RU2140981C1 - Avirulent strain vibrio cholerae of non 01-serogroup (variants), method of strain preparing (variants), vaccine - Google Patents

Abstract

FIELD: microbiology. SUBSTANCE: invention relates to avirulent strains of Vibrio Cholerae (CVD 112 and CVD 12RM) of non 01-serogroup that have no the RS1-sequence and core sequence of choleraic enterotoxin. Strains have sequences encoding the resistance to mercury and sequences encoding B-subunit of choleraic enterotoxin repeatedly inserted into chromosome. Invention relates to also methods of preparing avirulent strains of Vibrio Cholerae (CVD 112 and CVD 112RM) of non 01-serogroup. Avirulent strains Vibrio Cholerae CVD 112 and CVD 112RM of non 01-serogroup are used for production of vaccine against cholera. EFFECT: improved method of preparing the strain and vaccine. 16 cl, 40 dwg, 14 ex

Description

 This application is a partial continuation of US patent applications 08/133438 and 08/133439, registered October 8, 1993, both of which are incorporated into this description by reference. Both of these US patent applications 08/133438 and 08/133439 are a partial continuation of US patent application 07/931943, registered August 12, 1992, which is a continuation of US application 07/821872 of the present applicant, registered January 16, 1992 and which is a continuation of US patent application 07/533315 of the present applicant, registered June 5, 1990, each of which is incorporated into this description by reference. US patent application 07/533315 is a partial continuation of US patent application 06/581406, registered February 17, 1984 and is a partial continuation of US patent application 06/472276, registered March 4, 1983, each of which is incorporated into this description by reference . US patent application 07/533315 is also a partial continuation of US patent application 07/363383 of the present applicants, registered June 5, 1989, which decided to grant a patent on December 12, 1989, which is a continuation of US patent application 06/867633, registered on May 27 1986, which is a continuation of US patent application 06/472276, registered March 4, 1983, each of which is incorporated into this description by reference. Research work, the results of which are described in detail in the materials of this application, was carried out with the assistance of the National Institute of Health and partly the Center for Medical Biotechnology of the Maryland Biotechnological Institute.

 Cholera vibrio (Vibrio cholerae) is a non-invasive enteropathogen of the small intestine, unable to penetrate the mucous membrane. Local Siga-mediated immunity of the intestinal mucosa thus acts as a protective mechanism. The pathogenic cholera vibrio (V. cholerae) of serotype 01 releases a protein enterotoxin (also known as cholera enterotoxin or cholerogen, or cholera exotoxin), which is responsible for the multiplication and excretion of cholera vibrio by the intestines, which leads to diarrhea, a clinical symptom of cholera infection. The genes responsible for the production of cholera enterotoxin belong to the ctx genes (also known to specialists as tox genes or toxin genes). Cholera diarrhea can have an extremely severe course, leading to a significant loss of water and mineral salts by the human body, dehydration, acidosis, shock and death without emergency therapeutic treatment.

 It is known that in the region of the V. cholerae chromosome containing ctx genes that are responsible for reproduction, a 2700 bp region called RS1 (in the form of a repeating sequence) can be detected. Mekalanos, Cell, v. 35, pp. 253-263 (1983).

 During the development of the present invention, it was also found that V. cholerae produces a second enterotoxin, referred to as zonula occludents toxin, as previously reported by Fasano et al. In the article "Vibrio cholerae Produces a Second enterotoxin Which Affects Interstinal Tight Junction ", Proc. Nat. Acad Sci. (USA) 88.5242-5246 (1991).

 The currently created cholera vaccines can be divided into two broad categories: vaccines that stimulate antitoxic immunity, and vaccines designed to induce antibacterial immunity. Model animal experiments confirm the protective role of antitoxic and / or antibacterial immunity. It has been suggested that if both immunities act simultaneously, a synergistic effect occurs. [Holmgren, J. et al., J. Infect. Dis 136 Suppl., S105-S1122 (1977); Peterson, J. W. Infect. Immun. 25, 594 (1979); Resnick, I.G. et al., Infect. Immun. 13, 375 (1980); Svennerholm, A.-M. et al. Infect. Immun. 13, 735 (1976)]. However, it turns out that in humans protective immunity is manifested without a synergistic effect, that is, either a defense mechanism against toxins or against a bacterial infection [Eubanks, E.R. et al., J. Infect. Immun. 15, 533 (1977); Fujita, K. et al., J. Infect. Dis. 125, 647 (1972); Holmgren, J., J. Infect. Dis., See earlier; Lange, S. et al., Acta Path. Microbiol. Scand Sect. C 87.145 (1978); Peterson, J.W., see previously (1979); Pierce, N.F. et al. Infect. Immun. 37, 687 (1982); Pierce, N.F. et al., Infect. Immun. 21, 185 (1978); Pierce, N.F. et al., J. Infect. Dis. 135.888 (1977); Resnick, I.G. and others, see earlier; Svennerholm, A.-M. and others, see earlier].

Killed whole-cell vaccines
1. Parenteral whole-cell vaccines
For almost a century, killed whole cholera vibrios have been used as parenteral vaccines; these vaccines are still available on the market. The experience gained in the use of vaccines derived from inactivated whole cholera vibrios was analyzed by Joo, I. in "Cholera vaccines." In the book Cholera. (Barua D. and Burrows W., eds.), Saunders, Phyladelphia, pp. 333-355 (1974) and Feeley, JD et al., In abstracts of Cholera and Related Diarrhears, 43rd Nobel Symp., Stockholm, 1978. (O. Oucherlong, J. Holmgren, eds) Karger, Basel, pp. 204-210 (1980). Such vaccines stimulate the formation of high titers of serum antibodies with vibriocidal properties. They also enhance the increase in the titer of Siga antibody in the intestine against the somatic O-antigen of V. cholerae under conditions of vaccination of the population of Pakistan, and not Sweden [Svennerholm, A.-M. et al. Infect. Immun. 30., 427 (1980); Svennerholm, A.-M. et al., Scan. J. Immun. 6, 1345 (1977)]. It has been suggested that such an immune response to the vaccine occurs in recipients in Pakistan because they are already immunologically stable as a result of their previous exposure to the antigen, while such an immune reaction does not occur in individuals living in non-endemic regions (e.g. Sweden ) Parenteral killed whole-cell vaccines, as evidenced by the results of their testing, have a high immune potential to protect the body from V. cholerae homologous serotype, but usually for a period of less than one year [Joo, I. see earlier; Feeley, JC, see earlier; Svennerholm, A.-M. and others, see earlier, (1980); Svennerholm, A.-M. and others, see earlier, (1977); Mosley, WH et al., Bull. Wld. Hlth., Org. 49, 13 (1973); Philippines Cholera Committee, Bull. Wld. Hlth. Org., 49, 381 (1973)]. There is some evidence to support the suggestion that a parenteral whole-cell vaccine based on the Inaba serotype cholera vibrio provides excellent short-term immunity to both the Ogawa serotype cholera vibrio and the Inaba serotype, while the Ogawa serotype vaccine is effective only against the Ogawa serotype. The use of adjuvants in the composition of the parenteral whole-cell vaccine, allowed to provide protective immunity by about 70% for up to one and a half years (see, for example, Soroso, JS et al., Bull. Wld.Hlth. Org., 56, 619 (1978)) . However, very severe side effects (including sterile abscesses) often occur at the vaccine site of the vaccine with adjuvant, which limits the use of such adjuvant vaccines by the generally accepted method.

2. Oral whole-cell vaccines
Killed whole vibrios administered orally enhance the production of secretory antibodies against the vibrio antigen. [Freter, RJ, Infect. Dis., 111, 37 (1972); Freter R. et al., J, Immunol., 91,724 (1963); Ganguly, R. et al., Bull. Wld. Hlth. Org., 52, 323 (1975)]. The results obtained by other researchers indicated the high efficacy of such vaccines, but in most cases they led to the development of cholera diarrhea after a subsequent provocative test by pathogenic vibrios [Cash, RA et al. J. Infect. Dis. 130, 325 (1974)].

Toxoids
The immunogens intended for cholera prophylaxis by enhancing antitoxic immunity include the following substances:
1) Formaldehyde-treated cholera toxoid;
2) Glutaraldehyde-treated cholera toxoid;
3) Purified b-subunit of cholera enterotoxin and
4) Thermally inactivated cholera enterotoxin (treated or untreated with formaldehyde).

1. Formaldehyde-treated cholera toxoid
In vitro treatment of purified cholera enterotoxin with formaldehyde completely eliminates its virulence, resulting in the production of an toxoid that exhibits biological activity with a slight toxic effect, but stimulates the formation of antitoxin antibodies after parenteral immunization of animals. However, when a primary toxoid of this type was administered to both monkeys and humans as a parenteral vaccine, it again became partially toxicogenic, causing undesirable side effects at the vaccination site [Northrup, RS et al., J. Infect. Dis., V. 125, 471 (1972)]. Formalized cholera toxoid with adjuvant based on alumina was administered parenterally to Bangladeshi volunteers, including nursing mothers, but without mass testing of this vaccine [Merson, MH et al., The Lancet 1.931 (1980)]. In addition, the formalin cholera toxoid obtained in the presence of glycine was tested by oral administration, but no evidence was obtained of the immunological efficacy of this vaccine [Ohtomo, N. In Proceedings of the 12 th Joint Conference on Cholera, US - Japan Cooperative Medical Science Program, Sapporo (Fukumi H., Zinnaka Y., eds), pp. 286-296 (1976); Noriki.H, In Proceedings of the 12 th Joint Conference on Cholera, US - Japan Cooperative Medical Science Program, Sapporo (Fukumi H., Zinnaka Y., eds), pp. 302-310 (1976)].

2. Glutaraldehyde-treated cholera toxoid
Methods have now been developed for the large-scale production of glutaraldehyde-treated cholera toxoid, which contains virtually no somatic antigen [Rappaport, ES et al. Infect, lmmun., V.l4, p. 687 (1976)]. The researchers hoped that this pure antigen could be used to evaluate the protective mechanism of antitoxic immunity. Mass testing of this toxoid administered as a parenteral vaccine was conducted in Bangladesh in 1974 [Curlin, G. In Proceeding of the 11 th Joint Conference on Cholera, US-Japan Cooperative Medical Science Program., Pp. 314-329, New Orleans , 1975)]. This toxoid enhanced the formation of high titers of circulating antitoxin antibodies in Bangladeshi recipients. Two outbreaks of cholera caused by the vibrio El Tor Inaba, and then El Tor Ogawa captured the area where the vaccine was mass tested, which allowed a fairly reliable assessment of its effectiveness. However, the protective effect of the vaccine could be demonstrated only in one age group of the population, and at the same time it was limited by the duration of the epidemic cholera of the Inaba serotype, as a result of which the effectiveness of the cholera toxoid treated with glutaraldehyde obtained in its pure form in the form of a parenteral vaccine was low and lower than the results of testing parenteral inactivated whole-cell vaccines in the same population.

 The possibility of using glutaraldehyde-treated cholera toxoid as an oral vaccine was investigated, based on the assumption that the indicated toxoid administered in this way should be more effective by enhancing the formation of antibodies against cholera enterotoxin [Levine, M.M. and others, Trans. Roy. Soc. Trop. Med. Hyg. 73, 3, (1979)]. To this end, two groups of volunteers were immunized by administering three doses of 2.0 mg or three doses of 8.0 mg of the indicated toxoid directly into the lumen of the small intestine (via an intestinal tube), every month. After that, volunteers who received the vaccine and non-immunized persons who served as control took part in experiments with control infection with their cholera vibrio. In no experiment with an antigenic stimulus, there was a significant decrease in the symptoms of the disease and the severity of diarrhea in vaccinees compared with the control. The lack of effectiveness of an orally administered cholera enterotoxin disinfected with glutaraldehyde can be due to the fact that the ability of B subunits to bind GM1 ganglioside is greatly reduced as a result of its neutralization with glutaraldehyde.

3. Purified Subunit B of cholera enterotoxin
Cholera enterotoxin consists of two subunits, A and B, encoded by the ctx AB operon. The A subunit induces enzymatic changes that cause fluid secretion into the intestinal lumen, while the non-toxic B subunit is an immunogenic group that binds toxin receptor (GM1 ganglioside) on intestinal epithelial cells [Holmgren, J. Nature, 292, 413 (1981)]. It has been demonstrated that a purified B-subunit, administered both orally and parenterally to Bangladeshi, enhances the formation of Siga antitoxin in the intestinal fluid, that is, the result obtained, possibly due to immunological resistance in the endemic cholera focus [Svennerholm, A. -M. and others. The Lancet I, 305 (1982)].

 Significant advantages of an oral vaccine from the B-subunit of cholera enterotoxin designed to enhance antitoxic immunity include its complete safety (the probability of reverse conversion to a toxin, as is the case with toxoids, is eliminated) and the ability to bind enterotoxin receptors on enterocytes. Animal studies indicate, however, that a purified B subunit has less potential than native holotoxin to enhance the production of toxin-neutralizing antibodies [Pierce, N.F., see earlier, (1982).

 It should be understood that the purified B subunit can be used, if at all, if necessary, in conjunction, for example, with oral inactivated vibrio vaccines as a combined oral vaccine designed to enhance the production of both antibacterial and toxin neutralizing antibodies.

4. Thermally inactivated cholera enterotoxin
The thermally inactivated cholera enterotoxin is a large molecular weight toxoid (molar mass of approximately 1,000,000) obtained by heat treatment of the cholera enterotoxin at a temperature of 65 ^° C. for at least five minutes [Finkelstein, RA et al., J. Immunol., 107, 1043 (1971)]. This toxoid has immunogenic ability, although it still retains less than 5% of the toxicogenic activity of native cholera enterotoxin. A longer heat treatment (for example, for 25 minutes) produces a less biologically toxic toxoid [Germanier, R. and others, J. lmmun., 13, 1692 (1976)], and its subsequent treatment with formaldehyde completely eliminates residual biological toxicity in it . The obtained formalinized thermally inactivated cholera enterotoxin has at least the same potential as the native enterotoxin in enhancing the formation of serum antitoxin antibodies after immunization of rabbits. Swiss volunteers developed a strong humoral immune response after parenteral immunization with their formalinized thermally inactivated cholera enterotoxin, obtained in doses of 10, 30 or 100 μg [Germanier, R., et al., J. Inf. Dis. 135, 512 (1977)]. No marked adverse reactions were noted.

As an oral antigen, thermally inactivated cholera enterotoxin is more immunogenic when taken in the form without treatment with formaldehyde. In dogs, tolerance to formaldehyde-free, thermally inactivated cholera enterotoxin is as good as with the oral vaccine; orally administered doses of it (using NaHCO ₃ as a carrier) up to 500 μg do not cause diarrhea. Oral administration of five doses of 500 mcg for approximately 42 days provides a significant increase in protective immunity in dogs against an oral sample with pathogenic V. cholerae. In groups of six and four adult volunteers who received it in doses of 50 μg and 200 μg, respectively, using NaHCO ₃ as a carrier, no side effects were noted.

 It should be understood that thermally inactivated cholera enterotoxin can be used in combination, for example, with live vaccines, killed vibrios or other related antigens that have the ability to stimulate antibacterial immunity, as a result of which the antitoxic immunity induced by thermally inactivated cholera enterotoxin is enhanced.

Combination vaccines
The main advantage of an oral vaccine from killed cholera pathogens is its safety. An oral vaccine consisting of a mixture of antigens designed to stimulate both antibacterial and antitoxic immunity is most likely successful for the following reasons: toxoid vaccines that stimulate only antitoxic immunity are ineffective, according to research results, to protect against human cholera disease, although they can be used to immunize model animals. In addition, oral and parenteral killed whole-cell vaccines that do not stimulate any immune response to enterotoxin provide significant protective immunity against human cholera, albeit for a short period of time. Moreover, mixtures of antigens (for example, crude cholera enterotoxin, or toxin along with liposaccharide), stimulating both antitoxic and antibacterial immunity, cause a synergistic protective effect.

To date, two more large-scale trials of combination vaccines have been carried out under practical conditions. In the first trial, nine volunteers who received glutaraldehyde-treated cholera enterotoxin (at a dose of 2 mg weekly for four weeks), along with El Tor Inaba killed cholera vibrios (10 ¹⁰ vibrios twice a week for four weeks), were infected one month after immunization , along with six non-immunized volunteers serving as control, pathogenic cholera vibrios El Tor Inaba in a dose of 10 ⁶ vibrios. Diarrhea occurred in only two out of nine vaccinees, against four out of six in the control (vaccine efficiency was 67%), while the disease was milder in two patients who took the vaccine. More important in this experiment, perhaps, is the fact that V. cholerae can be directly cultured from isolated excrement in only two out of nine vaccinated, against six out of six in the control. This suggests that immunological mechanisms inhibit the proliferation of vibrios. In another study, three doses of a combination vaccine consisting of the B-subunit of cholera enterotoxin and the killed whole vibrio cholera were given to adult volunteers, who then participated in a control infection experiment to evaluate the effectiveness of the vaccine. The combination vaccine was taken on days 0, 14, and 28. Each of the three doses of the vaccine contained 0.5 mg of purified B-subunit of cholera enterotoxin and 2 • 10 ¹¹ killed V. cholerae (5 • 10 ¹⁰ classical vibrio of the Inaba serotype, 5 • 10 ¹⁰ classical vibrio of the Ogawa serotype, and 1 • 10 ¹¹ vibrio El Thor Inaba).

 A group of eleven volunteers immunized with this combination vaccine was given a month after the last dose of the vaccine, pathogenic V. cholerae El Tor Inaba, along with seven volunteers serving as control. Diarrhea occurred in seven of the seven volunteers in the control, and only four of the eleven vaccinated (p = 0.01). Symptoms of the disease in four of those receiving the vaccine were less pronounced.

 Thus, the results of evaluative studies of oral combined vaccines based on the indicated toxoid and killed whole cells demonstrate a comparable degree of their effectiveness. The effectiveness of such vaccines for the stimulation of protective immunity, however, is relatively low (55-65%), and several revaccinations are necessary to induce the mechanism of anti-infection protection.

Vaccines from attenuated V. cholerae
Studies conducted with the participation of North American volunteers indicate that both the so-called classic cholera infections and those caused by the clinical strain of El Tor vibrio stimulate a high degree of protective immunity for at least three years [Cash, RA and others, see previously (1974): Levine, M.M. and others, see earlier (1979; Levine, M.M. et al., Volunteers studies in development of vaccines against Cholera and enterotoxigenic Escherichia coli: a review article in Acute Enteric Infections in Children: New Prospects for Treatment and Prevention. (T. Holm, J. Holmgren, M. Merson, and R. Mollby, eds.) Elsevier, Amsterdam, pp. 433-459 (1981); and Levine, M.M. et al., J. Infect. Dis. , 143, 818 (1981)]. Based on the results of monitoring the status of these volunteers, it can be assumed that the most promising approach to solving the problem of immunological protection against cholera may be associated with attenuated non-virulent strains of V. cholerae, used as oral vaccines .

 1. Natural strains of serogroup 01 V. cholerae.

 Non-virulent strains of serogroup 01 V. cholerae isolated from environmental sources in India and Brazil were tested on volunteers as potential candidates for vaccines, and the results were disappointing. These strains did not colonize the intestines in humans, or were minimally colonized; the reactions of vibriocidal antibodies to the antigen were weak, and in addition, they did not cause protective immunity in experimental studies with control vaccination [Cash, R.A. and others,. Infect. Immun. 10, 762 (1974); Levine M.M. and others, J. Infect. Dis. 145, 296 (1982)]. In these strains, as shown by hybridization measurements using a radiolabeled DNA probe, the toxin gene was lost [Kaper, J.B. and others. Infect. Immun. 32, 661 (1981)].

 2. Mutagenic attenuated strains.

 Classical Inaba 569B was mutated using nitrosoguanidine (NTG) and a hypotoxicogenic mutant was isolated. [Finkelstien, R.A. and others, J. Infect. Dis. 129, 117 (1974); Holmes, R.K. and others, J. Clin. Invest. 55, 551 (1975). This mutant strain, M13, was administered to volunteers. Diarrhea did not occur, but the strain had a weak colony forming ability. Studies with control infections have shown that some protective effect was achieved through immunization with multiple doses [Woodward, E. and others, Develop. Biol. Stand. 33.108, (1976)].

 The strain of cholera vibrio El Tor Ogawa 3083 was also mutated [Honda, T. et al., Proc. Nat. Acad Sci., No. 76, 2052 (1979) As a result of intensive screening and analysis of thousands of colonies, one isolate was obtained that continued to produce an immunogenic B subunit, but without detectable A-subunit production or holotoxin, the only isolate, Texas Star-SR, met the criteria. The Texas Star-SR strain produces normal or increased B-subunit production, but detects a negative reaction in the test samples for holotoxin activity or A-subunit activity.

Large-scale trials of the Texas Star-SR vaccine strain were performed with the participation of volunteers (see, for example, Levine M.M. et al. Acute Enteric, earlier (1981). Groups of five volunteers received two doses of 10 ⁹ microorganisms, each with an interval of a week, and another eighteen volunteers received two doses of 2 • 10 ¹⁰ microorganisms, each after a week.Some diarrhea was noted in 16 of 68 people who received the vaccine (24%), and only one of the vaccinees exceeded the total stool volume 1.0 liter (1464 ml) In typical cases caused by the administration of the diarrhea vaccine I was accompanied by two or three meager liquid stools, not exceeding 400 ml in total.

Vaccine microorganisms were isolated from collaborative cultures, from about half of the recipients who received the vaccine. In the case when the contents of the jejunum were cultured (recipients receiving the vaccine at a dose of 10 ⁸ or more microorganisms), cultures showed a positive reaction in 35 of the 46 vaccinated (76%). Hundreds of Texas Star mutant clones isolated from co-cultures and jejunum cultures were tested for activity of the cholera enterotoxin subunit domain with a serological test using adrenocytes and Y-1; in no case was a seropositive reaction observed.

 A significant increase in serum toxin-neutralizing antibodies was detected in only 29% of those receiving the vaccine; 93% of vaccinees showed a significant increase in the serum of the vibriocidal antibody and their titers were almost close to the titers, as in the case after infection with pathogenic V. cholerae.

 In experimental studies with antigenic stimulus conducted on volunteers, a mutant Texas Star-SR strain was found to reliably elicit an immune response to vibrio antigens from both El Tor Ogawa and El Tor Inaba. The administration of one or two doses of an oral vaccine from an attenuated Texas Star-SR strain provides good protective immunity against cholera caused by El-Tor vibrio.

 It is clear that the use of attenuated strains has only their inherent advantages, since such strains provide an imitation of immunity after infection (post-vaccination immunity). However, Texas Star-SR strains are not without some drawbacks. First, mutagenesis (for example, using nitrosoguanidine) causes numerous mutations, not all of which are necessarily recognized. In addition, the exact mechanism of genetic damage, which is believed to be responsible for attenuating the virulence of Texas Star-SR, is not known. In addition, as with any pathogen mutated by nitrosoguanidine, Texas Star-SR can restore its virulence. 3. Natural 01-serologically untypical strains of V. cholerae.

 Cholera vibrio serotype 01 is usually responsible for the occurrence of epidemic cholera. Vibrio cholerae not belonging to the serotype 01 class is mainly responsible for sporadic cases of gastroenteritis and extraintestinal infectious diseases. More recently, however, the press reported that several strains of V. cholerae were isolated from patients during an outbreak of cholera infection [Ramamurthy et al. The Lancet, vol. 341, 703-704 (1993), the entire contents of which are incorporated herein by reference]. The serological characteristics of the large number of clinical strains obtained during this recent epidemic indicated that these strains did not elicit an agglutination reaction with antiserum 01, or with any of the tested monoclonal antibodies growing against factor A, B or C of V. cholerae serogroup 01 As a result, these vibrios were identified as 01-serologically atypical.

 In addition, with the exception of only one 01-serologically non-typeable strain in the above test sample, all other non-serogroup 01 strains tested during a new outbreak in the outbreak could not be identified by typing on a panel of 138 antigens created for V. cholerae non-01 serogroups at the Japan National Institute of Health, suggesting that the strain associated with this new outbreak is of a previously unrecognized type, or a recent, non-01 serogroup capable of causing Epidemic cholera.

 When analyzed by DNA hybridization, all strains isolated during this epidemic were crossed with both ctx and zot gene-specific probes, but not a single case of hybridization with DNA probes specific to the heat-resistant enterotoxin V. cholerae not serogroup 01 (NAG-ST). In addition, cholera enterotoxin production was reliably detected by enzyme-linked immunosorbent assay. The amount of enterotoxin produced by these new isolate strains was reported to be similar to that of enterotoxin in the clinical strains of V. cholerae serogroup 01. Most of these strains were reported to be resistant to, for example, streptomycin and furazolidone, but were sensitive to other commonly used antibiotics , including tetracycline. Their resistance to ampicillin, or its derivatives, has not been reported.

 In another recent epidemic, although the total number of cases of cholera did not increase, a wide variety of V. cholerae isolates obtained from identified patients with cholera-like diarrhea did not belong to the 01-serogroup, and like the above strains, it was impossible to identify with test samples and using the standard panel for typing.

 Another case of a non-01-serogroup cholera vibrio, a powerful outbreak of epidemic cholera that affected an adult population, was reported [Albert et al. The Lancet, vol. 341, 704 (1993), the entire contents of which are incorporated herein by reference]. Of the smears taken from the rectum, as reported in this publication, approximately 67 percent of the V. cholerae isolates did not belong to the 01 serogroup after the standard test, and not one was typed as V. cholerae serogroup 01.

 It was also reported that V. cholerae, responsible for this outbreak, resembles V. cholerae 01 serogroups, both biochemically and colony morphology, but at the same time, as noted, it does not cause an agglutination reaction to V. cholerae antiserum. The V. cholerae strains in the test sample did not show reactivity with monoclonal antibodies specific for V. cholerae factor A serogroup 01.

 In this second epidemic recently reported, all tested 01-untyped strains respond positively to cholera enterotoxin production in the agglutination reaction with Y-1 adrenocytes, and, as noted, are neutralized by the rabbit polyclonal antiserum against cholera enterotoxin.

 A polymerase-amplified blot hybridization test using non-01-serogroup cholera-specific primers, as also described, caused an amplification of the cholera enterotoxin sequence.

 The selected isolates were also analyzed by rabbit ileum ligation and, as described, they caused watery diarrhea when entangled, similarly to the case associated with V. cholerae 01.

 The strain V. cholerae isolated during this second outbreak was reported to be sensitive to certain antibiotics, including tetracycline, but was resistant to other vibriostatic agents. This result is in contradiction with the assumption that the currently prevailing strains in this area, most of which are isolates, are tetracycline resistance.

 Due to these outbreaks that caused outbreaks of V. cholerae non-01-serogroup infection in epidemic foci, there is an urgent need for vaccines specific to these microorganisms. In addition, it would also be desirable to obtain associated vaccines that are effective for both 01 and non-01 serogroups, especially for use in regions where virulent strains of both V. cholerae serogroups can be found.

Summary of the invention
Based on the proposed new methods, the applicants obtained and isolated mutants of the virulent strain of Vibrio cholerae non-01 serogroups, which are suitable for use as a vaccine for the prevention of cholera disease after the introduction of the virulent strain V. cholerae.

According to the invention, the avirulent strain of Vibrio cholerae CVD112 non-01 serogroup is used for vaccination against cholera. The specified strain is a strain from serogroup 0139 and has a genotype including ctxA ^- , zot ace ^- , mer, ctxB. The specified strain is characterized in that the DNA encoding the resistance to mercury and the DNA encoding the B subunit of the cholera enterotoxin are inserted into the site of the hemolysin gene in the chromosome.

The specified strain deposited as strain ATCC 55752 or its derivative. The avirulent strain of Vibrio cholerae CVD112 RM non-01 serogroup, used for vaccination against cholera, is also claimed according to the invention. The specified strain is a strain from serogroup 0139 and has a genotype including ctxA ^- , zot ^- ace ^- , recA-, mer, ctxB. The strain is characterized in that DNA encoding mercury resistance and DNA encoding the B subunit of cholera enterotoxin are inserted into the hemolysin gene site on the chromosome.

 The strain V. cholerae 1837, which belongs to the 01-serologically atypical strain 0139, is used as the initial strain for producing one mutant of the invention. Declare mutants contain deletions in the core region (“core”, core region) of V. cholerae enterotoxin. These deletions were obtained using restriction endonucleases from a newly identified, characterized and cloned core region at the ctx locus of the original strain, although other suitable deletion methods known in the art can also be used.

 To induce immunogenicity, sequences encoding the B subunit of cholera enterotoxin were reintroduced into the vaccine strain. In addition, sequences encoding resistance to heavy metals, such as mercury, were inserted into the mutant strains and which allow the detection of a vaccine strain without the use of potentially therapeutically antibiotic resistant markers.

 01 serologically untyped vaccine strains of V. cholerae of the present invention, thus, were purposefully transformed using recombinant DNA methods to eliminate their virulence, without affecting other components necessary for the production of immunity.

One avirulent strain of V. cholerae non-01-serogroup of the present invention is a strain of V. cholerae CVD112 (ser ^- , zot ^- , ace ^- , orfU ^- , ctxA ^- , ctxB, mer, HylA ^- , attRS1 ^- , RS1 ^- ). Another avirulent strain of V. cholerae non-01 serogroup is strain CVD112RM (ser ^- , zot ^- , ace ^- , orfU ^- , ctxA ^- , ctxB, mer, HylA ^- , recA ^- , attRS1 ^- , RS1 ^- ).

 Studies were performed on the characterization of the ctx locus of the virulent strain V. cholerae not of the 01 serogroup, which resulted in new data indicating that the isolated strain contains two core regions (core regions) and four repeating sequences (RSl sequences). A plasmid is constructed in which there is a deletion in the entire, or almost the entire core region of the cholera enterotoxin, but at the same time, long flanking (limiting) DNA sequences of the V. cholerae chromosome at the ctx locus (i.e., the segment of the ctx gene) remain. As a result of the conjugative gene transfer of the indicated plasmid into the virulent V. cholerae of the non-01-serogroup with subsequent homologous recombination and the second recombination event, the V. cholerae of the non-01-serogroup received a DNA sequence with only one core region and one SR1 element. The remaining sequence in the core region and the RSl element were then removed using the newly cloned and then deliberated 0139 chromosomal DNA.

 Sequences encoding the B subunit of cholera enterotoxin and sequences encoding resistance to heavy metals were then reintroduced into the chromosome of the V. cholerae mutant by homologous recombination.

 01-Untyped, non-toxicogenic deletion mutants of the invention are capable of colonizing the small intestine and stimulating local protective immunity against the introduction of a bacterial cell. After a transient episode of colonization, the vaccine induces protective immunity against subsequent infection with virulent toxicogenic strains of V. cholerae non-01 serogroup.

 The invention also provides methods for producing avirulent strains of V. cholerae non-01 serogroup, and a vaccine obtained from these strains.

In one aspect, the invention provides a method for producing a first avirulent strain of V. cholerae non-01-serogroup containing a deletion of the core region (core region is the main part of the nucleosome) of cholera enterotoxin and RS1 sequences, but expressing the B subunit of cholera enterotoxin and sequences encoding heavy metal resistance product, which comprises the steps of:
(a) constructing a first plasmid containing DNA having a core region of the Vibrio cholerae cholera enterotoxin core and flanking sequences of sufficient length to promote recognizable in vivo recombination ligated to the gene encoding the first selectable foreign marker, which causes resistance to the selective factor, wherein the first plasmid is incapable of extrachromosomal DNA replication in V. cholerae;
(b) crossing the virulent strain of Vibrio cholerae non-01-serogroup with the first microorganism carrying the first plasmid;
(c) selecting and isolating Vibrio cholerae expressing a first selectable marker;
(d) growing V. cholerae isolated in step (c) in the absence of said selective factor;
(e) screening for V. cholerae grown in step (d) for loss of expression of said first selectable marker;
(f) constructing a second plasmid containing the chromosomal DNA sequences of Vibrio cholerae non-01-serogroups that limit the cholera enterotoxin locus deleted (deleted) from the DNA containing the region of the cholera enterotoxin core and RSl sequence ligated with a second selectable foreign marker, which causes resistance to the second electoral factor;
(g) crossing the product screened in step (e) with a second microorganism carrying said second plasmid; and
(h) selecting a Vibrio cholerae that expresses a second selectable marker;
(i) growing the selected product of step (h) in the absence of a second selective factor;
(j) screening the V. cholerae product of step (i) for the loss of said second selectable marker; and
(k) isolating the product screened in step (j).

(l) constructing a third plasmid that contains enough V. cholerae chromosomal sequences of sufficient length to promote recognizable in vivo recombination, flanking sequences of the cholera enterotoxin B subunit sufficient to induce immunogenicity and sequences encoding a heavy metal resistance product, and ligated with a third selectable foreign marker, wherein said third plasmid is incapable of extrachromosomal DNA replication in V. cholerae;
(m) crossing the screened product of step (j) with a third microorganism carrying said third plasmid;
(n) selection according to the phenotype of Vibrio cholerae, which expresses a third selectable marker; and
(o) isolating the selected product of step (1).

The specified method for producing an avirulent strain of V. cholerae non-01 serogroup is characterized in that said virulent strain of Vibrio cholerae non-01 serogroup is a strain V. cholerae 1837, which refers to 01-serologically atypical strain 0139. According to the invention, said selected product is isolated stage (o) is a strain
V. cholerae CVD112.

 V. cholerae non-01-serogroup, obtained in accordance with the specified method, is a product with a deletion of the entire core region of the cholera enterotoxin and RS1 sequence at the ctx locus, but expresses sequences of the B subunit of the cholera enterotoxin sufficient to cause immunogenicity, and sequences which cause resistance to heavy metals.

In another aspect, the invention provides a second avirulent V. cholerae non-01 serogroup, which contains a deletion of the core region of the cholera enterotoxin and RS1 sequences of V. cholerae non-01 serogroup, but expresses re-inserted cholera enterotoxin subunit B sequences sufficient to induce immunogenicity, and sequences encoding a product that induces resistance to heavy metals. The said V. cholerae non-01-serogroup mutant of the present invention is a negative phenotype strain that further comprises a deletion in which the recA locus product is inactivated or lost (absent). The proposed method includes the steps of:
(a) constructing a first plasmid containing DNA having a Vibrio cholerae cholera enterotoxin core region and flanking sequences of sufficient length to promote recognition of in vivo recombination ligated to a gene encoding a first selectable foreign marker that induces resistance to a selective factor, wherein said first the plasmid is incapable of extrachromosomal DNA replication in V. cholerae;
(b) crossing the virulent strain of Vibrio cholerae non-01-serogroup with the first microorganism carrying the first plasmid;
(c) phenotype selection and isolation of Vibrio cholerae expressing the first selectable marker;
(d) growing V. cholerae isolated in step (c) in the absence of said selective factor;
(e) screening for V. cholerae grown in step (d) for loss of expression of said first selectable marker;
(f) constructing a second plasmid containing Vibrio cholerae non-01-serogroup chromosomal sequences that limit the cholera enterotoxin locus deleted from DNA containing the core region of the cholera enterotoxin and RS1 sequence ligated with a second selectable foreign marker that causes resistance to the second selective factor;
(g) crossing the product screened in step (e) with a second microorganism carrying said second plasmid; and
(h) selection according to the phenotype of Vibrio cholerae, which expresses a second breeding marker;
(i) growing the selected product of step (h) in the absence of a second selective factor;
(j) screening the V. cholerae product of step (i) for the loss of said second selectable marker; and
(k) isolating the product screened in step (j).

(l) constructing a third plasmid that contains V. cholerae chromosomal sequences of sufficient length to promote recognizable in vivo recombination that limits the cholera toxin B subunit sequences sufficient to induce immunogenicity, and sequences encoding a heavy metal resistance product, and ligated with a third selectable foreign marker, wherein said third plasmid is incapable of extrachromosomal DNA replication in V. cholerae;
(m) crossing the screened product of step (j) with a third microorganism carrying said third plasmid;
(n) selecting a Vibrio cholerae that expresses a third selectable marker;
(o) isolating the selected product of step (n);
(p) constructing a fourth plasmid containing V. cholerae chromosomal sequences of sufficient length to promote recognizable recombination at the recA locus, flanking sequences with a deletion at the recA gene locus to inactivate the rec A gene expression product, and ligated with a fourth selectable marker, wherein the fourth plasmid is incapable of extrachromosomal DNA replication in V. cholerae;
(q) crossing the screened product in step (o) with a fourth microorganism carrying the third plasmid;
(r) selection by Vibrio cholerae, which expresses a third selectable marker; and
(s) isolating the selected product in step (r).

 The deletion mutants Vibrio cholerae of the present invention can be used as vaccines to develop protective immunity against cholera, the causative agent of which is V. cholerae not 01-serogroup, as well as in methods for creating cholera vaccines.

 One strain of Vibrio cholerae of the invention, designated as CVD112, induces reliable post-vaccination immunity in humans upon subsequent exposure to a similar strain of non-01-serogroup. Another strain of Vibrio cholerae of the invention, designated as a culture of CVD112 RM, can induce significant post-vaccination immunity in humans when a similar strain of non-01 serogroup is vaccinated, and is also incapable of recA-mediated homologous recombination.

 The invention is also a vaccine for protection against cholera disease when exposed to Vibrio cholerae non-01 serogroup, which, according to the invention, contains the avirulent strain CVD112 or CVD112 RM non-01 serogroup.

Brief Description of the Drawings
FIG. 1. Strain V. cholerae N 16961 (pJBK55) (Ap ^r )
FIG. 2. Methods of cross-linking and conjugative gene transfer for the construction of V. cholerae JBK56.

 FIG. 3. V. cholerae JBK56.

 FIG. 4. Design diagram of the JBK21.

 FIG. 5. Design scheme pJBK54.

 FIG. 6. Design scheme V. cholerae JBK56.

 FIG. 7. In vivo recombination by cross-linking and elimination of the ctx gene.

 FIG. 8. Design scheme pJBK51.

 FIG. 9. Design scheme pCVD14 and pCVD15.

 FIG. 10. Design scheme pJBK108.

 FIG. 11. Design scheme pJBK107.

 FIG. 12. DNA sequences and corresponding amino acid sequences (upper part) of the XbaI and ClaI restriction sites that define the ends of the excised XbaI-ClaI fragment (550 bp in length) of the A subunit in Ogawa 395 serotype, and for the (lower part) the junction zone in CVD101 after deletion of this fragment and insertion of the XbaI linker.

 FIG. 13 (A and B). Effect of V. cholerae culture supernatant on ileum ligation (Isc) and tissue ionic conductivity (Gt). The indicators are given for 6 animals at each point depending on time; brackets - standard error 1A, Effect of V. cholerae 395 supernatants on Isc (solid line) and Gt (dashed lines). B, Effect of V. cholerae 395 culture supernatants (solid line), CVD101 (long dotted line) and 395N1 (dotted line) on Gt. The medium used as a control (short dashed line) includes a culture medium with uninfected cells.

 FIG. 14. Quantitative characteristics of the variability of the zone of tight contact ZO in tissues under the influence of culture supernatants or broth culture as a control.

 FIG. 15. Reversal of Gt variations induced by V. cholerae culture supernatant 395. V. cholerae culture supernatants (triangles) and uninfected cell medium (squares) were added and removed at the time indicated by arrows.

 FIG. 16. (A, B, C and D) CVD109 design diagram. Zot and ctx genes are linked to each other on the V. cholerae chromosome and are located in the V. cholerae chromosome region, which contains multiple repeats of the 2700 bp DNA fragment. called an RSI element (repeating sequence). RSl elements are located on both sides of the zot and ctx genes in the virulent strain V. cholerae E7946 (El Tor biotype, Ogawa serotype). Zot and ctz genes are depicted in the figure by a large unshaded or shaded arrow. RS1 sequences are depicted in the figure by a smaller solid arrow.

 FIG. 17 (A and B). Enterotoxin enterotoxin zot gene DNA sequence of the tight contact zone (zonula occludens), ranging from 1 to 1428 nucleotides. Capital letters on top of the DNA sequence indicate the predicted amino acid sequence of the ZOT protein encoded by the zot gene.

 FIG. 18. Scheme for the construction of plasmids pCVD621 and plasmids pCVD622.2B.

 FIG. 19 (A, B, C and D). CVD110 Design Diagram.

The abbreviation figures used for restriction sites have the following meanings:
A = AccI restriction endonuclease site
B = BgIII restriction endonuclease site
C = ClaI restriction endonuclease site
E = EcoRI restriction endonuclease site
H = HindIII restriction endonuclease site
P = PstI restriction endonuclease site
S = SalI restriction endonuclease site
X = XbaI restriction endonuclease site
K = KpnI restriction endonuclease site
Other abbreviations in the figures or anywhere in the description have the following meanings:
Ap = ampicillin resistance gene
Ap ^r = phenotype of ampicillin resistance,
Ap ^s Ampicillin-sensitive phenotype
Chrom = Chromosome
Cm = chloramphenicol resistance gene
CT = cholera enterotoxin
ctx = cholera enterotoxin gene (cholerogen)
CTA = A cholera enterotoxin subunit
ctxA = A cholera toxin subunit gene A
CTB = B subunit of cholera enterotoxin
cCxB = gene B subunit of cholera enterotoxin
hylA = hemolysin gene
kb = thousand base pairs
mer = mercury resistance gene
p = Plasmid
Su = sulfonamide
Su ^r = sulfonamide resistance phenotype
Tc = tetracycline
Tc ^s = tetracycline sensitive phenotype
Tp = Trimethoprine
zot = gene of the zone of tight contact (zonula occludens) of cholera enterotoxin
ACE = auxiliary protein gene at the cholera enterotoxin locus
ser = potential of the coding sequence for the proposed, although not yet designed, encoded core.

 FIG. 20. A map showing the relative positions of the ctx, zot, and ace genes, OrfU, and flanking sequences of RS1 elements (RS1 are shown by large arrows). Two blocks with vertical stripes correspond to two open reading frames in which ACE activity is initially localized. Currently, ACE activity is believed to be localized relative to the open reading frame at the zot gene locus. The figure shows a fragment of the plasmid pCVD630.

 FIG. 21 (A and B). Activity of V. cholerae CVD110 and CVD110 strains containing plasmid pCVD630, measured in the Ussing chamber. The panel in FIG. 21A on the left shows the changes induced in the short circuit flow (ISC), and the panel in FIG. 21B on the right shows changes in potential difference (PD).

 FIG. 22. A DNA sequence of 2.9 kbp length EcoRV fragment containing ACE activity (GATATC at the beginning and end of the sequence is the EcoRV restriction site). The complete sequence of 395 amino acid residues of a classic biotype strain is shown (SEQ ID N: 1). Below this is shown the insert in this region from El Top strain E7946 (SEQ ID N: 4) - only those amino acid residues of E7946 are shown which differ from sequence 395. If this sequence is identical for two strains, only sequence 395 is shown. (Dotted lines in residues 236-239 indicate that strain E7946 contains an AGT insert that is not in sequence 395). Above the line of the primary DNA structure is shown the amino acid sequence (in the form of only one uppercase letter), predicted based on the sequence 395. Two translation sites ORFs; classic 395 OrfU contains a fragment of amino acid residues from 1034 to 2218 (SEQ ID N: 3); classic 395 ace contains a fragment of amino acid residues from 2221 to 2508 (SEQ ID N: 2); El Top OrfU includes a fragment from 1037 to 2221 (SEQ ID N: 6); and, El Top ace contains a fragment of residues from 2224 to 2511 (SEQ ID N: 5).

 FIG. 23. Illustrates the location of the locus of cholera enterotoxin in the chromosome of strain 1837 V. cholerae non-01 serogroup.

 FIG. 24. Illustrates the location of the cholera toxin locus of V. cholerae strain 1837 (first line), strain 1837.1 (second line), strain 1837.2 (third line) and strain CVD112 (fourth line).

 FIG. 25. Illustrates plasmids pLC13, pLC14, pLC15, and pLC16.

 FIG. 26. Scheme for inserting sequences encoding the B subunit of cholera enterotoxin and sequences encoding resistance to mercury in the nucleus of the chromosome in the hylA locus of strain V. cholerae 0139 1837.2.

 FIG. 27. Illustrates the recombination that takes place at the A. cholerae gAA locus in the generation of strain CVD112RN.

 FIG. 28 (A, B, C and D). Permeability analysis using wheat germ agglutinin - horseradish peroxidase (WGA-HRP), measured on rabbit ileum homogenate under exposure to culture supernatant of various V. cholerae strains. A, control medium; B, strain V. cholerae 395: C, strain V. cholerae 395N1; D, strain V. cholerae CVD101.

 FIG. 29 (A and B). The study of the frozen fraction from rabbit ileum tissue under the influence of the culture supernatant V. cholerae: A, intact ZO with numerous intersections (wedge-shaped) between M-threads, microvilli. B, damaged ZO from the ileum tissue under exposure to strain V. cholerae 395; the structure of reticulin, apparently, is simplified due to a rather significant decrease in the frequency of intersection of threads.

Detailed disclosure of the invention
The present invention claims V. cholerae strains, a method for their preparation and methods for their use. The inventive strains of Vibrio cholerae, suitable for use as vaccine strains, are purposefully transformed by recombinant DNA technology to obtain an avirulent strain without significant effects on other cellular components necessary to provide protective immunity. This attenuation was achieved by restriction enzyme digestion of plasmids carrying the corresponding V. cholerae sequences, with a specific complete deletion of the locus of the genes encoding the cholera enterotoxin, or a part thereof. Conjugative gene transfer of these plasmids carrying the deliberate cholera enterotoxin genes into virulent host cells of V. cholerae is carried out by genetic selection of in vivo recombinants, resulting in avirulent strains that are not completely or partially containing cholerogen. It should be understood that the methods of the present invention are suitable for the isolation of other deletion mutants of the virulent V. cholerae, or for the isolation of strains containing fully or partially deleted sequences, which are then reinserted into the V. cholerae cell nucleus.

 Virulent strain Vibrio cholerae N 16961 of serogroup 01 was used as a starting material for vaccines. Strain N 16961, as shown, causes both typical symptoms of enteroinfection and powerful protective immunity against subsequent infection [Levine, M.M. and others. Acute enteric, see earlier, 1981]. The chromosome region of the bacterial cell, which is found to be responsible for the production of cholera enterotoxin, was cloned into plasmid pBR325, after screening the V. cholerae HindIII DNA hydrolyzate using a heat-resistant E. Coli probe for the enterotoxin gene [Kaper et al., Amer. Soc., See previously; Kaper and others, Symposium, see earlier]. It was established that the chromosomal fragment of the HindIII cholera vibrio contains all the genes necessary for the production of cholerogen. Subsequently, this region of chromosomal DNA was analyzed and the locus of the enterotoxin gene was mapped [Kaper, J.B. and others. Lancet II, 1162 (1981)]. Restriction enzymes were used to excise DNA fragments containing these genes, and the insertion of a DNA fragment encoding a selectable marker (for example, ampicillin resistance) was performed by ligation (crosslinking). Then, the ampicillin resistance gene with the flanking DNA sequences of vibrio was cloned into a derivative of plasmid pRK290, which can be used to transfer DNA from E. Coli to V. cholerae. The resulting plasmid pJBK55 was transferred from E. Coli K-12 to strain V. cholerae N 16961 by conjugation.

The obtained strain V. cholerae N 16961 (pJBK55) (Ap ^r ) contained a region with an intact chromosome in the extrachromosomal plasmid containing the same region with deleted genes for enterotoxin and replaced with genes encoding ampicillin resistance (See Fig. 1). At a low repetition rate, possibly at 10 ⁶ to 10 ⁸ , identical regions that confine the chromosomal cholera enterotoxin genes and the extrachromosomal (plasmid) ampicillin resistance gene usually either exchange or cross-recombine, or undergo in vivo recombination such that in the DNA region containing the resistance gene, there is a replacement for the enterotoxin gene on the chromosome (Fig. 2). This rare result is selected by a test sample of a mixture of mutated and non-mutated cells according to the phenotype of cells that are able to act as a host for the introduction of a foreign plasmid [Ruvkun, GB and others. Nature, 289, 85 (1981)]. Plasmids are divided into AW groups, the elements of which cannot stably coexist with each other. For example, a plasmid of incompatibility group P cannot be stably retained in the same cell as another plasmid of group P (Inc P). Thus, Inc P plasmids, such as R702, which are responsible for sulfonamide resistance, cannot be stored in a cell that has another Inc P plasmid, such as PRK 290, pJBK45, or pJBK55. Therefore, plasmid R702 can be stably maintained in a strain whose ampicillin resistance gene is recombined into the chromosome, rather than in the strain where the Inc P plasmid (e.g. pJBK55) is replicated extrachromosomally. When crossing E. coli strain containing Inc P-R702 plasmid (sulfide-resistant) and plasmid pJBK55 V. cholerae (ampicillin-resistant) and selection on V. cholerae, which are resistant to both ampicillin and sulfonamide, cell colonies are isolated in which resistance to sulfonamide is provided outside the chromosome by plasmid p702, and to ampicillin is achieved extrachromosomally by replacing cholerogen with the ampicillin resistance gene (Fig. 3).

 One such strain designated V. cholerae JBK56 was isolated and, when tested as a producer of enterotoxin, it was found to be avirulent.

 The final product of the vaccine strain, JBK70, was produced by replacing the ampicillin resistance gene, the therapeutically used antibiotic, with the mercury resistance gene. This replacement was carried out by cloning the mercury resistance gene directly to the locus of the ampicillin resistance gene of plasmid pZBK55, which leads to the inactivation of the ampicillin resistance gene and mercury resistance. The resulting plasmid, pJBK66 was also incompatible with R702 and was transferred to the plasmid pJBK56 V. cholerae.

 A mutant was then selected in which the mercury resistance gene was recombined into the chromosome using Inc P plasmid R702 and selecting V. cholerae, which were sensitive to ampicillin and resistant to mercury and sulfonamide.

 A spontaneous deletion mutant formed from pR702 was then selected. The final mutant, JBK70, did not show virulence and was only resistant to mercury.

 The strain of the vaccine JBK70 V. cholerae is one of the Inaba serotype. Another major serotype of V. cholerae is the Ogawa serotype. It is hoped that a vaccine prepared from one serotype of a cholera vibrio will induce protective immunity against another serotype (34). Live vibrio vaccine can also be prepared from the Ogawa serotype and tested with volunteers (Levine, M.M. et al. Enteric Acute, see earlier (1981). The exact mutations obtained in the Inaba strain JBK56 V. cholerae were then reproduced in strain E7946 by targeted transfer of a region of the chromosome containing ampicillin resistance to the cholerogen locus in JBK56 in E7946 through genetic recombination mediated by P, the sexual factor V. cholerae [Parker, C. and others, see earlier]. P-factor, which incompatible with Inc P-plasmid, then transferred to JBK56 followed by cross-breeding with a rifampin-resistant mutant E7946. By selecting a mutant that was resistant to ampicillin and rifampin, an Ogawa serotype isolate with completely deleted enterotoxin genes was obtained.

 In the case of insufficient antibacterial immunity for protection, the antitoxic component can be added by introducing back genes to produce B and not A cholera enterotoxin subunits. This was achieved by cloning the B subunit gene into the pMS9 cloning vector. The resulting plasmid, pJBK51, produces high levels of the B-subunit, and then it was introduced into the non-virulent vaccine strain V. cholerae JBK70 to obtain the attenuated vibrio vaccine of strain JBK70 (pJBK51), not capable of producing the A-subunit.

 The vaccine strains of the present invention are obtained essentially from V. cholerae N 16961, related to the Inaba serotype. It should be understood that other strains or other biotypes and serotypes can be used instead of strain N 16961 to obtain vaccine strains having specific deletions at the ctx locus of the gene or genes, or at other sites on the V. cholerae chromosome. Since this isolate object from such vaccine strains should mimic the infection process without causing pathologies, the site-directed mutagenesis of virulent strains, as described in this application, provides significant immune potential for preventive protection against cholera infection.

 For example, the authors of the present invention received another vaccine strain CVD101 V. cholerae, characterized by deletion of most of the A subunit gene in 2 copies from the ctx gene. When constructing the strain CVD101 in accordance with the general principles mentioned earlier, for example when constructing JBK70, the resulting strain CVD101, except that it did not have resistance markers, did not require correction. At the final stage, when isolating the second and found in vivo recombinant, the selection procedure for antibiotic sensitivity, for example, sensitivity to tetracycline, was included, however, the parent strain was inserted into the locus of the A subunit gene from the tetracycline resistance CT gene.

 It must be understood that such antibiotic sensitivity is another example of a selectable marker gene.

The production of vaccine strains can be accomplished by a wide variety of methods, including: Vibrio cholerae is subcultured from uterine cultures on cardiac extract agar (BHIA) and incubated at 37 ^° C overnight.

Identity is checked using group- and type-specific antiserum, and twenty to thirty cell colonies are suspended in broth from a cardiac extract. Pre-incubated agar plates with a cerebral traction are inoculated with a BHI suspension. After incubation for 5-6 hours, a supernatant culture is collected from each plate with the addition of 5 ml of sterile physiological saline, buffered with pH = 7.2 ± 0.1. The collected microorganisms are centrifuged refrigerated at a speed of 750 rpm / 10 min, resuspended and washed twice with four times the initial volume of culture. The resulting suspension is standardized spectrophotometrically and diluted to obtain the dose of microorganisms necessary for immunization (approximately 10 ⁶ , which varies depending on the results of studies involving volunteers). Re-plating is performed on a four-well inoculum plate before and after inoculation to confirm the inoculum content. The final inoculum is analyzed with Gram stain and agglutinated with homologous antiserum before administration.

The Vibrio cholerae strains of the present invention can be administered orally. Two grams of NaHCO _{3 are} dissolved in five ounces of distilled water. Volunteers drink four ounces of an aqueous solution of NaHCO ₃ ; after one minute, volunteers receive vibrios suspended in the remaining ounce of NaHCO ₃ / water. Volunteers before and after vaccination do not accept anything.

 Regarding the safety of this vaccine strain, it should be noted that it "does not switch back" to a toxicogenic state (that is, it produces an intact cholera enterotoxin), which can cause disease. To determine the activity of enterotoxin, two main methods of analysis are used: analysis using adreno-sensitive VI-type cells [Sack, D. A. and others. Infect. Immun. 11, 334 (1975)] and enzyme-linked immunosorbent assay (ELISA) [Sack, D.A. and others. J. Clin. Micro., 11, 35 (1980)]. The vaccine strain (JBK 70) was repeatedly tested using these two assay methods and a negative reaction was detected each time. A much more significant point in these analyzes, however, is that they were carried out for the presence of cholerogens.

 A radioactive label can be introduced into the DNA of the cholera enterotoxin genes and used as a site-specific probe to recognize other cholera enterotoxin genes in the strain, in accordance with the Southern method, E. M. J. Mol. Bio 98, 503 (1975). In a test sample using this method, the vaccine strain of the invention does not contain any detectable genetic material that would include cholera enterotoxin. The vaccine was also tested on model mice "suckers" in accordance with the method of Baselski, V. and others. Infect. Immun., 15, 704 (1977). After repeated (ten) serial tests, no accumulation of serous fluid was noted, which indicates the absence of signs of the disease. As expected, the JBK70 strain was capable of colonizing the intestines of a mouse sucker.

 To avoid unwanted side effects of vaccine strains, such as diarrhea with vomiting, seizures, and other symptoms of the disease, vaccine strains can also include a second restriction DNA fragment with a deletion of the sequence encoding the toxin (zonula occludens) toxin (ZOT).

 The Vibrio cholerae culture includes a Vibrio cholerae strain containing a first restriction DNA fragment removed to ensure avirulence and colonization of the small intestine of the host animal and a second restriction DNA fragment with a deletion of the tight contact zone (zonula occludens) toxin (ZOT) to attenuate residual diarrhea in the host animal. The first DNA fragment with a deleted sequence can encode V. cholerae or part of it, for example, the A subunit. One isolated deletion mutant has a deletion at the ctx gene locus bounded by AccI restriction sites and a deletion at the zot gene locus. Another isolated deletion mutant contains a deletion at the ctx locus of the gene determined by the XbaI and ClaI restriction sites, and a deletion at the zot gene locus determined by the StuI and AccI restriction sites.

A method for isolating such deletion mutants of Vibrio cholerae comprises the steps of:
(a) constructing a first plasmid comprising the flanking DNA sequences of a Vibrio cholerae containing one or more deleted fragments at the restriction endonuclease site and a gene for a selectable foreign marker, crosslinked with said flanking sequences to replace and insert them at the site of said deleted fragment, wherein the sequences are long enough to promote recognition of in vivo recombination;
(b) crossing a virulent strain of Vibrio cholerae with a first microorganism carrying the first plasmid;
(c) selecting a Vibrio cholerae expressing a first selectable marker;
(d) crossing the product selected in step (c) with a second microorganism carrying the second plasmid with a second selectable marker, said second plasmid being incompatible with the first plasmid;
(e) selecting a Vibrio cholerae expressing a first selectable marker and a second selectable marker;
(f) constructing a third plasmid containing the flanking DNA sequences of Vibrio cholerae from one or more restriction enzyme-deleted fragments homologous to those described in step (a) but characterized by the absence of a selectable foreign marker;
(g) crossing the selected product in step (e) with a third microorganism carrying the third plasmid described in step (f); and
(h) selecting a Vibrio cholerae no longer expressing said first selectable marker.

 This method can only be used for strains of the ZOT phenotype or to obtain a derivative of the ZOT strain from any strain with a deletion of cholera enterotoxin genes.

 Another culture of Vibrio cholerae includes a strain of Vibrio cholerae strain having a region of chromosomal DNA with a deletion of cholera enterotoxin and toxin of the zone of close contact (zonula occludens) toxin ZOT.

A method for isolating such deletion mutants of Vibrio cholerae includes the steps of (a) constructing a plasmid containing the DNA sequences of Vibrio cholerae encoding the genes of the cholera enterotoxin and enterotoxin of the tight contact zone (zonula occludens) and the gene of the selectable foreign marker, where the plasmid is not capable of replication in Vib outside the chromosome;
(b) crossing the microorganism carrying the plasmid with a virulent strain of Vibrio cholerae containing the indicated sequences for the promotion of recognizable in vivo recombination:
(c) screening for Vibrio cholerae expressing said selectable marker;
(d) growing the product isolated in step (c) in the absence of a selective factor;
(e) screening for Vibrio cholerae no longer expressing such a selectable marker; and, as a consequence of this, having a region of chromosomal DNA, with a deletion of the genes of cholera enterotoxin and enterotoxin of the tight contact zone (zonula occludens). Stage (b) may include manipulating:
b) crossing the microorganism carrying the plasmid with a virulent strain of Vibrio cholerae containing the indicated DNA sequences inserted between flanking identical copies of the second sequence, for example, RS1 sequence elements of sufficient length to promote recognition of in vivo recombination.

 Vibrio cholerae deletion mutants of the present invention are suitable for immunization against cholera.

 The new toxic factor disclosed in the present invention, obtained on the basis of V. cholerae, increases the permeability of the small intestine mucosa due to the impact on the structure of the intracellular tight contact zone or zonula occludens (ZO) (transcellular ion transfer path). The degree of production of this factor produced by V. cholerae closely correlates with the results of studies of its enteropathogenicity conducted with the participation of volunteers. If the mechanism of normal metabolism in the small intestine is violated through the transcellular pathway, this factor is probably responsible for the manifestation of residual diarrhea induced by deletion mutants of the ctx gene of V. cholerae and may contribute to the development of severe cholera diarrhea, which differs from other diseases accompanied by intestinal disorders.

Studies have been conducted on changes in the function of the small intestine under the influence of three strains of V. cholerae, one wild-type and two attenuated vaccine strains. Strain V. cholerae 395, a classic biotype, Ogawa serotype, is a highly virulent strain whose characteristics were studied in large-scale studies conducted at the Vaccine Development Center with the participation of volunteers. This strain causes diarrhea with a total average stool volume of approximately 5.5 liters (ranging from 0.3 to 4.4 liters) in more than 90% of volunteers who received it at a dose of 10 ⁶ microorganisms [Levine, M.M. and others, Infect. lmmun. 56, 161-167 (1988)]; [Levine, MM, Cholera and Related Diarreas, 195-203] (Karger, Basel, 1980). Cholera diarrhea is mainly due to the enzymatic effect of the A subunit of cholerogen on the mucosa of the small intestine. The cholera enterotoxin A subunit encoded by the ctx gene stimulates adenylate cyclase and leads to the secretion of plasma network fluid into the lumen of the small intestine. Gill DM Adv. Cyclic Nucleotide res., 8, 85-118 (1977). The vaccine strain V. cholerae CVD101 is a mutant of strain 395 with a deletion of the ctx gene, in which 94% of the DNA sequences encoding the A ₁ peptide of cholera enterotoxin were deleted. The surprising fact, however, was that although CVD101 no longer produced active cholera enterotoxin, this strain caused mild to mild diarrhea (average stool volume was 0.9 L, varying from 0.3 to 2.1 L) in 54 % of volunteers who received this microorganism. The second mutant 395, the vaccine strain 395 N1, obtained by Mekalanos gene technology, and others. Nature, 306, 551-557 (1983), lacks, according to our calculations, approximately 77% of the DNA sequences encoding the A ₁ peptide. In contrast to strain CVD101, 395N1 caused mild diarrhea (stool volume 0.3 l) in only 1 out of 21 volunteers (P = 0.002, compared with 13 out of 24 volunteers with diarrhea receiving CVD101). [Herrington, DA and others. J. Exp. Med. 168, 1487-1492 (1982)]. Since these strains were similar in their ability to colonize the small intestine, the authors of the present invention believe that this strain CVD101 produces a secretogenic factor that is poorly expressed or not expressed at all when exposed to 395N1 and that this factor is responsible for diarrhea observed in volunteers. receiving vibrio strain CVD101.

 These strains were studied using a rabbit small intestine tissue preparation placed in the Ussing chamber, a traditional method for studying the processes of active transport through the mucosa of the small intestine. V. cholerae culture supernatants were added to the tissue preparation and the potential difference (PD) and short circuit current (Isc) were measured. PD means EMF measured on the mucous side relative to the serous side of the tissue under investigation and Isc is the current required to set PD in state 0. As a result of these measurements, the tissue conductivity (Gt) was calculated in accordance with Ohm's law: Isc = PD • Gt . Initially, the effect of the culture supernatants of wild-type strain 395 on these parameters was first investigated using a non-inoculated culture medium added to compatible ileum tissue of the same animal as a negative control. In FIG. 13A shows the obtained variations of the values of Isc and Gt. The initial peaks in Isc and PD, which appeared both in the negative control and in the test samples, are most likely due to the combined transport of Na and nutrients entering the culture medium. In the control, a return to the initial values of Isc and PD was observed after about an hour, and then the values of Isc, PD, and Gt remained unchanged throughout the remaining time of the experiment. In contrast, tissue preparations exposed to culture supernatants of strain 395 showed a significant increase in Gt, reaching a maximum value after 2-hour incubation. In the test samples, there was not a single case of a return of the Isc value to its initial values, but a steady state period was noted in the interval between 40 and 60 minutes. Since the Isc value corresponds to PD • Gt and the observed PD values after 60 minutes did not differ from the initial values (data not shown), a significant increase in the Isc value in tissue preparations treated with strain 395 during this period of time can only be caused by an increase in the Gt value. (See Fig. 13A for 60 min.) (12). After 60 min, the Isc value began to rise again with an increase in PD in tissues treated with strain 395. This second phase apparently reflects the effect of cholera enterotoxin on ion fluxes, since the purified CT increases Isc in rabbit ileum after a lag time of at least 40 minutes. These data indicate that there are two factors expressed by strain V. cholerae 395, which can change the mechanism of ion transport in Ussing chambers. One such factor, cholera enterotoxin, causes a significant increase in the values of Isc and PD, starting approximately 60 minutes after the addition of the culture supernatant, while the second factor induces an instantaneous increase in tissue conductivity, which can be observed within 20 minutes after the addition of the supernatant culture.

 Variations of Gt induced by culture supernatants of attenuated V. cholerae CVD101 and 395N1 strains were another aspect of the study. Strain CVD101 caused an instant increase in Gt, which is comparable with the results obtained in the case of strain 395 (Fig. 13B). In contrast, strain 395N1 did not cause an instant increase in Gt; the variation in Gt in tissue samples treated with strain 395 N1 was similar to the results obtained in the control with an incompatible broth culture and significantly lower than that observed with strains 395 and CVD101 during almost 100-minute incubation. After this period, the change in Gt in tissues exposed to strains 395, CVD101 and 395N1 was similar. These results indicate that strain 395N1 produced a lesser or less active form of the factor responsible for this increase in Gt.

 Variations in transepithelial electrical conductivity are a clear reflection of changes in tissue permeability through the intracellular space, since the resistance of the plasma membrane is relatively high. Due to the fact that ZO is the main barrier in this transcellular pathway and variations in the Gt value are the most accurate criterion for evaluating the ZO function, changes in the ZO morphology induced by the culture supernatants of V. cholerae strains 395, CVD101 and 395N1 were studied. With the introduction of a low molecular weight electron-dense marker, such as agglutinin from germ wheat and radish peroxidase (WGA-HRP) on the mucosal side of the epithelial layer, it usually does not extend beyond the ZO zone [Alberts, B. et al., Molecular Biology of the Cells, 2nd edition, 1989]. WGA-HRP was added to the mucosal side of the small intestine treated with culture supernatant of strains 395, CVD101, 395N1 or uninoculated broth as a control for 60 minutes. Tissue preparations treated with uninoculated culture medium were impervious to WGA-HRP, while tissue preparations treated with strains 395 and CVD101 showed staining in the transcellular space. In tissue preparations exposed to the culture supernatant of strain 395N1, no changes were noted, since the intracellular space remained closed enough to prevent the passage of WGA-HRP into it (Fig. 14). These results were confirmed and refined using the electron microscopy method of the frozen fraction, where the number of filaments lying parallel to ZO are in correlation with the data on transepithelial electrical conductivity. In tissue preparations exposed to the culture supernatant, a mixture of the changed ZO and the non-transformed ZO was observed, accompanied by a simplification of the structure of the thread. The strands located perpendicular to the relatively long axis of ZO turned out to be predominantly lost, thereby reducing the number of jumpers (intersections) between the strands. The degree of change in the morphology of ZO exposed to the culture supernatant of each strain was evaluated by measuring the density of the bridges between the strands. As can be seen from FIG. 14, in tissues exposed to the culture supernatant of strains 395 or CVD101, there was a significant decrease in the number of filaments and complexity of the ZO reticular tissue compared to tissues treated with uninoculated broth medium or culture supernatant of strain 395N1.

 Changes in the morphology of ZO induced by strains 395 and CVD101 are in parallel with the increase in tissue conductivity caused by these strains. The function of the ZO tight contact zone of the small intestine is to regulate the transcellular pathway and to limit or prevent the diffusion of water-soluble molecules through the intracellular space back into the intestinal lumen. This diffusion mechanism is triggered by concentration gradients through transepithelial transport processes. As a result of the restructuring of the mechanism of the transcellular pathway, the intestinal mucosa becomes more permeable, and water, Na and Cl leak into the intestinal lumen, thereby leading to diarrhea.

 Restructuring in the transcellular pathway induced by V. cholerae 395 and CVD101 is specific for the small intestine; the replacement of the tissue sample of the small intestine of the rabbit with the tissue of the cecum did not lead to any change in the value of Gt induced by the supernatant of the culture strain 395 (data not shown). This fact confirms for the first time the presence of a bacterial factor, which is capable of weakening the closure of the tight contact zone in the intact tissue of the small intestine and can be a new mechanism that causes bacterial diarrhea. The Clostridium difficile exotoxin A subunit, influenza viruses and vesicular stomatitis (VSV) viruses are reported to weaken the tight contact area in tissue monolayer culture, but no activity was reported anywhere on the intact tissue or their association with diarrhea.

 Thus, V. cholerae strains 395 and CVD101 produce a factor that may be responsible for the occurrence of diarrhea observed in volunteers receiving deletion mutants of the V. cholerae ctx gene. Diarrhea caused by these ctx mutants is comparable to the test results of numerous enterotoxicogenic E. Coli strains. This toxin-secreting factor, which the authors of the present invention called ZOT as the enterotoxin of the tight contact zone (zonula occludens toxin), induces an increase in the Isc value and tissue conductivity, which is not associated with the effect of CT on ion fluxes, in the initial phase. This increase in Gt is associated with a weakening of the closure of the tight contact zone, an effect with a quick reversal to its original state after removal of the culture supernatant (Fig. 15). The rapid reversal of this effect is in complete opposition to the long-term effects of cholera enterotoxin. These results are not comparable with previously made unexplained observations from Fields, et al., J. Clin. Invest., 51, 796-804 (1972), which noted an instant increase in the Isc value caused by raw and not yet purified cholera enterotoxin preparations. These results may be comparable to those of Nishibuchi and others, lnfect. Immun., 40. 1083-1091 (1983), which noted an early phase of fluid accumulation (FA) not related to delayed CT-induced FA in sucker mice injected with V. cholerae. The ability of a negatively reacting V. cholerae enterotoxin to cause diarrhea in volunteers correlates with the results of ZOT production by two attenuated strains obtained from the same parent cell line; strain CVD101 (causing diarrhea) produces ZOT, while strain 395N1 (not inducing diarrhea) causes ZOT production with little or no detectable activity.

Another culture of Vibrio cholerae includes a strain of Vibrio cholerae strain having a chromosomal DNA region with a deletion of the cholera enterotoxin locus and the enterotoxin of the ZOT tight contact zone and having an insertion of the mercury resistance gene and the DNA sequence encoding the Vibrio cholerae enterotoxin B subunit. A method for isolating such deletion mutants of Vibrio cholerae comprises the steps of:
(a) constructing a plasmid containing the DNA sequences of Vibrio cholerae encoding the genes of the cholera enterotoxin and enterotoxin of the tight contact zone (zonula occludens) and the gene of selectable foreign marker, where the plasmid is unable to replicate to Vibrio cholerae outside the chromosome;
(b) crossing the microorganism carrying the plasmid with a virulent strain of Vibrio cholerae containing said DNA sequences inserted between flanking identical copies of a second sequence having a sufficient length for promotion of recognized in vivo recombination;
(c) screening for Vibrio cholerae expressing said selectable marker;
(d) growing the product isolated in step (c) in the absence of a selective factor;
(e) screening for Vibrio cholerae no longer expressing such a selectable marker; and, as a consequence of this, having a region of chromosomal DNA, with a deletion of the genes of cholera enterotoxin and enterotoxin of the tight contact zone (zonula occludens).

(f) constructing a second plasmid containing the mercury resistance gene and a DNA sequence encoding the Vibrio cholerae enterotoxin B subunit and a second selectable foreign marker gene, said plasmid being incapable of replication outside the chromosome in Vibrio cholerae, and a sequence having sufficient the length for promotion recognized in vivo recombination, limit the region of said mercury resistance gene and the DNA sequence encoding the Vibrio cholerae enterotoxin B subunit;
(g) crossing the microorganism carrying said second plasmid with said Vibrio cholerae disclosed in step (e) containing sequences homologous to said sequences of sufficient length to promote recognizable in vivo recombination;
(h) selecting a Vibrio cholerae expressing said second selectable marker;
(i) growing the product selected in step (h) in the absence of a second selected factor;
(j) selecting a Vibrio cholerae not expressing more than a second selectable marker; and
(k) screening said Vibrio cholerae disclosed in step (j) on Vibrio cholerae that contain a mercury resistance gene and a DNA sequence encoding the B subunit of cholera enterotoxin and have a region in the chromosomal DNA with a deletion of the dense cholera enterotoxin gene and enterotoxin contact (zonula occludens).

 This method of isolating deletion mutants of Vibrio cholerae having a region of chromosomal DNA sequences encoding a cholera enterotoxin and deletion of the tight contact zone gene and insertion of the mercury resistance gene and the DNA sequence encoding the Vibrio cholerae B subunit can be used in step (f) revealing flanking sequences of sufficient length containing a gene that can be removed without affecting the colonizing ability and immunogenicity of Vibrio cholerae. An example is the hemolysin gene. Strain CVD110 V. cholerae, constructed in accordance with the proposed method, have a chromosomal DNA region with sequences encoding the A and B subunits of cholera enterotoxin and deletion of the enterotoxin of the tight contact zone (zonula occludens), and also have a mercury resistance gene and a DNA sequence encoding the B-subunit of Vibrio cholerae enterotoxin inserted at the site of the hemolysin gene. Other examples of sequences of sufficient length include his gene (Hone, Microbial Pathogenesis 5, 35 pp. 407-478 (1989)) and nanH gene (Vimr, J. Bacter., 170, pp. 1495-1504 (1988)) .

 The inventive strain CVD112 has a region with a deletion of the entire nucleus of cholera enterotoxin, which includes the genes sulfur, ace, zot, orfU, and ctxAB, as well as a deletion of RSI elements and attRS1 sites [Pearson et al., Proc. Natl. Acad Sci. (USA), 90, pp. 3750-3754, 1993] together with a locus in the ctx gene chromosome. In addition, this strain has sequences encoding a sufficient portion of the B-subunit of cholera enterotoxin to ensure immunogenicity, re-inserted into the chromosome of the vibrio. Insertions additionally introduced into the chromosome are DNA sequences that cause resistance to heavy metals, such as mercury.

 Another V. cholerae non-01 serogroup strain of the present invention, namely CVD112 RM, has identifying characteristics of the above non-01 serogroup strain, and furthermore has a deletion at the Vibrio cholerae recA locus, as a result of which the resulting strain loses the gene in homologous recombination.

 In the examples that further illustrate the present invention, any of the methods, reactions, and isolation methods are well known to those skilled in the art. All enzymes, unless otherwise specified, are available from one or more commercial sources, for example. New England BioLabs - Beverly, Massachusetts; Collaborative Research - Waltham, Massachusetts; Miles Laboratories -Elkhart, Indiana; Boehringer Biochemicals Inc., Indianapolis, Indiana; and Bethesda Research Laboratory - Rockville, Maryland, which are listed here as a few representatives. Buffer solutions and reaction conditions for restriction enzyme hydrolysis are used according to the recommendations provided by the manufacturer for each enzyme, unless otherwise specified. Partial hydrolysis by restriction enzymes is carried out using a lower concentration of the enzyme, which must be determined in advance from trial experiments for each batch of enzymes. A standard technique for other enzymatic reactions, gel electrophoresis separation methods, and E. coli transformants can be found in Methods in Enzymology, vol. 68, edited by Ray Wu, Academic Press (1979). You can also refer to another well-known source - Maniatis, T. and others. Molecular Cloning, Cold Spring Harbor (1982). Bacterial cells were grown according to the methods described by Miller, in Experiments on Molecular Genetics, Cold Spring Harbor Laboratory (1972). Vibrios of cholera were propagated according to the general methods described by Lennett, E.A. and others, eds. Manual of Clinical Microbiology, 3rd Edition, American Society for Microbiology, Washington in (1980). E. Coli and V. cholerae were crossed according to the general procedure described by Johnson, Steven R. and others. J. Bact., 137, 531 (1979); and Yokata, T. and others, J. Bact., 109, 440 (1972).

 The strains of the present invention were deposited with the American Type Culture Collection, for example, located in Rockville, Maryland, prior to the filing of this application. The strains of V. cholerae JBK56, V. cholerae JBK70, V. cholerae N1696, V. cholerae JVK70 (pJBK51), V. cholerae 395 of the Ogawa serotype, strains CVD101, CVD109 V. cholerae E7946, and E. Coli SM10 lambda pirCV were deposited V. cholerae CVD110, and E. Coli SY327 lambda pir (pCVD622.2B), which have access number ATCC 39,317, 39,318, 39,315, 39,316, 39,541, 39,540, 55,057 (deposited on June 4th, 1990), 55,056 (deposited on 4 June 1st, 1990), 68,335 (deposited on June 5th, 1990), 55188 (deposited on June 3rd, 1991), and 68630 (deposited on June 3rd, 1991), respectively.

Example 1
Construction of a plasmid with a selectable marker gene inserted to replace cholerogens.

 Plasmid JBK16 contains a PstI-BgIII fragment of length 4, etc. bases from a chromosome containing enterotoxin genes. Enterotoxin genes are restricted to AccI sites and contain an internal AccI site. JBK16 was completely hydrolyzed with AccI and AccI restriction enzymes, and the resulting fragments containing enterotoxin genes were separated from the remaining plasmid.

 Overlapping of the plasmid residues or the sticky ends of the AccI site turned into blunt ends, filling the gap with a Klenov fragment obtained from E. Coli polymerase polymerase (i.e., single-stranded DNA remaining after AccI hydrolysis received double-stranded with short ends). The gene encoding resistance to ampicillin was purified from plasmid pREG153 (pREG153 is a derivative of plasmid pREG151 [Weiss, A. et al. J. Bact., 152, 549-552], modified by replacing the trimethoprine resistance gene with the ampicillin resistance gene and attaching cos- sequences) and the sticky ends were filled with a gap as described above. The resulting fragment was then ligated with the DNA of the vibrio, as a result of which the resistance genes for AP were located exactly in the same place as the deleted enterotoxin genes flanked by the same DNA sequences of the cholera vibrio. The resulting plasmid was constructed as pJBK21 (FIG. 4) containing a region with a deletion of a cholera enterotoxin and an ampicillin resistance gene.

Example 2
Introduction of flanking homologous sequences, followed by conjugative gene transfer in V. cholerae.

 To ensure specific insertion into the chromosome from the deleted sequence in pJBK21, a DNA fragment of approximately 7000 base pairs in length was ligated with each end of the PstI-Bg1II fragment from plasmid pJBK21. (The probability of the occurrence of a homologous recombination event increases with increasing length of flanking homologous sequences). To achieve this, a fragment of approximately 18 kb was cloned from the chromosome of strain N 16961. This clone designated as pJBK44 contains a fragment of the 4 kb PstI-Bg1II enterotoxin gene limited to a flanking DNA sequence of approximately 7 t .by. at both ends (see Fig. 5). Plasmid pJBK21 was partially hydrolyzed at the PstI site, as a result of which only one of the Pst sites was excised (an additional Pst site was inserted within the locus of the ampicillin resistance gene) after restriction enzyme digestion of the Bg1II site with isolation of the Pstl-Bg1II fragment 4 kb in length containing a region with a deletion of the enterotoxin gene and a region of resistance to Ap.

Plasmid pJBK44, containing a vibrio DNA fragment approximately 18 kb in length, was partially digested with restriction enzyme at the Bg1II site, resulting in only one of the 4 Bg1II sites being excised. After this partial hydrolysis, complete hydrolysis was carried out by restriction enzyme of the PstI site and the obtained fragments were separated by electrophoresis through 0.3% agarose gel. The isolated fragments were then purified and analyzed, and only one fragment, as shown by the analysis, contained the complete pJBK44 sequence with the exception of the 4 kbp PstI-Bg1II gene fragment of the cholera enterotoxin. (see Fig. 5.). This fragment, representing the flanking DNA sequence, was then mixed with ligation with the Pstl-Bg1II fragment from plasmid pJBK21 containing the ampicillin-resistant gene. The resulting plasmid, PJBK54, contained a Vibrio chromosomal DNA sequence of approximately 17 kb in length with the ampicillin-resistant gene replaced by the deleted enterotoxin genes. The modified region in the chromosomal DNA was then cloned into a plasmid, which can be easily activated in V. cholerae. Plasmid pRK290 [Ditta, G. et al. Proc. Nat. Acad Sci. 77, 7347 (1980)] refers to plasmids of the incompatibility group P and has only one EcoRI site into which the plasmid pJBK54 was cloned (Fig. 6). The resulting plasmid pJBK55 was then crossed with strain V. cholerae N16961 using the conjugative plasmid pRK2013. As a result, V. cholerae N 16961 (pJBK55) (Ap ^r -ampicillin-resistant phenotype) was obtained.

Example 3
In vivo recombination
Mutant cholerogens, after conjugative gene transfer, as described in Example 2, were now present in an extrachromosomal state in strain V. cholerae N 16961 (see Fig. 1). At a very low frequency of repeatability (possibly from 10 ^-6 to 10 ^-8 ), homologous flanking DNA sequences form a conjugation pair on the chromosome (see Fig. 7). This rare result leads to the replacement of the region with deleted enterotoxin on the plasmid by ctx genes on the chromosome. To select this rare result, the plasmid incompatibility phenomenon was used [Ruvkin, GB, see earlier]. Plasmids can be divided into incompatibility groups from A to W, based on their ability to stay in a stable state together in the same cell. If two plasmids cannot stably exist together in the same cell, they are incompatible and belong to the same incompatibility group, possibly because they use the same replication mechanism in the cell. With the selective use of the antibiotic resistance factor present on one plasmid, and not on the other, it is possible to select the type of these two incompatible plasmids that will be compatible. Plasmid pJBK55, due to what is derived from plasmid pRK290, also belongs to the P group (Inc). R702 also belongs to group P (Inc) and contains a marker of resistance to kanamycin, tetracycline, sulfonamide, and streptomycin, but not to ampicillin. When pR702 (Su ^R ) was crossed in strain N16961 (pJBK55) (Ap ^r ) and selected on a medium containing both ampicillin and sulfonamide, cells were selected in which the ampicillin resistance marker was inserted into the chromosome and the sulfonamide resistance marker remained on plasmid R702, since pR702 and pJBK55 are incompatible (see FIG. 2). The resulting JBK56 strain (Fig. 3) contained an ampicillin resistance marker and enterotoxin, a negative reaction detected in the analysis using adreno-sensitive Y-1 cell types and using Gm ₁ in an enzyme-linked immunosorbent assay (ELISA). In addition, when chromosomal DNA was hybridized with DNA probes containing a clone of the cholera enterotoxin genes, JBK56 detected a negative reaction, which confirms the assumption of a complete deletion of enterotoxin genes.

 The antibiotic resistance factor encoded on R702 was eliminated (deleted) by selection of a spontaneously corrected copy of the plasmid lost in the indicated plasmid (this event occurred at a frequency of approximately 1 in 2000).

Example 4
The elimination of breeding marker from example 1
To eliminate ampicillin resistance factor, a plasmid derived from pJBK55 plasmid was constructed in which genes encoding mercury resistance (Hg ^r ) from R100 were cloned at the PstI site of the Ap gene, thus inactivating the insertion of ampicillin resistance factor. This derivative plasmid was then crossed in V. cholerae JBK56, in the presence of pR702, and selection was performed as previously described for the Hg ^r marker, Ap ^s V. cholerae. The final product, strain V. cholerae JBK70, was sensitive to all proven antibiotics, resistant to mercury, and toxin-negative by phenotype. In his chromosomal DNA, there was no detectable hybridization ability with DNA probes containing cholera enterotoxin genes. Briefly, DNA sequencing of the full chromosome, JBK70, does not seem to have significant differences from the parent strain N16961, with the exception of deletion of the enterotoxin genes and insertion of the mercury resistance gene and inactivation of the ampicillin-resistant gene. Such a strain does not reverse to a toxicogenic state, because the enterotoxin genes underwent not only mutations, but were completely lost (deleted).

Example 5
Conjugative gene transfer to provide antitoxic immunity.

In the case when both antibacterial immunity and antitoxic immunity are necessary for synergism, a new plasmid can be constructed from plasmid JBK70 to produce only the B subunit of cholera enterotoxin. For this purpose, an enterotoxin gene product was obtained that produces only the B subunit and in which the A subunit genes are lost (Fig. 8). The HpaII fragment excised from the pJBK16 plasmid containing the B structural gene was cloned into a phage cloning vector, M13mp7, inserted between the BamHI site and EcoRI on either side of the gene (Fig. 8). The fragment flanked again by BamHI sites was cloned into plasmid pMS 9, which contained a very active trp promoter. Placing B-subunit genes in the region of transcription regulation of a powerful promoter allows for high production of B-antigen. Of the investigated clones, approximately 50% did not synthesize any antigen. This established fact reflects two possible orientations of the cloned gene insertion - one transcriptionally active, the other the opposite. One plasmid derived from pJBK51 that produced the B subunit was crossed in the Vibrio cholerae JBK70 strain and was found to produce a significantly higher amount of B antigen than the parent strain N 16961, resulting in the JBK70 mutant (pJBK51). Other only B-secreting mutants were obtained using various promoters, including the P ₁ promoter, and any significant difference in their in vivo expression could be evaluated using appropriate models.

Example 6
Colonization of the small intestine of a newborn mouse using strain JBK70 without reversion to toxicogenicity.

Sucker mice (weighing 2.0-3.5 g) were weaned from their mothers and held on an empty stomach for about 3 to 8 hours. Then, four of them were injected with the inoculum for 1 day per os (through the mouth) into the abdomen using a 22-gram microsyringe needle. The inoculum contained approximately 10 ⁸ CFU (colony forming units / mouse) of JBK70 strain in a volume of 0.05 ml to 0.1 ml. The inoculum was prepared in a cardiopulmonary broth almost according to the procedure described by Baselski, V. and others, see earlier. The inoculum also contained approximately 0.01% Evans blue. The presence of this dye in the abdomen of the animal, as could be seen through the walls of the abdominal cavity, indicated the proper delivery of the inoculum. The introduction of Evans blue was interrupted every other day in order to exclude the inhibition of JBK70 activity.

 Subsequent inoculations were performed with the mouse-in-mouse (MXM) method, or alternatively, mouse-on-tablet-in-mouse (MXMX), however, different procedures were necessary for preparing the inoculum compared to the Baselski vaccination schedule for 1 day.

To prepare the MXM inoculum, the cecum was dissected from the abdomen to the anus, subject to sterility. The sample was weighed, placed in a beaker with a homogenizer, and approximately 0.5 ml of cardiac extract broth was added. The resulting mixture was quickly triturated to a homogeneous state with a teflon pestle until complete tissue lysis. The resulting cell suspension was used to inoculate approximately 10 ⁸ CFU in each newborn mouse. The purity test was checked by bar-wiring on MEA agar plates (with meat extract) without the addition of Evans Blue dye.

To prepare the MXRXM inoculum, a sterile loop was used to transfer cells from the MEA agar plate to a cardiac extract broth. Approximately 10 ¹¹ CFU / ml was added to 1 ml of BHI broth to obtain a thick cell suspension. The mixture was brought to a homogeneous state, and a dose of 0.05-0.1 ml (approximately 10 ¹⁰ CFU) was inoculated per os in each newborn mouse. Evans blue dye was added.

For all series of vaccinations, mice were kept in beakers at room temperature 73-76 ^o F (Fahrenheit). The glasses were placed in a plastic box, which was loosely closed to maintain the temperature slightly above room temperature, 78 ^o F.

 As can be seen from the results, a sufficient number of infected cells were present in the intestine for vaccination of the next animal, when the cells were examined by hatching on MEA plates. Thus, the strain Vibrio cholerae JBK70 has the ability to colonize the small intestine of newborn mice. In addition, the accumulation of liquid content (FA) did not increase, since there was no significant increase in the FA index (FA index is greater than or equal to 0.065 - accumulation of seropositive fluid).

Example 7
Construction of strain V. cholerae CVD 101 with deletion of the restriction fragment at the locus of the gene encoding the A-subunit.

 Another classic strain selected for attenuation was Ogawa serotype 395 Vibrio cholerae strain (alternatively designated “395”), which, like strain N 16961, was tested in large-scale experiments involving volunteers, and which has been found to induce strong protective immunity [ Levine, M. M. Immunity to cholera as evaluated in volunteers, "in Cholera and Related Diarrheas: 43rd Nobel Symposium, Stockholm, 1978. (O. Ouchteriong & J. Holmgren, eds.) Basel: S. Karger, pp. 195-2-3 (1980); Levine, MM et al. Acute Enteric, see earlier (1981)]. The technique used to attenuate strain 395 did not differ significantly from that used used to attenuate strain N 16961 (as described previously in Examples 1-5). At the first stage, two copies of cholera enterotoxin strain 395 were cloned and mapped. As a result of Southern blot hybridization, 2 HindIII fragments of approximately 16 and 12 p in length were detected .O., Each of which hybridized with the cloned genes of cholera enterotoxin. These fragments were purified by agarose gel electrophoresis and then cloned into a hydrolyzed plasmid pBR325 with HindIII restriction enzyme treated with alkaline phosphatase (Fig. 9). The resulting recombinant plasmids containing enterotoxin genes were designated pCVD14 and pCVD15.

Plasmids pCVD14 and pCVD15 were then mapped to restriction endonucleases. As a result of mapping, an approximately 550 bp XbaI-ClaI fragment was found containing the complete base sequence of the A ₁ subunit, with the exception of the codons of the first 10 amino acid residues A ₁ . This XbaI-ClaI fragment was excised in vitro from both plasmids pCVD14 and pCVD15 by the series of manipulations shown in FIG. 10 by the example of plasmid pCVD15. As a result of the first partial hydrolysis of ClaI, a population of linear DNA molecules was obtained in which only one of the 5 ClaI sites was excised. In the next step, the ends of the linear molecules were filled with a gap using DNA polymerase to obtain blunt ends. XbaI linkers were crosslinked at the blunt ends of ClaI sites to yield a population of molecules to which XbaI restrictase was added to remove the linker. Then, the tetracycline resistance gene was introduced on the XbaI fragment and ligated. After transferring the genes to E. Coli strain K-12 and selecting for tetracycline resistance, the contents of the plasmid were examined to determine the amount of transformants obtained. A large number of deletion mutations have been found that contain deletions in one or more XbaI - ClaI fragments. One deletion mutant was selected that lacked only a 550 bp ClaI-XbaI fragment containing the A ₁ gene. This deletion mutant, designated pCVD25, was purified, hydrolyzed with XbaI restriction enzyme and re-ligated to remove the tetracycline resistance gene. The resulting clone, pcVD 30. showed a negative reaction to holotoxin, as measured in the analysis using adrenosensitive Y-1 cell type [Sack DA et al., See earlier, (1975)], but positive for the production of the B-subunit measured by the solid-phase method ELISA [Sack DA et al., see earlier (1980)], in which the A ₁ resistance genes were lost, as evidenced by the results of DNA hybridization using a labeled A ₁ probe. A HindIII fragment containing a mutation with enterotoxin deletions was then cloned into pJBK85, a Tc-sensitive, Cm-resistant plasmid derived from pJBK108. The resulting plasmid was designated as pJBK108.

The absence of a selectable marker in the deletion mutation of enterotoxin in PJBK108 required modification of the method previously used to attenuate El Tor strain N16961. To perform the elimination of Ai genes from strain 395, the HindIII fragment excised from the plasmid pCVD15 was cloned into PJBK85, resulting in the plasmid pJBK88 (Fig. 11). The tetracycline resistance gene on the XbaI fragment was then cloned into the XbaI site in the region of the A ₁ gene of plasmid PJBK88 to obtain a derivative of plasmid pJBK107. This tetracycline resistance marker gene was then recombined into the chromosome of strain 395, according to the procedure previously used to obtain V. cholerae pJBK56. PJBK107 (Tc ^r , Cm ^r ) was transferred to strain 395 and then the second Inc P plasmid, pR751 (Tp ^r ), was introduced. Colony selection by Tc ^r , Tp ^r , Cm ^r yielded V. cholerae JBKI 13 strain, which contained tetracycline resistant genes in both chromosomal copies of enterotoxin. PJBK 108 containing the deletion mutation was then introduced into V. cholerae JBK 113. Homologous recombination of the deletion mutation on the chromosome results in loss of A ₁ gene sequences, a result detected by the loss of tetracycline resistance factor. Since recombination occurs even at a very low frequency of repeatability, a tetracycline-sensitive cell enrichment procedure was used in a population of tetracycline-resistant cells. In this enrichment technique, the fact that tetracycline belongs to bacteriostatic antibiotics was taken as a basis, while ampicillin and D-cycloserine belong to bactericides. Accordingly, a culture of strain V. cholerae JBK113 containing plasmid pJBK108 was incubated for 3 hours at 37 ^° C in 1 L of broth containing 2 μg / ml tetracycline, 50 μg / ml ampicillin and 50 μg / ml D-cycloserine . By 3 hours, most of the tetracycline-resistant cells were killed, and tetracycline-sensitive cells were detected by plating the culture on L-agar and obtaining a replica on L-agar with tetracycline. Tetracycline-sensitive colonies were then tested for the presence of A ₁ genes by DNA hybridization. One tetracycline-sensitive strain with a deletion of both copies of the A ₁ subunit gene was designated V. cholerae CVD101 and tested for B-subunit production activity by ELISA [Sack, see earlier]. V. cholerae CVD101 was found to reliably produce the enterotoxin B subunit at levels equivalent to the parent virulent strain 395 V. cholerae.

Example 8
Enterotoxin DNA gene sequence.

The complete DNA sequence of the V. cholerae Inaba 62746 enterotoxin genes was determined, some of which were reported by Lockman, et al. J. Biol. Chem. 258, 13722 (1983). Mapping of plasmids pCVD14 and pCVD15 by restriction with an endonuclease showed that the sequences found in strain 62746 are also present in the enterotoxin genes of strain 395. Predicted crosses after deletion of a 550 bp Xba1-Cla1 fragment, but with the introduction of an Xba1 linker, shown in FIG. 12. The Xba1 site of the cholera enterotoxin sequence covers 10 and 11 amino acid residues of the structural gene A ₁ (not counting 18 amino acids of the main sequence A ₁ ).

Example 9
Construction of Strain V. cholerae containing a deletion of the area of tight contact (zonal occludens) of enterotoxin in CVD101.

 A mutant with a deletion of the zot gene of V. cholerae was obtained by a similar method as described previously in example 7 for strain CVD101. The Zot gene is contained in the recombinant plasmid pBB68. PBB68 consists of a chromosomal DNA fragment at the EcoRI-PbrI site excised by V. cholerae 569B, which contains the zot gene and ctx genes having a 550 bp XbaI-ClaI fragment deletion. The StuI-AccI fragment of 575 bp in length was cut in vitro from the plasmid pBB68 by hydrolysis with StuI restriction enzymes and the AccI site to obtain blunt-ended molecules by filling the gaps with DNA polymerase. (The result is a deletion of 48% of 1199 base pairs of the zot gene). One half of the sample was ligated with the tetracycline resistance gene (foreign origin), resulting in a selectable marker.

An in vitro deletion zot gene mutant as described above was inserted into the V. cholerae CVD101 chromosome according to the procedure used to construct the ctx deletion mutant in strain CVD101. The tetracycline resistant clone obtained above was cloned into the Inc P-plasmid pJBK85. This plasmid (Tc ^r , Cm ^r ) was transferred to strain CVD101, followed by selection of the Tc ^r genotype. Then, the second Inc P plasmid, pR751 (Tp ^r ), was introduced. As a result, colony selection for Tc ^r , Tp ^r , and Cm ^r resulted in strains of V. cholerae, where cross recombination between the Tc ^r gene and the zot gene occurred.

A plasmid containing the StuI-AccI deletion mutant without the Tc ^r gene was then transferred to the V. cholerae strain with the T ^r gene. Homologous recombination of the deletion mutant into the chromosome led to the loss of zot gene sequences, the result being revealed by the absence of colonies with Tc ^r . Tc ^r colonies were selected and screened for loss of zot sequences by DNA hybridization using the StuI-AccI fragment as a probe.

Example 10
Construction of Strain CVD109 V. cholerae with deleted sequences encoding enterotoxins V. cholerae and enterotoxin tight contact zone Zonula Occludens.

 The construction of attenuated V. cholerae CVD109 (ATCC # 55057) involves manipulating the insertion of the cloned Vibrio DNA sequences together with the sequences encoding a selectable marker on the chromosome of the virulent strain V. cholerae. An initial in vivo recombination event of homologous sequences from a recombinant plasmid to a chromosome provides a selectable marker at this site.

The second in vivo recombination event between homologous flanking DNA sequences leads to the excision of the trained genes from the chromosome to obtain the final product, which is a deletion mutant. In FIG. 16 shows a design diagram of a CVD109. Zot and ctx genes are located next to each other on the V. cholerae chromosome. Multiple copies of 2700 bp DNA sequences, named as RS1 elements (in the form of a repeating sequence) contain zot and ctx genes at both ends in the virulent strain V. cholerae E7946 (El Tor biotype, Ogawa serotype). In FIG. 16A, sequences at the zot and ctx loci of the genes are shown by a large unshaded or hatched arrow. RS1 sequences are shown by a small, solid arrow. The recombinant plasmid pCVD51 (Fig. 16A) contains cloned zot / ctx sequences (open hatched) that are homologous to the chromosomal zot / ctx sequences (shown in Fig. 16A by a dashed arrow) and contain a selectable marker, the ampicillin-resistant gene (Ap ^r ). The vector of the plasmid into which the Vibrio DNA sequence was cloned is plasmid pGP704 (Miller and Mekalanos J. Bact. 170, 2575-2583 (1988)). This plasmid is not capable of replication in the extrachromosomal state in V. cholerae, but can be replicated together with compatible strains of E. Coli. PCVD51 was crossed with a microorganism from E. Coli with transfer to V. cholerae E7946. Since this plasmid is not capable of replication in the extrachromosomal state in V. cholerae, strains were obtained by selection of colonies with ampicillin-resistant cells in which the plasmid carrying the Ap ^r marker was homologously recombined into the chromosome at the site of the zot / ctx sequences. (The exact recombination site, whether it is the locus of the zot gene or ctx gene, is unknown). As a result of the occurrence of only one cross-recombination event (and not double cross-recombination), the structure of the so-called joint association, which is shown in FIG. 16B.

RS1 — sequences flanking the zot and ctx genes are of sufficient length to allow for recognition of in vivo recombination; intramolecular recombination between homologous RS1 elements leads to the loss of all sequences between them. The strain V. cholerae with an integrated plasmid containing the Ap ^r marker gene was grown in the absence of ampicillin and then colonies with ampicillin resistance factor Ap ^s were selected. The recombination of RS1 elements flanking ctx and zot resulted in the loss of the insertion sequences zot and ctx along with the plasmid vector containing Ap ^r (Fig. 16C).

Colonies of V. cholerae with factor Ap ^s grown in the above steps were screened by DNA hybridization on a zot sequence. The DNA probe contained a fragment with a 575 bp Stul-AccI restriction site formed from a cloned zot gene. Colonies that did not hybridize with the indicated DNA probe were selected and then tested for the presence of ctx genes by DNA hybridization using a probe with ctx gene. Hybridization results confirmed the loss of zot and ctx gene loci. One representative strain was rescued and designated as strain CVD109. In FIG. 160 shows the chromosome of cells of the CVD109 line in which zot and ctx sequences are deleted, but one copy of the RS1 element is saved. (The plasmid shown in Fig. 160 was lost in the final strain containing Ap ^s , but is shown only as an illustration of the second cross-recombination event. This intermediate was lost spontaneously because this plasmid is not capable of replication in the extrachromosomal state in V. cholerae) .

Example 11
Construction of the V. cholerae CVD110-a strain with deletions of sequences encoding A and B of the V. cholerae enterotoxin subunit and tight contact zone enterotoxin and insertion of the mercury resistance gene and the DNA sequence encoding the V. cholerae enterotoxin B subunit .

 CVD110 (ATCC # 55188) was constructed directly from V. cholerae CVD109, which was previously described. In strain V. cholerae CVD109, both the A- and B-subunit of cholera enterotoxin (CT), as well as the zot gene and mercury resistance gene, were deleted. A gene fragment containing the marker gene of the B-subunit of cholera enterotoxin (ctxB) and a marker of mercury resistance were constructed in vitro. This construct was then inserted into the chromosome of strain CVD109, and targeted to the hemolysin gene. Thus, the final vaccine strain CVD110, induces the production of the B- but not the A-subunit of cholera enterotoxin, is resistant to mercury and does not produce the type of HlyA protein (hemolysin).

 Construction of the B-subunit of cholera enterotoxin: ctxB and ctx promoter fragments were obtained from plasmid pCVD30, which is described in example 7. This plasmid pCVD30 contains a deletion of the ctxA gene. A 1.4 kb fragment containing the ctxB gene and ctx promoter but the zot gene was lost was obtained by hydrolysis of pCVD30 with the restriction enzyme HinP1 and HaeIII. The resulting fragment was treated with T4 DNA polymerase to obtain it with blunt ends and synthetic KpnI linker sequences were crosslinked with the indicated fragment. The fragment was then cloned into the plasmid vector pCVD315 [Galen, et al. Advances in Researchon Cholera and Related Diarrheas, vol. 7 (Sack et al., Eds.) Pp. L43-153 (1990)]. The plasmid vector pCVD315 does not have any significant differences from the vectors used for this purpose except that it contains the KpnI site. The resulting vector plasmid containing the ctxB gene was designated as pCVD621 (Fig. 18).

 Genes for resistance to mercury: as a gene for resistance to mercury (mer), a marker similar to the one used in the construction of V. cholerae JBK70 was used. A 4.2 kb NcoI-StuI fragment containing the mer gene was obtained from the plasmid pDB7 [Barrineau et al., J. Molecular and Applied Genetics (1984) vol. 2, p. 601, 619]. The fragment was treated with DNA polymerase (Klenov fragment) to obtain it with blunt ends and synthetic KpnI linker sequences were crosslinked with the indicated fragment. Then the fragment was cloned into the vector of plasmid pCVD43.2 (source unknown), which is a derivative of plasmid pCVD43 [Kaper and others. Advances in Researchon Cholera and Related Diarrheas, vol. 6 (Ohtomo et al., Eds.) Pp. 161-167 (1988)]. PCVD43 contains the cloned hemolysin genes (hlyA) of V. cholerae with a 400 bp deletion of the HpaI fragment. inside the hlyA locus. Deletion of an HpaI fragment of 400 bp in length. leads to inactivation of the indicated [Kaper and others. Advance, see earlier, vol. 6]. The plasmid pCVD43.2 is identical to pCVD43 except that the synthetic KpnI linker is ligated to only one HpaI site of plasmid pCVD43. The combined clone of mer genes inserted at the hylA gene locus was designated as pCVD43.3.

 Insert ctxB and mer genes into CVD109: to transfer these genes into the CVD109 chromosome, the plasmid vector pGP704, which is described in Example 10, was used. ClaI-Bg1II fragment from plasmid pCVD43.3, 8.1 bp in length, containing mer and hylA genes was cloned into plasmid pGP704 to obtain plasmid pJMK12 (Fig. 18). pJMK12 was partially hydrolyzed by the restriction enzyme KpnI to yield a population of linear molecules in which only one of the 3 KpnI sites was excised. A fragment of plasmid pCVD621, a length of 1.4 p. (described above) containing the ctxB gene was then ligated with plasmid pJMK12 to obtain plasmid pCVD622.2B. The relative position and orientation of the inserted genes is shown in Fig. 18.

Plasmid PCVD622.2B was then transferred to V. cholerae CVD109 by conjugation from E. coli host cells. As already indicated in Example 10, pGP704 is not capable of replication in an extrachromosomal state in V. cholerae, but is capable of replication in allowing E. Coli cells. Since pCVD622.2B is unable to replicate in an extrachromosomal state in V. cholerae, strains in which the plasmid pCVD622.2B carrying the Ap ^r marker is homologous were obtained by selection of colonies with ampicillin-resistant cells [pGP 704 contains the gene encoding resistance to ampicillin] recombined into the chromosome at the site of the hylA gene. [She is not capable of recombination between ctx or zot sequences due to the loss of these genes]. As a result of a single cross-recombination event (not double cross-recombination), a so-called jointly integrated structure or “Merodiploid” (these terms are used interchangeably) is obtained, which is shown in FIG. 19B.

A second cross-recombination event can occur between homologous hylA sequences flanking the pCVD-622.2B plasmids joined together. This second cross-recombination event occurs spontaneously and is detected by selection of colonies in which the Ap ^r phenotype is lost. This second cross-recombination event may have one of two possible outcomes, depending on the exact recombination site. Both possible results lead to the loss of the sequences of the plasmid vector pGP704, as shown in FIG. 19C and 19D. One result simply restores the original situation, that is, a strain similar to CVD109, in which the genes ctx, zot, and mer are lost. The second outcome of the event may lead to the loss of pGP704 sequences, however, mer and ctx sequences within the region of hlyA sequences are not lost. Two possible outcomes of the event are easily detected by DNA hybridization using the radiolabeled ctx sequence as a probe. To isolate the target recombinant, a CVD109 cell culture containing co-integrated plasmids pCVD-622.2B was grown in L-broth without the addition of antibiotics. These cultures were seeded on L-agar plates without a selectable marker and the grown colonies were replicated on ampicillin-containing agar plates. Ampicillin-sensitive colonies were then hybridized with a ctx probe and colonies with ctx sequences were isolated. One such colony was designated V. cholerae CVD110. This strain, as confirmed by DNA hybridization analysis, contains the ctx and mer sequences, as well as the loss of the pGP704 fragment and 400 bp HpaI fragment. within the hlyA gene region. The strain V. cholerae CVD110, as also confirmed by the results of enzyme-linked immunosorbent assay, causes the production of the B subunit of cholera enterotoxin [Sack, DA and others, see earlier (1980).

 DNA sequence of inserted genes: exact DNA sequences of inserted ctx and mer genes are known from the literature. The exact location of the hlyA gene site into which these genes are inserted is also known.

Example 12
Descriptions of ACE (Cholera Impurity Enterotoxin)
As indicated earlier (Example 10), the zot and ctx genes are located in the region with a 4.3 kb DNA sequence, which is flanked by copies of RS1 sequences in most strains of the El Tor cholera vibrio. As for the vaccine strain CVD110, the complete DNA sequence in this area is deleted. As for the locus of the zot or ctx gene, there are DNA sequences encoding a third enterotoxin, ACE. In FIG. 20, a map of this area is shown. Fragment EcoRV, length 2.9, etc. (SED ID N: 1) was cloned into the plasmid vector pCVD315 [Galen et al. Advances in Research on Cholera and Related Diahrreas, Issue 7, (Sack et al., Revised), pp. 143-153 (1990) 1 to obtain clone pCVD630 (shown in Fig. 20). Plasmid pCVD630 was then transferred to V. cholerae strain CVD110 and the activity of this strain was measured in a Ussing chamber (Ussing chambers described previously). Cultural supernatants of strains CVD110 and CVD110 containing pCVD630 were tested according to the procedure described previously. In FIG. 21A shows examples of obtained culture supernatant fractions that contained DNA molecules weighing less than 10 kilodaltons and fractions containing DNA molecules weighing more than 10 kilodaltons. Essentially, no Ussing activity was observed in fractions <10 kilodaltons. In the fraction> 10 kilodaltons, CVD110 caused some changes in the indicators of short circuit current (Isc) but not more than in the negative control, PBS (phosphate buffered saline). However, CVD110 culture supernatants (pCVD630) did not cause a significant increase in the Isc value. This difference was statistically significant (p <0.02) compared with the results obtained only with strain CVD110. As indicated earlier, an increase in the value of Isc can be due to both an increase in the potential difference (PD) and an increase in the electrical conductivity of the tissue (Gt). The increase in Isc induced by the genes contained in pCVD620 is caused by an increase in PD, as evidenced by the results shown in FIG. 21B.

 Thus, the 4.3 kb region covered by RS1 elements in El Tor strains contains 3 conditional enterotoxins. The same section with a length of 4.3 bp present in classical strains, however, RS1 repeats are found on only one side. Each of these three enterotoxins can cause an increase in Isc in the Ussing chamber when using rabbit ileum tissue samples, and their activity is comparable to the result of their ability to cause diarrhea in humans. Two toxins, cholera enterotoxin and ACE, act with increasing PD, while a third, ZOT, begins with an increase in tissue conductivity. It is advisable to exclude all three of these active factors from attenuated vaccine strains of V. cholerae to prevent the reactivity that is observed in strain CVD101 and other attenuated vaccine strains. Strain CVD110, which lacks all these three activities, as can be seen from FIG. 21 causes changes in the Ussing chamber, comparable to the results of the variations induced by PBS (that is, essentially no changes occur).

 The DNA sequence of the fragment recognized by EcoRV is 2.9 in length. etc. o (SED ID N: 1) as part of plasmid pCVD620 is shown in FIG. 22. There are two open reading frames (ORFs) with instant transcription of the zot gene in the 3'-5 'direction. The smaller open ORF reading frame with instant transcription of the zot gene in the opposite direction is a 297 bp nucleotide sequence with a codon (ACE) that can potentially encode a protein of 11 kilodaltons and the large ORF with instant transcription of the gene in the opposite direction of ORF, 297 bp in length, is a 1185 bp nucleotide sequence with a codon (OrfU) that can potentially encode 44 kilodaltons protein. Ace enterotoxin activity is believed to be localized to a 297 bp open reading frame.

Example 13
Construction of V. cholerae CVD112 - vaccine strain of V. cholerae 0139 serogroup.

 Strains of V. cholerae are divided into a large number of groups 0. Strains 0 of group 1 cause cholera with the potential for widespread transmission of the infection to the foci of a pandemic. V. cholerae from the other 0 groups, the so-called non-01 V. cholerae, can cause disease, but until recently, it did not lead to epidemic cholera. Recently, there have been reports of tens of thousands of infections with this V. cholerae, not belonging to group 01. It is believed that current vaccine strains based on non-01 serogroup cholera vibrio may be ineffective for immunity against non-01 vibrios serogroups. The new clinical strain was designated as the new serogroup 0139.

 V. cholerae 1837 was used as the starting strain for the vaccine strain V. cholerae strain non-01 serogroup. This strain was selected from serotype 0139 and isolated from a patient with cholera in Bangladesh. In FIG. 23 shows the location on the chromosome of the locus of cholera enterotoxin in strain 1837.

 Plasmid pCVD51 was transferred to strain 1837 according to the procedure described in Example 10. This plasmid underwent recombination on the bacterial chromosome, and the second recombination event caused the loss of one copy of the enterotoxin genes and 3 copies of RS1 elements. The resulting strain V. cholerae 1837.1 is shown on the second line of FIG. 24. For simplicity, FIG. 24, the gene sequences of ser, orfU, ace, zot, ctxAB are indicated together as the "core" (core, core portion) of V. cholerae enterotoxin. The locus of the V. cholerae chromosomal DNA includes one or more core regions and the RS1 elements associated with them.

 The remaining sequences of the chromosomal DNA of strain 1937.1 were deleted as follows. Chromosomal DNA was obtained from strain 1837.1, which was partially hydrolyzed by restriction endonuclease Sau3a with clipping fragments of about 30 to 40 kb in length. These fragments were cloned into the cosmid plasmid vector pHC79 (Hohn and Collins, Gene, 11, 291-298, 1980) and the resulting clones were screened after transfer to E. Coli by DNA hybridization using ctx gene sequences as a probe. Clone containing cxt genes on an insert of approximately 30 kb in length has been identified and isolated. The resulting plasmid was partially mapped. The insulated structure is indicated in FIG. 25 as pLC13. This plasmid was digested with EcoRI restriction enzyme and cross-linked, resulting in a decrease in insert size to approximately 25 kb. The resulting plasmid was designated as pLC14. Plasmid pLC14 was partially hydrolyzed with HindIII restriction enzyme and then cross-linked to remove a recognizable HindIII fragment of approximately 14.6 kb in length, containing the core region, RS 1 sequences, and an unsequenced V. cholerae DNA fragment of approximately 7.4 t. by. The resulting plasmid with a deletion of the core region and the RS1 sequence was designated as pLC15. A similar recognizable HindIII fragment was excised during the construction of the attenuated strain according to the method described by Pearson and others, P.N.A.S. (US), 90, 3750-3754, 1993. The resulting plasmid pLC15 was then hydrolyzed with restriction enzymes Sal1 and EcoRI, and vibrio DNA was cloned into the killer vector plasmid pGP704 according to the procedure described in Example 10. The resulting plasmid was designated as pLC16.

 The killer vector plasmid pLC16 containing the chromosomal DNA sequences of V. cholerae strain 1837.1 that span the ctx locus was transferred to V. cholerae strain 1837.1 using the procedure described in Example 10. Recognition of homologous in vivo recombination of vector sequences into the chromosome was performed by screening on ampicillin-resistant because the ampicillin resistance factor is encoded by vector-mediated DNA sequences.

 Ampicillin-resistant V. cholerae obtained in accordance with this method contains plasmid DNA sequences integrated into the bacterial chromosome, then grown in the absence of ampicillin, ampicillin-sensitive colonies were selected. The second recombination event led to the loss of the phenotype ctxA, ctxB, zot, ace, orfU, ser, and RS1 elements along with the plasmid vector encoding the ampicillin resistance factor. This final strain was designated as 1837.2. In FIG. 24, the third line, is shown in schematic form the chromosome of the vibrio in strain 1837.2.

 The gene encoding the B-subunit of cholera enterotoxin together with the gene encoding the mercury resistance factor was transferred to strain 1837.2 according to the procedure described in Example 11. As the killer vector pCVD622.2B, the same vector was used that was used to obtain the strain CVD110. The event leading to the insertion of the DNA sequences of the B subunit of the cholera enterotoxin is depicted in FIG. 26. The final product, strain CVD112, contains deletions of all genes of ctx, zot, ace, ser, and RS1 elements, expresses only the B subunit of cholera enterotoxin and the mercury resistance factor.

 Obtaining the drug strains CVD112 and Al1837 to study the possibility of use as a vaccine.

CVD112 and Al1837 strains of V. cholerae were stored in TSB containing 18% glycerol at a temperature of -70 ^° C. Before administration to volunteers, the contents of the ampoule of the corresponding strain were thawed and streaked onto blood agar, TCBS, and BI agar. After incubation for 24 hours at 37 ^° C., colonies of cells that agglutinated the appropriate sera were selected and seeded on BHI agar, then incubated at 37 ^° C. for approximately 20 hours. The strain V. cholerae was then re-seeded and incubated for 5 hours at a temperature of 37 ^o C and the resulting cell harvest was collected in sterile saline. The concentration was determined by the optical density of the solutions, which were compared with known standards. Then a quantitative analysis was carried out using the replica transfer method before and after the introduction of volunteers into the body.

 Clinical trials of vaccines.

 A group of adult volunteers aged 18 to 40 years was selected, with whom a conversation was held and their written consent was obtained. Volunteers were carefully examined to ensure that they were in excellent physical and mental condition. A group of 14 volunteers was hospitalized and allowed to adapt to their surroundings for two days, during which the examination was completed and blood samples were taken as reference samples to determine the level of antibody formation.

Volunteers were examined with a double blind method to obtain one of the following doses of the vaccine on the third day:
(i) CVD112 at a dose of 5 • 10 ⁸ cfu using a buffer (n = 6)
(ii) CVD112 at a dose of 5 • 10 ⁶ cfu using a buffer (n = 6)
(iii) just a buffer solution
Two vaccinated volunteers who received only buffer solution were with recipients who received the vaccine to determine the possibility or impossibility of transferring infection from one person to another under experimental conditions. Vaccinated were continuously monitored for 5 days, and then they were treated with tetracycline for five days.

After about 5 weeks, the volunteers were re-infected with the pathogenic strain Al1837 V. cholerae serogroup 0139 to evaluate the effectiveness of the vaccine against the homologous antigen. The provocative dose contained approximately 10 ⁶ cfu of the V. cholerae 0139 strain Al1837. The volunteers were monitored continuously for approximately 120 hours after the provocation. All volunteers received tetracycline (at a dose of 500 mg every 6 hours for 5 days), starting from 5 days after immunization and from 4 days after infection control to exclude aftereffect of vaccine strains and provocation.

Eight vaccinees carried out a control infection, while one group of four received a dose of 5 • 10 ⁸ , and the other group of four received a dose of 5 • 10 ⁶ A group of fifteen unvaccinated volunteers who served as a control also received a dose of provocative test. One of eight who received the vaccine (13%) and twelve people in the control (80%) had diarrhea after provocation, as a result, the vaccine was 84% effective. One of the vaccinees in whom diarrhea was noted received a vaccine strain CVD112 at a dose of 5 • 10 ⁶ .

 Three (38%) of the eight vaccinated and 14 (93%) in the control did not show the isolation of the strain used for infection, although the peak excretion was 100 times lower than that of the vaccinated, indicating that the vaccine strain multiplied effectively in the intestines vaccinated.

 These results are clear evidence of the safety and efficacy of strain V. cholerae CVD112 for use as a vaccine to induce post-vaccination immunity against cholera caused by non-01 serogroup 0139 serotype vibrio strains.

Example 14
Construction V.cholerae CVD112 RM-attenuated, recA ^- mutant of V. cholerae serogroup 0139.

The strain V. cholerae CVD112 is the initial strain for the construction of a weakened strain of V. cholerae, rec ^- , 0139-serogroup. Mutation of the recA gene is believed to be necessary to reduce the already low probability of reversing the virulence of the attenuated strain through recombination with wild-type ctx genes in vivo. The RecA mutant was constructed by a similar technique described by Ketley and others, Infect. Immun., 58 (5), 1481-1484, 1980 and Goldberg et al., J. Bacter, 165 (3), 715-722, 1986.

 Plasmid pCVD831 carrying a 7 kb NarI-NheI fragment with the recA gene of V. cholerae El Tor strain N16961, was cloned into a broad-spectrum host plasmid pCVD316, which is a derivative of the previously described IncR plasmid pRK290. Plasmid PCVD831 was digested with restriction enzymes XbaI and PvuII to cut a fragment of approximately 50 bp. within the DNA sequences encoding the recA gene. The ends of the restricted DNA fragment were then filled with a gap to obtain it with blunt ends, and the resulting plasmid was re-crosslinked. Removal of a fragment of approximately 50 bp in length provides inactivation of gsa-protein. The resulting plasmid was designated as pCVD832.

Plasmid pCVD832 was transferred to strain V. cholerae CVD112, and as a result of homologous recombination, the native gesA gene is replaced by the gesA mutant. The result of a homologous recombination event is determined by screening for a methyl methane sulfonate-sensitive phenotype (MMS) in accordance with the method described by Kettler et al., See earlier (1980). The strain obtained as a result of these manipulations has a deletion of all ctx, ser, orfU, ace loci and RS1 elements and an inactivated mutation at the chromosomal rec-A locus (that is, it is a recA ^- strain). The result of the cross-recombination event that occurs at the V. cholerae recA locus in the generation of strain CVD112 RM is shown schematically in FIG. 27.

 Strains V. cholerae CVD112 and CVD112 RM can be effectively used as a vaccine that causes protective immunity against the virulent strain of serotype 0139 and other non-serogroup 01 strains after subsequent infection with these virulent strains.

 Strains CVD112 and CVD112 RM are also suitable for use with one or more other V. cholerae vaccine shams, toxoids, thermally inactivated cholera enterotoxin, etc., as a combined (associated) vaccine to protect against both virulent strains 01 of serogroup and virulent strains of non-01 serogroups, for example, strains of V. cholerae of the Ogawa or Inaba serotype.

 More recently, there have been reports that the locus of the ser gene, which encodes the nucleus encoded in the chromosome of the cell nucleus, can act as an additional factor in colonization. However, the cep gene product is not believed to have a significant effect on the ability of the Vibrio cholerae strains of the present invention to colonize the small intestine of the host organism, in particular the human intestine. However, if desired, DNA sequences, including the ser gene, or any other suitable colonization factors, can be inserted again into the V. cholerae chromosome, in accordance with the invention, following the methods and methods disclosed in the present description. As an example, when re-inserting DNA sequences encoding the B subunit of cholera enterotoxin and DNA sequences encoding the mercury resistance factor into the chromosome at the hemolysin gene locus, one can easily transfer the gene of interest that is actively associated with the expression signal to the plasmid which is used in the recombination technique.

 The strain resulting from this cross-recombination synthesizes the B-subunit of cholera enterotoxin, that is, a protein that induces resistance to mercury and other heavy metals, and the additional gene described in this description.

Claims (16)

 1. Avirulent strain of Vibrio cholerae CVD 112 non-01 serogroup used for vaccination against cholera. 2. The avirulent strain of Vibrio cholerae according to claim 1, characterized in that said strain is a strain from serogroup 0139 and has a genotype comprising ctxA, zot ace ^- , mer, ctxB.  3. The avirulent strain of Vibrio cholerae according to claim 2, characterized in that the DNA encoding the resistance to mercury and the DNA encoding the B subunit of the cholera enterotoxin are inserted into the site of the hemolysin gene in the chromosome.  4. The avirulent strain of Vibrio cholerae according to claim 3, characterized in that the strain is deposited as strain ATCC 55752 or its derivative.  5. Avirulent strain of Vibrio cholerae CVD 112RM non-01 serogroup used for vaccination against cholera. 6. The avirulent strain of Vibrio cholerae according to claim 5, characterized in that said strain is a strain from serogroup 0139 and has a genotype comprising ctxA ^- , zot ^- , ace ^- , recA-, mer, ctxB.  7. The avirulent strain of Vibrio cholerae according to claim 6, characterized in that the DNA encoding the resistance to mercury and the DNA encoding the B subunit of the cholera enterotoxin are inserted into the site of the hemolysin gene in the chromosome.  8. A method of obtaining an avirulent strain of V. cholerae non-01 serogroup containing a deletion of the core regions of the cholera enterotoxin and RS1 sequences, but expressing the B subunit of the cholera enterotoxin and sequences encoding a product that causes resistance to heavy metals, characterized in that it includes stages : (a) constructing a first plasmid containing DNA having a core region of a cholera enterotoxin V. cholerae and flanking sequences of sufficient length to promote recognition recognized in vi vo recombination, ligated with a gene encoding a first selectable foreign marker that causes resistance to selective factor, said first plasmid being incapable of extrachromosomal DNA replication in V. cholerae; (b) crossing the virulent strain of V. cholerae non-01 serogroup with the first microorganism carrying the first plasmid; (c) selecting and isolating Vibrio cholerae expressing a first selectable marker; (d) growing V. cholerae isolated in step (c) in the absence of said selective factor; (e) screening V. cholerae grown in step (d) for loss of expression of said first selectable marker; (f) constructing a second plasmid containing the chromosomal DNA sequences of Vibrio cholerae non-01 serogroups that restrict the cholera enterotoxin locus deleted from DNA containing the region of the cholera enterotoxin core and RS1 sequences ligated with a second selectable foreign marker that causes resistance to second electoral factor; (g) crossing the product screened in step (e) with a second microorganism carrying said second plasmid; and (h) selecting a Vibrio cholerae that expresses a second selectable marker; (i) growing the selected product of step (h) in the absence of a second selective factor; (j) screening the V. cholerae product of step (i) for the loss of said second selectable marker; (k) isolating the product screened in step (j); (l) constructing a third plasmid that contains V. cholerae chromosomal sequences of sufficient length to promote recognizable in vivo recombination that limits the cholera enterotoxin B subunit sequences and sequences encoding a heavy metal resistance product and crosslinked with a third selectable foreign a marker, wherein said third plasmid is incapable of extrachromosomal DNA replication in V. cholerae; (m) crossbreeding the product screened in step (j) with a third microorganism carrying the third plasmid; (n) selecting a Vibrio cholerae that expresses a third selectable marker; and (o) isolating the selected product of step (n).  9. A method of producing an avirulent strain of V. cholerae non-01 serogroup according to claim 8, characterized in that said virulent strain of Vibrio cholerae non-01 serogroup is a strain of serogroup 0139.  10. A method of producing an avirulent strain of V. cholerae non-01 serogroup according to claim 9, characterized in that said virulent strain of V. cholerae non-01 serogroup is strain 1837.  11. A method for producing an avirulent V. cholerae non-01 serogroup according to claim 8, characterized in that said isolated selected product in step (o) is a V. cholerae CVD 112 strain.  12. A method of obtaining an avirulent strain from a serogroup weakened by recombination V. cholerae non-01, containing a deletion of the core region of the cholera enterotoxin and RS1 sequence, but expressing the B subunit of the cholera enterotoxin and a sequence that encodes a product that causes resistance to heavy metals, characterized in that it comprises the steps of: (a) constructing a first plasmid containing DNA having a core region of the cholera enterotoxin Vibrio cholerae and flanking sequences sufficient th length to promote detectable in vivo recombination, ligated to a gene encoding a first selectable marker of foreign origin which confers resistance to a selective agent, wherein said first plasmid is incapable of replicating extrachromosomally in V. cholerae; (b) crossing the virulent strain of Vibrio cholerae non-01 serogroup with the first microorganism carrying the first plasmid; (c) selecting and isolating Vibrio cholerae expressing a first selectable marker; (d) growing V. cholerae isolated in step (c) in the absence of said selective factor; (e) screening V. cholerae grown in step (d) for loss of expression of said first selectable marker; (f) constructing a second plasmid containing the Vibrio cholerae non-01 serogroup chromosomal sequences that limit the cholera enterotoxin locus deleted from DNA containing the region of the cholera enterotoxin core and RS1 sequences ligated with a second selectable foreign marker that causes resistance to the second selective factor; (g) crossing the product screened in step (e) with a second microorganism carrying said second plasmid; and (h) selecting a Vibrio cholerae that expresses a second selectable marker; (i) growing the selected product of step (h) in the absence of a second selective factor; (j) screening the V. cholerae product of step (i) for the loss of said second selectable marker; and (k) isolating the product screened in step (j); (l) constructing a third plasmid that contains V. cholerae chromosomal sequences of sufficient length to promote recognizable in vivo recombination that limits the cholera enterotoxin B subunit sequences and sequences encoding a heavy metal resistance product and crosslinked with a third selectable foreign a marker, wherein said third plasmid is incapable of extrachromosomal DNA replication in V. cholerae; (m) crossbreeding the product screened in step (j) with a third microorganism carrying the third plasmid; (n) selecting a Vibrio cholerae that expresses a third selectable marker; (o) isolating the product selected in step (n); (p) constructing a fourth plasmid containing V. cholerae chromosomal sequences of sufficient length to promote recognizable recombination at the rec A locus, flanking sequences with a deletion at the rec A gene locus to inactivate the rec A gene expression product, and ligation with a fourth selectable marker, when this fourth plasmid is incapable of extrachromosomal DNA replication in V. cholerae; (q) crossing the screened product in step (o) with a fourth microorganism carrying the third plasmid; (r) selecting a Vibrio cholerae that expresses a third selectable marker; and (s) isolating the product selected in step (r).  13. A method of obtaining an avirulent strain of V. cholerae non-01 serogroup according to claim 12, characterized in that the virulent strain of V. cholerae non-01 serogroup is a strain of serogroup 0139.  14. A method of obtaining an avirulent strain of V. cholerae non-01 serogroup according to claim 13, characterized in that the virulent strain of V. cholerae non-01 serogroup is strain 1837.  15. A method of obtaining an avirulent strain of V. cholerae non-01 serogroup according to claim 12, characterized in that said isolated selected product in step (s) is a strain of V. cholerae CVD 112RM.  16. A vaccine for protection against cholera disease when exposed to Vibrio cholerae non-01 serogroup, characterized in that it contains an avirulent strain of CVD 112 or CVD 112RM non-01 serogroup.

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