BACTERIAL MUTANTS, THE PROCESS TO PRODUCE THEM, AND LIVE VACCINES CONTAINING THEM.

Description

TITLE MUTANTS AND! ATT Yes CI r PROCEDURE TO PRODUCE THEM AND LIVE VACCINES THAT THEY CONTAIN. INV. DES AND THEY ARE NOW RON PRIORITY ISRAEL OOM. PATENT, N. 59663 OF MARCH 19, 1980 Rome, there SEPTEMBER 1986 Register A Protocol No. 20393 A / MINISTRY OF INDUSTRY, TRADE AND CRAFTS Provincial Office of Industry, Commerce and Crafts of Milan ΓΟΡΙΛ DLL VEKBALE 01 PATENT DEPOSIT FOR INDIVIDUAL INVENTION: In the year 1981, the eighteenth day of the month of March, jjWiUa* RAMOT UNIVERSITY AUTHORITY FOR APPLIED RESEARCH AND INDUSTRIAL, DEVELOPMENT LI®, of Israeli nationality, Sto&c in Tel* Aviv (Israel): MODIANO & ASSOCIATI Sas, di dr.ing. G, MODIANO & C. Vl fl through an agent (Italian Patent and Trademark Agency) and with domicile for legal purposes in Milan - Via Meravigli IC at the agent has presented to me, the undersigned: - Stamped application for the granting of a patent for an industrial invention having for Τ IT 0 L Ο ί BACTERIAL MUTANTS, THE PROCESS TO PRODUCE THEM, AND LIVE VACCINES CONTAINING THEM. Designated inventor: Eliora Z. RON. Priority of the patent application in: Israel No. 59663 of March 19, 1930. accompanied by: - Description in duplicate of n. pages of writing. - fkJteemsjtaHkxRxxxxxxxx^KMiuptBXXx - Letter of assignment - Priority document and Italian translation (with reservation) • Inventor designation document. - Proof of payment on postal order no. 00668004 made out to the Tax and Concession Registry Office Rome of L.99,000, issued by the Post Office of Milan 15 on 17 / 3 / 1901 n. 474 • Revenue stamp of L. 2,000, The depositor refuses to sign the declaration referred to in circular no. 149 for the following reason: he does not believe he should, on principle, make declarations not required by law. Lb The application, descriptions and drawings listed above have been signed by the applicant and stamped with the office stamp of the applicant and countersigned by me. lAEPOSITANTE .0, THE OFFICIAL Pietro Mcssiuco p, the D\jj>Kore (Salvatore Ravalli) & HEAD OF THE BREVEI OFFICE y 72410 / fz ίέοη,ΐο MINISTRY OF INDUSTRY, TRADE AND CRAFTS 2 0 3 8 3 A / 81 _Central Patent Office “ROME- f U, Γ: Γ / Γλ'μΓ 1 The Company '· ----RAKOT UNI VERSITY AUTHORITY IOR APPIJED RESkARCR. . . , ( AND industrialists, MVELORMENT LTD. UbLOÌ 02IS of Hieraellan nationality JESHeSEe Tel-Ανιν (leradie? ----LM* -·-......-..mm... by Agent MODIANO & ASSOCIATI Sas, of Dr.Ing.G.MODIANO ά C.(Italian Patent and Trademark Agency) and with domicile for legal purposes in Milan Via Meravigli 16, requests a patent for 1 invention entitled: BACTERIAL ULTRACTANTS, PROCEDURE FOR PRODUCING THEM AND LIVE VACCINES CONTAINING THEM. Designated inventor: Eliora Z. RON. Priority of patent application in: Israel No. 59663 of March 1980. Attached documentation: a) Description in duplicate, b ) DixxjgXBtxiii>Lrt>ixxxx?LtimEÌ)C <ix[XJdxipliX3®iXX!83teQ:x c) Letter of appointment - XtfiteriatierDtmBapzxxaai^ d) Priority document with Italian translation (with reservation) e) / aajtxaaixxBX&mìesuBìcxabbBicòhctt^ f) Act of designation of the inventor. g) Proof of payment of the required taxes, h) Revenue stamp of L. 2,000, Date / / N: f241O / VEN / fz (59663) BACTERIAL MUTANTS, THE PROCESS TO PRODUCE THEM, AND LIVE VACCINES CONTAINING THEM. €Sk y $ Inventor Designated; Eliora Z. RON. Filed on 1 8 MAR. 1381 at No. 2 0 3 9 3 fi / gj SUMMARY The present invention relates to a live vaccine for the vaccination of fowl and mammals against a variety of gram-negative pathogenic germs belonging to the Enterobacteriaceae, comprising a bacterial suppression mutant derived from a non-pathogenic strain or race of B. coli, and a process for obtaining a mutant bacterial race for use in a live vaccine according to claim 1, which comprises exposing a non-pathogenic race of E. coli to a mutation-inducing agent, and selecting races free of O-ontigens and culturing them to obtain the bacteria to be used in such live vaccine; the new race E. coli LR-2 is a process for immunizing fowl and mammals against Enterobacteriaceae, which comprises applying an effective dose of such vaccine to the fowl or mammal by the oral route or as an aerosol. DESCRIPTION The present invention relates to novel bacterial mutants, a process for producing them, and live vaccines containing them. The invention relates to broad-spectrum vaccines based on novel bacterial mutant strains. <V\ Fig. )g| ti is a procedure for vaccinating mammals and poultry using this live vaccine. The vaccine is particularly valuable in animal husbandry and provides long-lasting immunity against a wide range of bacteria. The new mutants are irreversible; they are essentially non-pathogenic and can be used for the effective immunization of mammals and poultry. Gram-negative bacteria, especially those belonging to the Enterobacteriaceae (enteric bacteria), pose an increasingly serious problem in medicine and animal husbandry. These bacteria cause a variety of infections in poultry, humans, and other mammals, and treating the diseases and infections they cause is becoming difficult because a large proportion of these bacteria have developed resistance to a large number of currently used antibiotics. This antibiotic resistance is likely due to the fact that these bacteria carry drug-resistant infectious plasmids. One possible solution to the problems caused by these bacteria, especially in animal husbandry (such as poultry and similar), is the use of effective vaccinations. These vaccinations should be long-lasting and should cover a broad spectrum of infectious agents. Because Enterobacteriaceae belong to a wide variety of serotypes, it is difficult to prepare a vaccine that covers a sufficient number of infectious agents. It has been demonstrated in laboratory experiments that it is possible to obtain bacterial mutants that lack the 0 antigen, that is, bacteria that can be defined as deep rough and that such bacteria have a no spectrum immunization, when used in heat-killed vaccines, ; which is quite broad, Such bacterial mutants, i.e. deep roughe bacteria can be used as heat-killed vaccines for immunization against a fairly broad range of Enterobacteria, but immunization by means of heat-killed vaccines is not possible.) It is highly effective: multiple immunizations are required, which must be administered by injection. Furthermore, this immunization is only short-term. Immunization by injection is not practical in human medicine and is practically prohibitive in animal husbandry when large numbers of poultry or similar animals must be vaccinated. The disadvantages of conventional vaccines, including heat-killed vaccines of the deep-rough type, are overcome by the present invention. The present invention relates to novel bacterial mutants that · can be used for effective vaccination of poultry and mammals. The invention relates to a process for preparing such mutant strains and vaccines based on live mutant bacteria thus produced. It also relates to novel vaccines based on such live bacteria and a process for vaccinating livestock using such vaccines. By preparing deeply crude mutants of predetermined bacteria, new isolated strains can be produced that completely lack the O antigen. Similar strains can be obtained that carry a non-regressive mutation in lipopolysaccharide biosynthesis. The mutants are suppressor mutants and can be conveniently cultured. No reversions have been observed in the laboratory or after re-culture. Injected animal strains. The strains are essentially non-virulent or non-pathogenic. Large numbers of these bacteria have been injected into laboratory animals without causing harmful effects. These mutants are the actual active constituents of live vaccines that provide a broad spectrum of protection against pathogens, the extent of protection depending on the type of bacteria used. According to a specific embodiment of the invention, there are Enterobacteriaceae suppression mutants which substantially lack the O antigen (i.e., they may be termed profoundly crude) and which are adapted to provide effective and long-lasting immunization against a broad range of bacteria belonging to such Enterobacteriaceae. The new vaccines can be used in medicine and are particularly valuable in animal husbandry. One of the notable advantages of these live vaccines is that they can be administered as an aerosol. Experiments have shown that chicks immunized by exposure to an aerosol of such a vaccine acquired long-lasting protection against a broad spectrum of bird pathogens, A preferred embodiment of the invention relates to .. a new mutant strain of Escherichia coli K-12 which is itself a non-pathogenic strain and in which it performs EK1 recombination experiments DNA. The new mutants are essentially avirulent and can be used in live vaccines. Such vaccines can be used in aerosol form for effective mammalian immunization. The new race is a mutant that lacks the O antigen as a result of non-regressing suppression. Since these O antigens are responsible for species-specific serotypes, a mutant that is defective in the biosynthesis of O antigens results in immunization to those antigens that are common to all enteric bacteria, namely KDO and lipid A. The mutant is effective in providing effective immunization against a broad range of Enterobacteriaceae. The new mutant of the K-12 race is called LR-2. It is characterized below. Mutagenesis was carried out using nitrous acid and by selecting the required mutant strain. The strain is non-regressive, with a regression rate of less than 10%. No regressors were isolated after repeated injections into animals. Mutagenesis can be induced by radiation, by suppression induced by phages, such as phage ^u, etc. The invention is applicable to a wide range of bacteria. It Ò is illustrated by way of example in the following reference to the production of a specific new mutant strain derived from Escherichia coli K-12. Mutagenesis was performed with nitrous acid which was prepared by mixing equal amounts of sodium nitrite (0.1M) and 0.1M acetate buffer, pH 4.6 to a final concentration of 0.0511 nitrite, respectively. A single colony of Escherichia coli K-12 grown overnight with shaking at 37°C in LB medium containing 10 g Bactotrypton, 5 g yeast extract, and 10 g sodium chloride per liter, was washed with an equal volume of acetate buffer and resuspended in 0.3 ml nitrous acid (0.05M). After incubation for 30 minutes at 37°C, the cells were washed, resuspended in 10 ml of LB medium, and incubated for 1 min. - 6 nights at 37°C. / The mutant was screened as follows: the overnight culture was diluted 1:100 in fresh LB medium, and grown at 37°C by shaking. After achoreogenesis had resumed, bacteriopagus T was added in 4 multiple injections (5) and incubation continued until Lysis had not occurred. The surviving bacteria were placed on LB-agar plates and the colonies were screened for antibiotic sensitivity. eie for lake resistance. The new mutant, designated LR-2, has the following characteristics: I i that: Nutrients required: Leucine, adenine, tryptophan, histidine, arginine, isoleucine, vesicle, methionine, thiamine. ι i The breed does not determine the fermentation of the following substances: » Lactose, galactose, xylose, mannitol, maltose. The breed is resistant to: Coli phage T|, Coli phage T^, Coli phage T?, Coli phage P^. / The sensitivity of the other phages was not tested and it is assumed that the breed is resistant to other Coli phages. Highly sensitive to: Crystal violet, gentian violet, methylene blue, eosin and other dyes; high molecular weight (hydrophobic) antibiotics: novobiocin, rifampicin, erythromycin and similar. The myoorganism was deposited at the German Collection Myoorganism (LSM), Goettingen, on 25 February 1991 under No. DSM 2051. - 7 The speed of reversion was examined and found to be I —11 less than 10: no regressors were isolated on plates and after injections ( multiple animals. It is considered likely that the mutant that blocks the formation of the O-antigen results in suppression. The mutation induction method used is a method known to produce a high yield of suppression. Chemical analysis of purified ribopolysaccharides obtained from the LR-2 strain demonstrated that the mutant contains lipid A and KDO; carbohydrates of the 0 antigens could not be detected. Vaccine production To produce the vaccine, a single colony of the LR-2 strain was grown overnight on LB medium and diluted 1:100 in the same medium for large volumes. Several cells were harvested at approximately 5 10 bacteria per ml, washed three times by centrifugation in sodium chloride, and resuspended in a solution. At this stage, the culture could be lyophilized to obtain a highly effective live vaccine. Immunization: 10 live bacteria per animal were injected into mice and chicks*. No negative reactions occurred and the survival rate was γ at 100$. Mice were injected with a vaccine containing 10' bacteria per mouse po (ip injections) and this resulted in protection lasting more than two weeks against lethal doses of K, pneumonia which was injected ip* After six ip injections a titer of 1:640 was obtained in the agglutination of ββη^θ·' ' -.V. ι.I'Ί· ' ..............·<·. . Ori antibodies formed after six injections interacted with lipo- polysaccharides (LPS) from E. coli 075, K.Pneumonia, and Enterobaoter aerogenes, γ Six injections (ip,) of 10 bacteria each provided effective immunization against iv injections of lethal doses of E, coli O75 (see Table 3). ✓ * Test for virulence: Chicks (1, 7, 14, and 28 days old) were exposed to large doses of the vaccine. A quantity of 10 bacteria was introduced directly into the chicks' air sacs or injected intravenously, and no harmful effects were observed. In various laboratory animals (mice, rats) q was injected A high number of bacteria (more than 10 per animal) and no harmful effects were observed. The live vaccine is safe and non-pathogenic and can be used to vaccinate mammals and poultry without any harmful side effects. Immunization of chicks: Two chicks were immunized by exposure to an aerosol of a vaccine containing 3 x 10 bacteria per ml. A quantity of 10 ml was aerosolized into a 20-liter volume containing 36 chicks, and the chicks were exposed to this aerosol for 10 minutes. Another experiment was carried out by applying the vaccine in , 8 drinking water. In this experiment, a quantity of 3x10 bacteria , / ml was supplied. YES a third experiment was carried out by injecting 2x10 bacteria per chick into the saoI fì iL'àrià. The results are summarized in Table 1. -9 Immunized chicks were exposed to E.coli 0 ì . . which are pathogenic germs for birds. fi*. and E.coli 0 WITH THE TABLE 1 PROTECTION OF CHICKS BY VACCINATION WITH LR-2 AGAINST INFECTION (AIR SAC INJECTION) WITH E. coli 0 The infection was with 10 bacteria applied in the air sac. There were 20 chicks in each group. Age at Immunization Method of Age at Immunization Increase in Weight (days) I. 1+7 spray, spray 28 50.5 1+7 spray, drinking water 28 57.6 1+7 control 28 20.4 II. 1+7 spray, spray 30 12 1+7 control 30 -3 TABLE 2 PROTECTION OF CHICKS BY VACCINATION WITH LR-2 AGAINST INFECTION WITH E. coli Ο„ Λ - θ Immunization was by exposure to an aerosol, 3x10 / mi, 10 ml in a volume of 20 liters for 10 minutes. Groups of 20 chicks were used. Age at immunization Age at immunization Weight gain after infection (days) (days) (%) I. 1+14 30 5.7 1+21 30 9.2 21 30 11.3 control 30 0.02 k V> * ««> I - ..>*,» «ir. *·>» ·,.·«*·-. * Ai· t . rffr. . . , k ► 4. > . II. 11+21 50 —6 1+14+40 50 6.9 40 50 6.1 control 50 -5 - 10 5 The infection was with 10 bacteria applied in the air sac. Mice were immunized with six ip vaccinations of 7 2x10 live bacteria for two weeks and he administered a supplement ip at the third and fourth week. The infection was applied at the end of the sixth week. TABLE 3 SURVIVORS AFTER INFECTION WITH E. coli 0 (2x107, iv) Immunization oon: E. coli 0 75 (heat-killed) 17 / 18 94% LR-2 22 / 31 70% P. aureginosa (heat-killed) 12 / 38 31% NaCl (1%) control 12 / 36 33% The above mutation induction procedure can be used with a variety of similar bacteria (such as other members of the Enterobacteriaceae and Pseudomonas) and the resulting mutants, which lack the 0 antigen, can be used for broad-spectrum immunization of mammals in the form of a live vaccine.* Immunization by application of an aerosol of the vaccine is a very convenient and effective system for immunization with this type of vaccine. Polyvalent vaccine Immunization with LR-2 protects mice against infections by a broad range of ENTEROBACTERIACEAE. follows Table ' . I * ' μ 1'· 4« Ut t?· y H.....Λ ι r' i J Immunization Day V 1»7 1.7 1,7,14,21 Check 1.7 Check Organism used for_infection K. pneumonia,1 II ' it P. Vulgaris II Infection Day Survivors _ 1.00 * 100 100 Both immunization and infection occurred ip in γ mice. C^HeB. Immunization - 2 x 10 bacteria per mouse, infection - 2Εϋ^θ

Claims

1. CLAIMS 1. A live vaccine for the vaccination of poultry and mammals against a variety of gram-negative pathogenic germs belonging to the Entebacteriaceae, comprising a bacterial suppression mutant derived from a non-pathogenic E. coli strain.

2. A live vaccine according to claim 1, wherein the bacterial mutant is a deep rough mutant derived from E. coli K12.

3. Live vaccine according to claim 1 wherein the mutant is E, coli LR-2.

4. A method for obtaining a mutant bacterial strain for use in a live vaccine according to claim 1, which comprises exposing a non-pathogenic strain of E. coli to a mutation-inducing agent and selecting strains devoid of O antigens, and culturing the same to obtain bacteria for use in such live vaccine. 5* Process according to claim 4 wherein the agent used is nitrous acid. 6, E. coli LR-2 mutant strain. 7· Live vaccine according to claim 1, wherein the bacteria belong to the mutant race E, eoli,LR-2.

8. A process for immunizing poultry and mammals against Enterobacteriaceae comprising applying to the poultry or mammal an effective dose of a vaccine according to any of claims 1 to 3 or 7* orally or by aerosol. 9· Process according to claim 8 wherein the vaccine is applied to poultry.

10. Live vaccine for the immunization of mammals or birds against Enterobacteriaceae comprising a suppression mutant of E. coli essentially as described above. The Authorized Representative: Yes