JPH0555486B2 - - Google Patents

Description

[Detailed description of the invention]

Escherichia coli is a ubiquitous pathogen that causes a wide variety of clinically and economically significant diseases in humans and animals. The main cause of death throughout the world is infant death, and diarrhea caused by E. coli in areas with poor sanitary conditions is one of the major causes of death. E. coli is estimated to be responsible for approximately 50% of traveler's diarrhea. E. coli often causes nosocomial infections and infections in patients with debilitating and traumatic generalized debilitation. Bacteremia and burn trauma infections are gradually caused by E. coli. E. coli is a major cause of urinary tract infections that cause acute and chronic cystitis and pyelonephritis. It is clear that developing methods to prevent diseases caused by E. coli can alleviate human misfortune and prevent huge economic losses. The applicant investigated a large number of E. coli strains collected from several human pathogenic bacteria, and found that most of them belong to a single immunological group called "E. coli type 1" glandular hairs. It has been proven that the company has It has been discovered that an easily defined hierarchy can be established between members of the group of immunologically cross-reactive glandular fungi. In other words, if a bacterium has glandular hairs and the glandular hairs can be isolated, purified, and utilized to produce antiserum containing antibodies against the glandular hairs, such bacteria are classified as having a superior-subordinate relationship. It is possible. Within the groups producing such immunological cross-reactivity in this study, the degree of cross-reactivity between the antiserum of the first strain and the glandular hairs of the second strain was We have found the surprising fact that the degree of cross-reactivity between antisera against the first strain and glandular hairs producing antibodies against the first strain is not the same. This surprising difference led to the discovery of a way to construct immunological groups that can be clearly ranked. In carrying out this method, cell materials and cell debris are removed from the glandular trichomes of a group of bacteria that have glandular trichomes. The glandular hairs are then presented to an immunocompetent organism, such as a mouse or rabbit, and antisera against the glandular hairs containing antibodies against the glandular hairs are isolated in the conventional manner. Known cross-reactivity experiments, such as ELISA tests, are performed on glandular hairs and antisera. Suitably, these results are tabulated in the form of a matrix, with the antisera listed vertically and the corresponding glandular hairs listed horizontally.
By such classification, the homogeneous reaction between a specific glandular hair and its corresponding antiserum can be read diagonally. The numbers on the diagonal are normalized to the same predetermined value and the corresponding titers for all non-paired glands for each antiserum are adjusted accordingly. The table showing the results shows the gradation relationship. Redrawing this standardization table from top to bottom, it becomes clear that there is a significant and consistent asymmetry between antiserum and glandular hair titers. The titer obtained between the glandular trichomes of a higher-ranking strain and the glandular trichomes of a lower-ranking strain is always greater than the titer obtained between the antiserum against the glandular trichomes of the superior strain and the glandular trichomes of the subordinate strain. big. This relationship also exists within the immunologically related family of glandular hairs. The conclusion drawn from this surprising result is that within such a group, the higher ranked microorganisms contain immune factors that provide an immune response against all microorganisms at the same or lower rank in the classification, and therefore provide protection against future infections. This means that vaccines can be provided. Conversely, the bacteria at the lowest rank contain factors that are common to all bacteria that are higher in the classification, and therefore the lowest rank bacterium is used for the purpose of detecting antibodies belonging to higher families. can be used. The methodology is
It was shown that E. coli type 1 bacteria of the family Adenophyllaceae can be classified from the highest to the lowest. Antisera directed against the top glandular hair type can produce extensive cross-reactivity with all other members of the family. Therefore, a single type of purification
Vaccines consisting of E. coli type 1 glandular trichomes may cause infections caused by E. coli in humans, i.e. strains with glandular trichomes of the type 1 family, especially strains with glandular trichomes that are subordinate to the glandular trichomes in the vaccine. Most infections can be prevented. There are other immunologically related glandular families among E. coli strains that are pathogenic to humans. Although these families are immunologically unrelated to type 1 glandular hairs, they are referred to as NMS glandular hairs. Vaccines derived from NMS are also useful in preventing E. coli infections. The present invention is capable of increasing antibody levels in humans to the level of prevention of infections caused by bacteria of the first group of glandular E. coli strains; different,
or glandular trichome formation by cross-reacting cells of a first group of bacteria consisting of strains that are appropriately subordinate to a serum having antibodies against glandular trichomes of a second group.
The present invention provides a vaccine composition comprising: b) glandular hairs obtained from a second group strain of B. coli; and b) a pharmaceutically acceptable carrier. In this application, examples are given of vaccines containing glandular hairs obtained from the same strain that are effective against infections caused by a single given strain of bacteria. Vaccines containing both Type 1 and NSR glandular hairs are also within the scope of the present invention, provided that the glandular hairs immunologically cross-react with at least one infectious disease agent. It is. An E. coli strain with significant enterotoxicity that has been shown to cause spontaneous disease when inoculated into non-immune humans was isolated from a human patient with diarrhea. Although this virulent strain had sparse glandular hairs, it was propagated and selected as a clone with good glandular hair formation. These clones were grown and maintained on minimal glucose-based agar;
The glandular hairs were separated from the cells, repeatedly crystallized in aqueous magnesium chloride, and then resolubilized in a low ionic strength, neutral buffer. Subjects were injected with glandular hairs. They were then given enteral doses of E. coli at doses known to cause infections in immunocompromised patients. Although the immunized group developed diarrhea, they recovered more quickly than the non-immunized group who received the same treatment. Culture of E. coli H10407 from a patient with severe diarrhea in Bangladesh.
A sample of the parent strain H10407(078:H11) was isolated.
The sample was passed through still broth, a colony was selected from it, and a clone with good glandular hair formation was obtained and named E. coli H10407 (ATCC31705).
Clones were then cultured on minimal glucose agar. The glandular hairs were separated from the cells by mixing with a low ionic strength neutral buffer such as phosphate buffer and saline at pH 7.0 and centrifuging. Glandular hairs were crystallized by adding concentrated magnesium chloride (water soluble) to the buffer to increase the strength of the buffer to 0.10M. The crystallized glandular hairs are placed in a low ionic strength neutral buffer such as 0.04M phosphate buffer PH 7.0 (saline) and reprecipitated with magnesium chloride as well. Preferably, the glandular hair crystallization cycle is repeated one to five times.
The method used here is from Brinton, Trans.NY
It is almost the same as that described in Acad.Sci.27, 103 (1965). Final purification of the glandular trichome vaccine consists of mixing the recrystallized glandular trichomes and passing through a sterile filter with a suitable sterile preservative such as merthiolate. The quality of the glandular hairs treated as described above is of a level that satisfactorily passes the general safety, sterility and pyrogenicity standards established by the Department of Applied Biology of the United States Federal Food and Drug Administration. Glandular hairs may be administered orally, such as by capsule, or by injection, such as subcutaneously, intradermally, or intramuscularly. When administering by injection, pharmaceutically acceptable suspensions can be used since the glandular hairs are solid. It has been found to be particularly useful to use a phosphate buffer suitably containing merthiolate as an excipient or suspending agent. Ionic strength of 0.005 to 0.1, optimally with 0.005 to 0.1%, optimally 0.01% merthiolate
Preference is given to using a phosphate buffer that is 0.04. The concentration of glandular hairs in the vehicle is not critical. The only requirement for the glandular hairs is that they be fine enough to pass the general criteria that the suspension can be injected into a syringe. 10ml suspension
about 20 with a concentration of 1 to 30, preferably 20 g per
mg of glandular hair protein is particularly preferred. It is generally considered best to administer the vaccine composition in portions at regular intervals. This time period is selected to allow the formation of appropriate antibody titers against glandular hairs in the injected subject. It has been found appropriate to administer the vaccine composition at least once before injection, preferably 60 days and again 30 days. There appears to be no upper limit to the dosage since vaccination does not produce local or systemic toxic effects. However, we have found it appropriate to administer between 1 and 100 micrograms of glandular hair per kilogram of body weight, optimally about 20 micrograms per kilogram of body weight per injection. E. coli infections occur in different species of mammals depending on the strain. We discovered that E. coli species with type 1 glandular hairs can cause infections in humans. Therefore, when glandular hairs obtained from related strains of this species group are administered, infection-protective antibodies are obtained in the body. Example Production of E. coli glandular hairs (type 1) 2 broths (Bactotryptone 1%, NaCl 0.8
%, yeast extract 0.1%, glucose 0.1%) using a culture prepared by resuspending glandular trichoform phase colonies grown on minimal glucose agar-based medium in liquid medium and solidified with agar. Trays containing the same growth medium were inoculated. After overnight incubation at 37°C, confluent bacterial growth was suspended in 0.01 molar phosphate buffered saline pH 7.0. Approximately 30cm×
Approximately 20 to suspend the grown bacteria in a tray with dimensions of 40 cm
Milliliters of buffer were used. Add 200 ml of the resuspended proliferating bacteria to a 400 ml cup of Sorveur Omnimixer at a time and stir for 5 minutes at a speed of 14,000 rpm.
Glandular hairs were separated from the cells. Then cells for 20 minutes
It was removed by centrifugation at 10,000 G to obtain a supernatant. The glandular hairs are made into 0.1 mol crystals by adding magnesium chloride (MgCl ₂ ). After the crystallization was completed, the pellet was separated from the suspension by centrifugation at 20,000 G for 60 minutes to obtain a pellet. The pellet containing crystalline glandular hairs was redissolved in 0.04 molar phosphate buffer (no saline) at pH 7.02. Suspension 20,000 for 60 minutes
The mixture was centrifuged twice to obtain a supernatant. 2 cycles of crystallization, centrifugation, redissolution, and centrifugation
The procedure was repeated 4 to 4 times to obtain a purified glandular hair suspension. Example Vaccine production a) Place 100ml aliquot (partial specimen) in a 200ml cup and mix at high speed at 14000rpm for 10 minutes using a Solvaall Omnimixer to loosen the glandular hair aggregates and divide the rods. b) The concentration was measured by UV and adjusted to a dose of 1800mcg/10ml. Removal of flagella The pH was adjusted to 12.3 by repeatedly adding sodium monohydroxide to the dissolved glandular hair preparation, and after leaving it for 30 minutes at room temperature with occasional stirring, 10% (u/v) saturated ammonium sulfate was added. The glandular trichomes were sedimented and centrifuged at 20,000 G for 30 minutes to obtain flagellar subunits separated in the supernatant. The precipitated glandular hairs were redissolved in phosphate buffer (0.04M). Examples Immunization Groups of 3 to 6 volunteers (total
21 patients) were sequentially administered 45, 90, 900, or 1800 mcg of purified glandular hair vaccine by injection into the triceps muscle. Then, on the 28th day, 15 of the 21 patients received the glandular hair vaccine.
A booster dose of 1800 mcg was given by intramuscular injection.
Volunteers who received the 1800mcg initial or booster vaccination were instructed to watch for local reactions caused by glandular hairs rather than the intramuscular injection itself. Neither the volunteers nor the doctors who examined them were told who had been given the vaccine and who had been given the saline solution. Challenge research for vaccine testing Approximately 1 month after the additional 18mcg (microgram) vaccination (2 months after the first vaccination), challenge research was conducted on 6 type-vaccinated subjects and 7 control subjects to evaluate the effectiveness of the vaccine. was evaluated. Example Production of inoculum E. coli used in past challenge studies
The H10407 parent strain was inoculated into Z broth (an aqueous solution consisting of CAYE and sterile water), cultured for 4 hours with shaking at 30°C, and frozen at -70°C with dimethyl sulfoxide (DMSO). Frozen stocks were later thawed and streaked onto Casamino-Yeast Extract (CAYE) agar plates. Inoculum and Vaccine Test After 15 hours of incubation at 30°C, 16 glandular hair colonies identified under a stereomicroscope were streaked on CAYE agar. After culturing at 37°C for 12 hours, 30 glandular hair colonies were placed on 6 CAYE agar plates and cultured at 37°C.
After 12 hours, the CAYE basal culture was dissolved in saline (0.85
%) and diluted with saline. EXAMPLE Administration of inoculum 2 gm of NaHCO ₃ was dissolved in 150 ml of distilled water and 120 ml was given to volunteers to drink. One minute later, the remaining 30 ml of suspended E. coli inoculum (5 x 10 ⁸ bacteria) was allowed to drink.
The size of the inoculum was determined by replicate pour plate method before and after vaccination. The presence of type 1 bacterial cells and NMS glandular hairs on the treated bacteria was confirmed by specific antiserum agglutination. Clinical Observations Immunization Volunteers vaccinated with the glandular hair vaccine were observed in an isolation ward for 2 days after vaccination. The temperature in the oral cavity is measured every 6 hours, and erythema, fever, and
The presence or absence of hardness and tenderness was examined. Challenge Volunteers were examined daily for 3 days prior to ingestion of virulent bacteria. Oral body temperature was measured every 6 hours, and if the temperature was 37°C or higher, the temperature was taken again within 5 minutes. Feces and vomit were collected on a plastic cholera sheet, and a nurse or doctor observed and measured the amount. Stool was evaluated in five stages. Grades 1 (normal stool) and 2 (soft stool) were considered normal. 3rd degree is concentrated liquid stool, 4th degree is cloudy liquid stool,
5 degrees is a stool that resembles rice soup. Diarrhea is defined as 3 or more diarrheal stools within 24 hours (3rd to 5th stool).
The condition was defined as a case where the patient had diarrhea of more than 250 ml at least twice within a 24-hour period.
Prior to discharge, all volunteers received neomycin (500 mg every 6 hours) orally for 5 days to eradicate fecal carriage of virulent ETEC strains. Results Clinical results None of the 21 volunteers who received the primary vaccination with each dose of purified glandular hair vaccine (Table 2) developed erythema, induration, fever, tenderness, fever, or malaise.
Of the 15 people who received the 1800mcg booster shot, none of them had a worsening of their general condition, but 6 people showed objective local reactions such as induration, fever, or erythema. (Table 2)
Subjects who developed local reactions after booster vaccination had primary doses of vaccine of 45(2), 900(2), and 1800(2) mcg. Local reactions became evident 24 hours after inoculation, but disappeared after 48 hours in all but one case. Volunteers described the reaction as mild to moderate. No nausea, vomiting, diarrhea, or high fever was observed in any case. Vaccine Efficacy Clinical Efficacy Six subjects who had received an intramuscular booster injection of 1800mcg of glandular hair vaccine one month earlier and seven immunocompromised controls participated in the challenge study. 5×
¹⁰ All seven controls developed diarrheal symptoms after ingesting the virulent E. coli H10407 bacteria (first
table). The three control subjects defecated copious amounts of rice-squeezed stool, and the total amount of cholera-like diarrhea stool was
It became 3.8 liters, 7.5 liters, and 9.9 liters. Two controls required intravenous fluid infusions to prevent dehydration. In comparison, only two of the six vaccinated subjects developed diarrheal symptoms. (p=0.04 Fisher Precision Test). Control subjects experienced general malaise (7 out of 7 subjects) and vomiting (6 out of 7 subjects), whereas vaccinated subjects did not complain of these symptoms, regardless of whether they had the symptoms or not. (Table 1). 2
The diarrheal symptoms exhibited by the vaccinated subjects were similar to those of the control group in terms of culture, total amount of diarrheal stool, and duration of diarrheal stool.

[Table] * Number of positive cases / number of doses + Little ** 〓Range〓

[Table] * Number of people who showed a reaction/number of people who received immunization Example
No. 187049) discloses a concept for the gradation of strains within a group of immunologically related glandular fungi. This application shows how to identify the superior bacteria in a group, and also shows how the superior bacteria protect against infections caused by the inferior bacteria. The disclosure of that application is hereby incorporated by reference. Therefore, the E. coli vaccine protects against infections caused by lower-ranking strains by using the composition obtained from higher-ranking strains. Table 3 shows a cross-reactivity table standardized based on the principles of the joint application. i.e. E25
(ATCC31706) Vaccine obtained from glandular hairs is H10407
(ATCC31705) and other lower-ranking strains can be prevented. Although the principle is shown below for type 1 glandular hairs, it is similarly applicable to NMS glandular hairs, and vaccines obtained therefrom are also clearly included in the scope of the present invention.

[Table] Examples a) Isolation and characterization of glandular trichome-forming phase E. coli (NMS glandular trichomes) The original H10407 parental culture was transferred to Casamino Acid-Yeast Extract (CAYE) agar and the resulting colonies were transferred to D- Mannose (NMS = Mannose insensitivity)
A screening was conducted to examine the hemagglutination reaction of human red blood cells, which are not sensitive to the inhibition of erythrocytes. If such a clone is detected, CAYE continues.
Transferred to agar. Glandular hair formation was confirmed by electron microscopy, and type 1 of H10407 was isolated from the same parent.
It was observed that they could be morphologically distinguished from the glandular hair-forming clones. NMS glandular hairs are rod-shaped with a diameter of 50-60A like type 1 glandular hairs, but they are more flexible and
It breaks apart to form a thin fiber (10−
20A diameter). When grown under these conditions, NMS colony types are smaller and less pigmented than Type 1 glandular or nonpiliated colonies. Continuous passage of NMS clones through CAYE plates resulted in little reversion after 10-14 hours at 37°C. H10407 exhibits two different hemagglutination reactions.
One type is a type 1 glandular hair, and it was confirmed that it is a type 1 glandular hair by participating in a mannose-insensitive hemagglutination reaction using guinea pig and human red blood cells.
Other hemagglutinins cause mannose-insensitive hemagglutination with human but not guinea pig red blood cells. Clones with the latter element are
It is called NMSP+. b) Proliferation Clones were maintained by serial subculturing on CAYE agar for 12 hours, as slight morphological differences in NMS colonies are best seen on 10- to 14-hour cultures. Five to six colonies were selected to avoid selecting atypical mutants. In addition, growth in liquid media severely depleted the culture medium of NMS-glandular trichome cells. Therefore, the culture medium for inoculating large growth trays (10-1/2 x 15 1/2 x 1 inch) had to be prepared on a solid medium using CAYE agar. Growth for inoculation was collected using 0.7% casamino acids in Petri dishes by scraping the agar surface with a bent glass rod. Pipette the suspended growth from the dishes, 2-3 per tray.
ml was used to inoculate large multiplication trays. After inoculation, the tray was covered with a tight aluminum lid and incubated at 37°C for 19 to 21 hours at 70% relative humidity in a floor-standing humidified incubator placed in an adjacent room. After inoculation, the multiplication tray was returned to the vaccine device for bacterial collection, and the tray was visually inspected for contamination. 5 to 8 ml of bacterial collection buffer was added to the culture medium, and grown bacteria were scraped off the agar surface using a clean glass plate. The suspension culture was aspirated into a sterile flask and kept on ice until one day's worth of bacteria had been harvested. The pooled bacteria were treated as follows. c) Isolation of NMS glandular hairs To remove glandular hairs from the cells, the collected suspension was mixed in 200 ml portions in an ice-cold cup using a Sorbol type omnimixer. 200ml each
Read it 3 to 4 times with a tachometer.
5′ mixing was performed at a speed of 11,700-13,000 r.pm. Approximately half of the 80 tray collection was mixed and the other half was centrifuged before mixing. The mixed collected bacteria were centrifuged at 15,380 g for 25 minutes to remove cells. The cell pellet was discarded. The supernatant of the mixed culture medium was pooled and the volume measured. Crystalline ammonium sulfate was slowly added with continued stirring to reach 20% saturation.
(Unlike type 1 glandular hairs, NMS glandular hairs contain M++
In some cases, crystallization could not be achieved. ) Flow birefringence became immediately visible. The purified product was allowed to stand overnight under cold air to crystallize. NMS glandular crystals were pelleted at 30,050 g for 60 minutes and the supernatant liquid was discarded. The crystalline hair pellet was dissolved by magnetic stirring for about 1 hour, then placed in solubilization buffer and left in the cold overnight. One liter of lysis buffer was used per batch of 80 trays. The dissolved preparation was centrifuged at 30000 g for 60 minutes to obtain a clear solution. The crystallization/dissolution cycle was repeated once more by weighing the correct amount of clarified supernatant and adding crystalline ammonium sulfate slowly with stirring until 20% saturation. The preparation was left in the cold overnight and centrifuged at 30,050 g to pellet the glandular hair crystals. Solubilization and clarification were repeated as before. The third cycle of purification was the same as the second cycle, but with a small amount of buffer added to dissolve the crystals.
I only used 500ml. All preparations were monitored during the purification process by dark field microscopy and SDS-PAGE. The gland preparation was allowed to equilibrate for approximately 20 minutes to reach room temperature. The pH was adjusted to 2.5 using 0.2N HCl with rapid stirring. 5 PH adjusted solution
After leaving at room temperature for ~8 minutes, immediately add 30,050 g to 60
Centrifuged for minutes. The supernatant containing glandular hairs was decanted and the pH was immediately readjusted to 7.0-7.2 with 0.2N NaOH. The brown flagella in the pellet were discarded. Ammonium sulfate crystals were added to the supernatant to achieve 20% saturation, and crystallization was allowed to occur overnight under cold air. At this point, preparations with the same degree of purification were pooled to form two pools of NMS glandular hairs for ease of handling. The flagellar removal step was repeated on these two pools until the amount of flagella was less than 1% as determined by SDS-PAGE. Example: Vaccine production Expand the prepared sample to 0.5 mg/m/142 mm Millipore disk with a 2 liter capacity cylinder.
Passed once through a 45μ Gelman filter using a filter unit. Hemerthiolate was added to a final concentration of 0.01%, and the final vaccine material was filled at once into a sterilization unit under positive pressure, over-sterilized, and collected into sterile bottles. Glandular hair vaccine has been shown to provide appropriate antigen to rabbits when administered at a rate of approximately 1 μg per kg of body weight. For an average adult male weighing 150 lbs.
Approximately 1.0 ml of 0.5 mg/ml vaccine is required.

[Table] Example: Prevention of neonatal E. coli infection in infants Pregnant mothers are vaccinated once again with the E. coli glandular vaccine described above, suitably 60 days before distribution, preferably 30 days. The neonate may be breastfed by the mother immediately after separation, or colostrum may be collected from the mother and given to the neonate for protection. It should be noted that the presence of antibodies in the colostrum can confer protection to newborn infants from immunocompetent mothers who are breastfeeding. Comparative Example 1 Production of Antigen and Antiserum In order to use pure glandular hairs for the production of antiserum, purified Meningococcus glandular hairs were subjected to the final purification step of CsCl density gradient centrifugation. This high-quality purified product is placed in front of a New Zealand White Rabbit weighing 3 to 5 pounds to collect blood.
It was administered by subcutaneous injection into the back of the neck. Rabbits have glandular hair and Freund's insufficiency adjuvant (Deifico) 1:
Approximately 300 to 500 μg of glandular hair protein was administered in a suspension of No. 1. Injections were repeated three times at intervals of approximately 10 days. Blood was drawn from the ear after the last injection. The rabbits were not anesthetized and blood was collected from the central ear artery. The hair behind the ears was shaved and the skin was rubbed with xylene to temporarily dilate the arteries. The blood was drawn into a sterile syringe fitted with a 25-gauge needle, and it was possible to collect 10-20 ml of blood in this manner. The blood was then transferred to sterile centrifuge tubes and allowed to aggregate for 30 minutes at room temperature. The coagulum was broken up and removed from the walls of the container, and all samples were refrigerated at 4° C. overnight. The tubes were then spun at 4000g for 30 minutes to remove serum. This antiserum preparation was divided into small portions, frozen in liquid nitrogen, and stored at -20°C.

Claims (1)

Claims: 1. capable of increasing antibody levels in humans to levels sufficient to prevent infection by members of the first strain group of glandular E. coli, (a) type 1; cells of the first group obtained from at least one member of the second group of glandular E. coli strains selected from the group of glandular trichomes and the group of NMS glandular trichomes; glandular hairs that can be agglutinated by serum containing antibodies against the glandular hairs of the second group, and wherein the first group consists of a strain that is the same as or different from the strains of the second group; A vaccine composition comprising a usable carrier. 2. A member of a second group of strains, each of said members of said second group being capable of producing antibodies that aggregate cells of at least one member of said first group in the presence of said members of said second group. A composition according to claim 1, consisting of glandular hairs obtained from more than one component. 3 The second group of strains is E. coli H10407
(ATCC31705) or E-25 (ATCC31706). 4. Composition according to any one of claims 1 to 3, characterized in that the injectable excipient consists of 1 to 30 mg of glandular hairs of the second group per 10 ml. 5. Composition according to claim 4, characterized in that the excipient is a physiologically acceptable saline solution. 6. The vaccine composition according to claim 1, wherein the strains of the first group are at the same level or lower than the strains of the second group.