration (mg / ml) was measured based on the protein content of standard albumin. Toxin titers were measured using the VTEC-RPLA kit (SENKA SEIKEN CO.). The toxin titer was adjusted from 1: 1,000 to 2,500 and the pH was 8.0, and after glutaraldehyde treatment (0.2 to 0.5%, 37 ° C., 2 hours), neutralization with lysine (0.2 to 1.0%) was made and used as toxoid antigen. It was stored in a 4 ° C refrigeration chamber until now. After adjusting the antigen concentration with sterile phosphate buffered saline (PBS) (toxin titer 1: 1,000 or so), 15% aluminum hydroxide gel (Al (OH) ₃ ) and 0.3% oil (ISA70) as an adjuvant Toxoid vaccine was prepared by adding and mixing for 2-3 hours using a magnetic bar.

Example 6 Protective Effect of Intestinal Hemorrhagic E. Coli (EHEC) Toxoid Vaccine in Experimental Animals

In the protective effect test of mice with cStx1, cStx2 and cStx1 + cStx2 toxoid vaccines, the protective effect was 100% from Stx1 producing strains and 70 to 80% from Stx2 and Stx1 + Stx2 producing strains (see Table 12). ).

The present invention is to determine the safety by inactivating the Shiga toxin characteristically produced by these strains using EHEC isolates prevalent in domestic cattle to prevent calf hemorrhagic diarrheal disease and use in the livestock It can be used for prevention measures such as inducing reduction of infection rate, and ultimately can prevent human hemorrhagic E. coli infection.

Claims (6)

Escherichia coli EC377 strain of Accession No. KACC 91240P, which produces Shigatoxin 1 (stx1) and is serotype 055 as a strain selected from Korean bovine hemorrhagic E. coli isolates. Escherichia coli EC611 strain of Accession No. KACC 91242P, which produces Shigatoxin 2 (stx2) and is serotype 06, selected among the domestic cow-derived E. coli isolates. Escherichia coli C381 strain of Accession No. KACC 91241P, which produces Shigatoxin 1 and 2 (stx1 + stx2) and is serotype 0153 as a strain selected among the domestic cow-derived E. coli isolates. The production of Shiga toxins (Stx1, Stx2), characterized in that 0.5 ~ 1.0ug / ml Mitomycin C (Mitomicin C) is added to the intestinal hemorrhagic Escherichia coli strain culture according to any one of claims 1 to 3. Promotion method. delete A toxoid for preventing hemorrhagic E. coli infections, comprising at least one of the Shigatoxin cStx1 and cStx2 toxoid antigens produced from the strain according to any one of claims 1 to 3 and inactivated therein as an active ingredient. vaccine.

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