ZA200507021B - Attenuated strains of Vibrio cholerae and lyophilised vaccines containing same - Google Patents

Description

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Attenuated strains of Vibrio cholerae with improved biological safety features in freeze dried form for oral vaccination

Technical sector.

The field of invention is that of biotechnology, in particular, the obtainment of

Vibrio cholerae live attenuated vaccine strains, more specifically, the introduction of defined mutations to prevent or limit the possibility of reacquisition and (or) the later dissemination of CTX® phage encoded genes by those live vaccine strains and a method to preserve them to be used as vaccines.

Previous art

First, definitions:

During the description of the invention will be used a terminology whose meaning is listed bellow.

By CTX® virus is meant the particle of protein-coated DNA produced by certain

V. cholerae strains, which is capable of transducing its DNA, comprising cholera toxin genes, to other vibrios.

By cholera toxin (CT) is meant the protein responsible for the clinical symptoms of cholera when produced by the bacteria.

By CTX®-encoded toxin genes are meant, in addition to CT genes, zot and ace genes that encode for the “zonula occludens toxin” and for the accessory cholera enterotoxin, respectively. The activity of ZOT is responsible for the destruction of the tight junctions between basolateral membranes of the epithelial cells and ACE protein has an activity accessory to that of the cholera toxin.

The term well tolerated vaccine or well tolerated strain refers to such strain lacking the residual reactogenicity that characterize most of the of non-toxigenic strains of V. cholerae. In practical terms, it means that it is a strain safely enough to be used in communities without or with limited access to healthcare institutions without risks for the life of the vaccinees. It should be expected a rate of diarrhea in 8% or less of the vaccinees and the diarrhea is characterized in that it does not exceed 600 ml (grs), only 1% of the vaccinees or less could suffer from headache, which should be minor and of

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® ® short duration (less than 6 h), and finally that it prompts vomits in less than 0.1% of the vaccinees, those vomits characterized for being a single episode of 500 ml or less.

By hemagglutinin protease (HA/P) is meant the protein secreted by V. cholerae manifesting dual function, being one of them the ability to agglutinate the erythrocytes of certain species and the other the property to degrade or to process proteins such as mucine and the cholera toxin.

By celA is meant the nucleotide sequence coding for the synthesis of the endoglucanase A. This protein naturally occurs in Clostridium thermocellum strains and has a B (1-4) glucan-glucano hidrolase activity able to degrade cellulose and its derivatives.

The term MSHA is referred to the structural fimbria of the surface of V. cholerae with capacity to agglutinate erythrocytes of different species and that is inhibited by mannose.

By reversion to virulence mediated by VGJ® is meant the event in which a previously attenuated strain obtained by the suppression of CTX® genes reacquire all the genes of this phage through a mechanism completely dependent and mediated by

VGJ® and the interaction with its receptor, MSHA.

The possibility of disseminating the CTX® phage in a process mediated by VGJ® is that in which the filamentous phage VGJ® form a stable hybrid structure (HybP®) through genetic recombination with the DNA of CTX® and disseminate its genome with active genes toward other strains of V. cholerae, which could be environmental non pathogenic strains, vaccine strains or other from different species.

Second, information of the previous art:

Clinical cholera is an acute diarrheal disease that result from an oral infection with the bacterium V. cholerae. After more than 100 years of research in cholera there remains the need for an effective and safe vaccine against the illness. Since 1817 man has witnessed seven pandemics of cholera, the former six were caused by strains of the

Classical biotype and the current seventh pandemic is characterized by the prevalence of strains belonging to El Tor biotype. Recently, beginning in January of 1991, this pandemic extended to South America, and caused more than 25 000 cases of cholera and over 2 000 deaths in Peru, Ecuador and Chile. By November 1992, a new serogroup of V. cholerae emerged in India and Bangladesh, the 0139, showing a great

® ® epidemic potential and generating great concern through the developing world. These recent experiences reinforce the need for effective cholera vaccines against the disease caused by V. cholerae of serogroups O1 (biotype El Tor) and 0139.

Because convalescence to cholera is followed by an state of immunity lasting at least three years, much efforts in Vibrio cholerae vaccinology have been made to produce live attenuated cholera vaccines, that closely mimics the disease in its immunization properties after oral administration, but do not result reactogenic to the individuals ingesting them (diarrhea, vomiting, fever). Vaccines of this type involve deletion mutations of all toxin genes encoded by CTX®. For example, the suppression of the cholera toxin and other toxins genes encoded in the prophage CTX® is a compulsory genetic manipulation during the construction of a live vaccine candidate (see inventions of James B. Kaper, WO 91/18979 and John Mekalanos WO 9518633 of the years 1991 and 1995, respectively).

This kind of mutants have been proposed as one dose oral vaccines, and although substantially attenuated and able to generate a solid immune responses (Kaper J. B. and Levine M. Patentes US 06,472,276 and 581,406). However, the main obstacle for the widespread use of those mutants has been the high level of adverse reactions they produce in vaccinees (Levine and cols., Infect. and Immun. Vol 56, No1, 1988).

Therefore, achieving enough degree of attenuation is the main problem to solve during the obtainment of live effective vaccines against cholera. There are at least three live vaccine candidates, which have shown acceptable levels of safety, i.e., enough degree of attenuation and strong immunogenic potential. They are V. cholerae

CVD103HgR (Classical Biotype, serotype Inaba) (Richie E. and cols, Vaccine 18, (2000): 2399-2410.), V. cholerae Peru-15 (Biotype El Tor, serotype Inaba) (Cohen M., and cols. (2002) Infection and Immunity, Vol 70, Not. 4, pag 1965 - 1970) and V. cholerae 638 (Biotype El tor, serotype Ogawa) (Benitez J. A. and cols, (1999), Infection and Immunity. Feb; 67(2):539-45).

Strain CVD103HgR is the active antigenic component of a live oral vaccine against cholera licensed in several countries of the world, the strains Pert-15 and 638 are other two live vaccine candidates to be evaluated in field trials in a near future.

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However, there is a second problem of importance to solve in those live attenuated vaccine candidates; one is the environmental safety, specially related with the possible reacquisition and dissemination of the cholera toxin genes by existent mechanisms of horizontal transfer of genetic information among bacteria. In accordance with this, the attenuated vaccine strains of V. cholerae, could potentially reacquire virulence genes out of the controlled conditions of the laboratory, in an infection event with CTX® phage (Waldor M. K. and J. J. Mekalanos, Science 272:1910-1914) coming from other vibrios and later on contribute to their dissemination. This process could become relevant during vaccination campaigns where people ingest thousands of millions of attenuated bacteria and keep shedding similar quantities in their stools during at least 5 days. Once in the environment, bacteria have the possibility of acquiring genetic material from other bacteria of the same or different species of the ecosystem.

For these reasons, at present it is desirable to obtain vaccine candidates with certain characteristics that prevent or limit the acquisition and dissemination of CTX, and especially of the genes coding for the cholera enterotoxin. As a consequence, this is the field of the present invention.

Bacterial viruses, known as bacteriophages, have an extraordinary potential for gene transfer between bacteria of the same or different species. That is the case of

CTX® phage (Waldor M. K. and J. J. Mekalanos, 1996, Science 272:1910-1914,) in V. cholerae. CTX® the genes of carries the genes that encode cholera toxin in V. cholerae and enters to the bacteria through interaction with a type IV pili, termed TCP, from toxin co-regulated pilus. TCP is exposed on the external surface of the vibrios. In accordance with published results, under optimal laboratory conditions the CTX® phage reaches titers of 10° particles or less by ml of culture in the saturation phase; this allows classifying it as a moderately prolific bacteriophage. Equally the expression of the TCP receptor of this phage has restrictive conditions for its production. In spite of these limitations, the existence of this couple bacteriophage-receptor, limits in some way the best acceptance of live cholera vaccines, that is why depriving the bacteria from the portal of entrance to this phage is a desirable modification.

There are two theoretical ways of preventing the entrance of CTX® into V. cholerae, 1) suppressing the expression of TCP or 2) removing the TCP sites involved in phage receptor interaction. None of the two forms has been implemented due to the

. ® essentiality of TCP for proper colonization of the human intestine and elicitation of a protective immune response. It should be noted that sites involved in the TCP-CTX® interaction are also needed for the colonization process. (Taylor R. 2000. Molecular

Microbiology, Vol (4), 896 -910).

Several strategies that counteract the entrance of the virus have been evaluated such as preventing the integration of the phage to the bacterial chromosome and its stable inheritance, consisting in the suppression of the integration site and in the inactivation of recA gene to avoid recombination and integration to other sites of the chromosome. (Kenner and cols. 1995. J. Infect. Dis. 172:1126 - 1129).

Also, it has been recently described that the entry of CTX® into V. cholerae depends on the genes TolQRA, however this mutation produces sensitive phenotypes not undesired in vaccine candidates of cholera and it has not been implemented. (Heilpern and Waldor. 2000. J. Bact. 182:1739).

Further methods that prevent the entrance of phages carryings essential virulence determinants to cholera vaccine strains or other vaccine strains have not been described.

The main subject of the present invention is related with the phage VGJ® and its capacity to transfer the genes coding for the cholera toxin, using the Mannose Sensitive

Hemagglutinin (MSHA) fimbria as receptor. Specifically, it consists in protecting the live attenuated vaccine strains from the infection with VGJ® by introducing suppression mutations or modifications that prevent the correct functioning of this fimbria.

In the previous knowledge of this fimbria, the following aspects can be summarized. The gene product of mshA was originally described to be the major subunit of a fimbrial appendage in the surface in V. cholerae that had the capacity to agglutinate erythrocytes of different species, this capacity being inhibited by mannose (Jonson G. and cols (1991). Microbial Pathogenesis 11:433-441). As such, the MSHA was considered a virulence factor of the bacteria (Jonson G. and cols (1994). Molecular

Microbiology 13:109-118). In accordance with the attributed importance, mutants deficient in the expression of the MSHA were obtained to study its possible role in virulence. It was demonstrated that MSHA, contrary to TCP, is not required for colonization of the human small intestine by the El Tor and 0139 V. cholerae (Thelin KH and Taylor RK (1996). Infection and Immunity 64:2853-2856). The MSHA has been also ,

® ® described as the receptor of the bacteriophage 493 (Jouravleva E. and cols (1998).

Infection and Immunity, Vol 66, Not 6, pag 2535-2539), suggesting that this phage could be involved in the emergence of the 0139 vibrios (Jouravieva E. and cols, (1998).

Microbiology 144:315-324). Later on it has been described that the fimbria MSHA has a role in biofilm formation on biotic and a-biotic surfaces contributing thus to bacterial survival outside of the laboratory and the host (Chiavelli D. A. and cols, (2001). Appl.

Environ Microbiol. Jul; 67(7):3220-25 and Watnick P. I. and Kolter R. (1999). Mol.

Microbiol. Nov, 34(3):586-95). It is evident from the previous data that several investigations related with the MSHA fimbria have been done, but none of them defines this pili as the receptor of a phage able to transduce in a very efficient way the genes of the cholera toxin and not only these genes but the complete genome of CTX®, what could notably contribute to their dissemination. Additionally, although an extensive search has been made no inventions related with this fimbria have been found, either as virulence factor or as a phage receptor mediating dissemination of CTX®.

On the other hand, it is common practices among those who develop live cholera vaccines to provide them freeze-dried. Thus, these preparations of the live bacteria are ingested after the administration of an antacid solution that regulates the stomach pH and so the bacterial suspension continues toward the intestine without being damaged in the stomach and achieves colonization in the intestine.

Elaboration of freeze-dried vaccines improves preservation of strains, facilitates preparation of doses, allows a long-term storage, limits the risks of contamination and makes the commercialization and distribution easier, without the need of a cold chain, generally not available in under developing countries.

Although Vibrio cholerae is considered a very sensitive microorganism to the freeze-drying process, some additives are known to enhance strain survival. Thus, for preservation of the vaccine strain CVD103HgR Classical Inaba, the Center for vaccine

Development, University of Maryland, United States, the Swiss Institute of Sera and

Vaccines, from Berne (ISSVB), developed a formulation, see (Vaccine, 8, 577-580, 1990, S.J. Cryz Jr, M. M. Levine, J. B. Kaper, E. Furer and B) that mainly contain sugars and amino acids. The formulation is composed of sucrose, amino acids and ascorbic acid, and after the freeze-drying process, lactose and aspartame are added.

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In a work about preservation by freeze-drying of the wild type strain 569B

Classical Inaba, published in Cryo-Letters, 16, 91-101 (1995) for Thin H., T. Moreira, L.

Luis, H. Garcia, T.K. Martino and A. Moreno, compared the effect of different additives on the viability and final appearance upon liophilization and after the storage at different temperatures of this V. cholerae strain. It was demonstrated that viability losses were less than 1 logarithmic order after 3 days of storage to 45° C.

The invention CU 22 847 claims a liophilization method where the formulations contain a combination of purified proteins or skim milk with addition of polymers and/or glycine, besides bacteriologic peptone or casein hydrolysate and sorbitol, with good results for the viability of Vibrio cholerae strains of different serogroups, biotypes and serotypes. The freeze-dried bacteria keep their viability after being dissolved in a 1,33% sodium bicarbonate buffering solution used to regulate the pH of the stomach.

Any vaccine formulation of cholera that it is supposed to be used in under developing countries should have certain requisites such as posses a simple composition, be easy to prepare and manipulate, be easy to dissolve and have good appearance after dissolved. Besides, It would be also desirable not to require low storage temperatures and to tolerate high storage temperatures at least for short periods of time, as well as the incidental presence of oxygen and humidity in the container. Additionally, it is also necessary an adequate selection of the composition of the formulation that allows the preservation of Vibrio cholerae of different serogroups, biotypes and serotypes. Finally, it is also remarkable that a formulation free of bovine derivate ingredients allows us to be in agreement with the international regulatory authorities related to the use of bovine components due to the Bovine Spongiform

Encephalopathy Syndrome.

Description of the invention.

The present invention propose a new generation of live attenuated vaccines to immunize against cholera by modification of their properties, specifically improving their biological safety during colonization of humans and later in the environment, outside the laboratories.

The present invention born from the necessity to protect live cholera vaccines from infection with the CTX® bacteriophage, which contains the cholera toxin genes,

® ® and also to impair the potential dissemination of this phage starting from live cholera vaccine candidates. Specifically it was born from the discovery and characterization of the VGJ® phage in our laboratory.

VGJ® is a filamentous bacteriophage isolated from V. cholerae 0139 but it has infective capacity on V. cholerae O1 of all serotypes and biotypes and also over other strains of V. cholerae 0139. The sequence of this phage was not described in the complete genome sequence of V. cholerae, indicating that this phage was not present in the strain N16961 (O1, El Tor Inaba). From a broad list of V. cholerae O1 strains existing in our laboratory, none of them had homologous sequences to VGJ®, while strains MO45, SG25-1 and MDO12C, of V. cholerae 0139 had.

The VGJ® phage infects V. cholerae through the MSHA fimbria. When this phage enters to the bacterium it can replicate or integrate into a specific chromosomal region. This is a very active phage that reaches 10" particles ml™ in the culture supernatants.

The most important characteristic in this phage, by virtue of which the following application of invention is issued, is their capacity to carry out a specialized transduction of the CTX® phage and consequently of the cholera toxin genes. This process occurs by a site-specific recombination between CTX® and VGJ® genome, followed by the encapsulation and exportation of both genomes into the VGJ® capsid. This hybrid viral particle was named HybP®. A culture of bacteria infected with both, CTX® and VGJo, produce 10" particles ml" of VGJ® and 107-108 particles ml" of HybP®, which is at least 100 times higher than the titers obtained with CTX® alone.

It is also important to understand, to the purpose of this application that the

CTX® phage receptor is TCP, which require special conditions for its expression, while the VGJ® receptor is MSHA fimbria, an antigen that is expressed abundantly in all culture conditions studied and that is also produced in the environment. Furthermore, other vibrios produce the MSHA what increase the risk of transmission, even to other bacterial species.

It is also important to know that once, a new host become infected with HybP®, a stable production of particles in the range of 107-10% ml” takes place in the saturation phase, thus this hybrid phage has a high potential to transmit and disseminate the cholera toxin genes.

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Another aspect of supreme interest to the purpose of this invention is that cholera toxin genes in HybP® are active enough to produce 50 ng mi” of toxin during in vitro culture and that the infection of an attenuated strain with HybP® revert it back to virulence as assessed by the infant mouse cholera model.

In accordance with these data, a primary objective of the present invention is to describe the additional mutations made to live cholera vaccines to prevent them to be infected with either VGJ® or HybP®, as well as the necessity to use live cholera vaccines from which the genome of VGJ® is absent to avoid the dissemination of CTX® mediated by VGJ®, in the case of reacquisition of CTX®.

An example of this mutation is a stable spontaneous mutation, conducive to the lack of expression of the MSHA fimbria in the cellular surface. This way, the VGJ® or its derivative phage, HybP® could not infect such vaccines.

Another example of this mutation is a suppressive mutation in the structural gene of the major protein subunit of this fimbria (MshA).

The use of live cholera vaccine candidates in which the genome of VGJ® is absent could be achieved simply by searching for hybridization of DNA in different strains to identify which have not homologous fragments to VGJ® described in this invention, although other well known methodologies could be applied to remove VGJ® from an infected strain.

Examples of live cholera vaccines to perform the specific mutations mentioned above are vaccine strains which are not able to react with a VGJ® specific prove and that have been demonstrated in the previous art, to have acceptable levels of reactogenicity in volunteers studies. The genotype of these strains includes suppressive mutations of CTX® phage leaving a remnant RS1 and the insertional inactivation of hap gene with the celA gene. Such strains are constructed by means of traditional methods of suppression of the CTX® prophage in epidemics strains of V. cholerae, followed by the inactivation of the hemagglutinin protease gene (hap) for the insert of the marker gene celA in their sequence. To see Robert's scientific article and cols., Vaccine, vol 14

No16, 1517-22, 1996), the scientific article of Benitez J. A. and cols, (1999), Infection and Immunity. Feb; 67(2): 539-45, and the application of invention WO9935271A3, of

Campos and cols, 1997. Other strains with these characteristic and that additionally

® ® have auxotrophy mutations are also useful to obtain the strains with the characteristics of interest of the present invention.

In accordance with the description in the above paragraph, a primary objective of this invention is to protect the use of suppressive or spontaneous mutations conductive to the absence of the MSHA fimbria in the surface of vibrios and in this way impede that live cholera vaccine reacquire and disseminate the cholera toxin genes by means of the infection with the hybrid phage, HybP®.

Among the preferred inclusions of this invention are any live cholera vaccine strain of the existing biotypes and serotypes or any non toxigenic strain of another emergent serotypes with genetic manipulations that suppress the genome of the CTX® phage, inactivate the hap gene, combined with any other mutation, for example the introduction of some auxotrophies (to lysine or metionine) and that also have the characteristics proposed in the present invention.

Among the preferred inclusions are also the use of the well tolerated live cholera vaccines, improved by the impossibility of acquiring the CTX® in an event mediated for

VGJ® and for the absence of VGJ® that diminish the risk of dispersion of CTX®, as a delivery system to present heterologous antigens to the mucosal immune system.

To obtain these mutants in the expression of the MSHA fimbria we have used several molecular biology techniques which are not object of protection of the present document.

The present invention also discloses the methods to preserve and lyophilizate these strains with the purpose of being able to prepare live vaccines that present a rapid and adequate reconstitution post lyophilization without affecting their viability when being reconstituted in a solution of sodium bicarbonate 1,33%.

It is also the object of the invention that by means of the adequate selection of components, the lyophilized formulations guarantee that live cholera vaccines does not decrease their viability less than 1 logarithmic order as consequence of the storage, independently of the serogroup, serotype or biotype or the mutations they have, even if they were lyophilized for separate or mixed as part of a same preparation.

Among the formulation components to be present are lactose (L), peptone (P), yeast extract (E) and sorbitol (S). The total concentration should not exceed the 10%.

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Example 1: Discovery and characteristics of the VGJ$ phage

VGJ¢ was discovered as an extrachromosomal transmissible element in total DNA preparations from Vibrio cholerae SG25-1, an 0139 strain isolated in Calcuta India, 1993 and kindly donated by professor Richard A. Finkelstein. Simple experiments showed the transmissibility of this element. Free-cell culture supernatants of the donor strain, carrying the element, was grown in standard condition like LB media (NaCl 10 g/l, triptone 10 g/l and yeast extract 5 g/l) and was able to transfer to a receptor strain that does not contain any extrachromosomal element, one genetic element of the same size and restriction map that the one was present in the donor strain. The property of transmission without the direct contact between donor-receptor is typical in phages.

Infection assays: Donor strains were grown until optical density to 600nm equal 0.2.

One aliquot from the culture was filtered through a 0.22 um- pore-size filter to remove the bacteria. The sterility of the filtrate was confirmed by growing one aliquot in LB plates and incubating overnight at 37°C. After checking the lack of colony forming units, 100 ul of free-cell supernatants or serial dilutions were used to infect 20 ul of a fresh culture of the receptor strain. The mixture was incubated for 20 min at room temperature and spread in solid or liquid LB media at 37 °C overnight. The infection was confirmed by the presence of replicative form (FR) and single strand DNA (ssDNA) of

VGJ¢ in the infected vibrios.

Purification of VGJ¢ phage: Purification of phage particles was done from 100 ml of the culture of 569B Vibrio cholerae strain (classic, Inaba) infected with VGJé. This strain was used because it contains a CTX¢ defective prophage. The cells were centrifuged at 8000 x g for 10 min. The supernatant was filtered through a 0.22 um membrane. Phage particles in the filtrate were precipitated by addition of NaCl and polyethylene glycol 6000 to a final concentration of 3 and 5 % respectively. The mixture was incubated in ice for 30 min and centrifuged at 12000 x g for 20 min. The supernatant was discarded, and the phage-containing pellet was suspended in 1 ml of phosphate buffered saline.

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Characterization of VGJ¢: VGJ¢ particles precipitated retained the capacity to infect 569B strain and were stable in PBS solution during at least 6 month at 4°C.

After phage particles purification, the genomic DNA was extract using phenol- chloroform solution. The analysis of this DNA showed resistance to digestion with ribonuclease H indicating that the genome is DNA and not RNA (data not shown) and it was also resistant to the treatment with different restriction enzyme but sensitive to treatment with Mung-Bean and S1 nuclease (data not shown), indicating that the phage genome consists of ssDNA. An electrophoresis analysis in the presence of acrydine orange demonstrated similar results to the previous ones. The acrydine orange intercalated in the double stranded DNA (dsDNA) fluoresce green, while fluoresce orange when intercalates in the ssDNA. As expected, the genomic DNA fluoresced orange indicating its single stranded nature (data not shown) and the plasmid DNA observed in the infected cells fluoresced green indicating that it consists of dsDNA.

Identity between the genome of VGJ¢_and the intracellular replicative form. Southern blotting analysis carried out using the genome of VGJ¢ as a probe showed a genetic identity between the extrachromosomal elements of the donor strain SG25-1 and the infected strain 569B. This result confirms that the ssDNA of the viral genome is produced by the cytoplasmic RF and at the same time suggests that VGJ¢ is a filamentous phage, which uses the rolling circle mechanism of replication to produce the genomic ssDNA that is assembled and exported in phage particles.

The RF, isolated from the infected strain 569B, was mapped by restriction analysis.

The map obtained showed that the phage genome size (about 7500 b) and the electrophoretic restriction pattern were different to those of the previously reported V. cholerae-specific filamentous phages. These results indicated that the phage isolated from SG25-1 was not described previously and it was designated VGJ¢.

Titration of VGJ¢. For tittering the phage suspensions the procedure was the same as the infection assay, but the indicator strain cells were plated onto an overlay of soft agar (0,4%) over solid LB plates. The plates were incubated overnight at 37°C and the observed opaque plaques (infection focuses) were counted.

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This assay revealed that a culture of 569B infected with VGJ¢ is able to produce until 3x10" phage particles per ml of culture, what is unusually high compared with other described filamentous phages of V. cholerae like CTX¢, which produces a maximum of 10° particles per ml.

Electron microscopy. Different quantities of VGJ¢ particles were negatively stained with a solution of 4% uranile acetate (m/v) and observed over a freshly prepared

Formvar grids in a transmission electron microscope JEM 200EX (JEOL, Japan). The observation confirmed that the phage particles had a filamentous shape (Fig. 1).

Construction and titration of VGJ-Kné. The RF of VGJ¢ was linearized by its unique

Xbal site. One DNA fragment containing the R6K replication origin and a kanamycin resistance cassette from pUC4K plasmid was inserted in the Xbal site of VGJ¢. This recombinant RF was introduced in V. cholerae 569B and the phage particles were designated as VGJ-Kn¢.

The donor strain, 569B infected with VGJ-Kn¢, was cultured until an ODgg=2.0. An aliquot of the culture was filtered through a 0.2 um-pore-size filter to eliminate the bacterial cells. The sterility of the cell-free suspension was checked by plating an aliquot of 50 ul in a solid LB plate and incubating overnight at 37°C. Aliquots of 100 ul of the cell-free phage suspension or dilutions of it were used to infect 20 ul of a fresh culture of the receptor strain (about 10° cells). The mixture was incubated at RT for 20 min to allow infection. Subsequently, the mixtures were plated onto solid LB supplemented with kanamycin (50 ug/ml) and the plates were incubated overnight at 37°C. The colonies that grow in the presence of antibiotic acquired their Kn-resistance due to the infection with the marked phage VGJ-Kn¢. Several of these colonies were checked for the presence of the RF of VGJ-knd by purification of plasmid DNA and restriction analysis of it.

Titration assay done by this method agreed with those obtained by that of opaque plaques with VGJ¢, showing that a culture of 569B infected by VGJ-kn$ produces about 2 x 10" particles of phage VGJ-kn¢ per milliter of culture.

Nucleotide sequence: The nucleotide sequence of VGJ¢ consisted of 7542 nucleotides and had a G+C content of 43.39%. The codified ORFs were identified and compared to protein data bases.

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The genomic organization of VGJ® was similar to that of previously characterized filamentous phage, such as phages of Ff group (M13, fd and f1) of E. coli and other filamentous phages of V. cholerae (CTX®, fs1, fs2 and VSK) and V. parahemolyticus (VH12, Vi33 and VfO3k6). VGJ® does not have a homologous gene to the gene IV of phages of Ff group which suggests that VGJ® could use a porine of the host for assembling and exporting its phage particles, similar to CTX® phage.

The nucleotide sequence of VGJ® revealed that VGJ® is a close relative of fs1 and

VSK phages, sharing several ORF highly homologous and exhibiting 82.8 and 77.8% of

DNA homology to VSK and fs1. However, there are genome areas highly divergent and

ORFs not share between them. Besides, the genome size is different and it has not been described before that fs1 or VSK being capable of transducing the genes of cholera toxin.

The nucleotide sequence of VGJ® also revealed the presence of two sites homologous to att sequences known to function in integrative filamentous phage. These sites of VGJ® are partially overlapped and in opposite directions. This arrangement was also found in phages Cflc, Cf16-v1 and ®LF of X. campestris as well as Vf33 and

VfO3k6 of V. parahemolyticus and VSK of V. cholerae. All these phages except Vf33 and VSK integrate in the chromosome of their hosts by the att site present in the negative strand of the replicative form of these phages.

Example 2. Identification of VGJ® receptor.

Filamentous phages generally use type IV pili as receptor to infect their hosts.

Previously reported V. cholerae-specific filamentous phages use TCP or MSHA pili as receptor. Therefore, two mutants of the El Tor strain C6706 for these pili, KHT52 (AtcpA10) and KHT46 (AmshA), were used to identify if any of them was the receptor of

VGJ®. While parenteral strain C6706 and its TCP-mutant KHT52 were sensitive to the infection with VGJ®, the MSHA-mutant KHT46 was fully resistant to the phage, indicating that MSHA was the receptor of VGJ®. Complementation of strain KHT46 with wild type mshA structural gene (from parental C6706) carried on plasmid pJM132 restored phage sensitivity, confirming that MSHA is the receptor for VGJ®. The

® ® resistance or sensivity to VGJ® was evaluated by the absence or presence of replicative form in cultures of receptor strain analyzed after the infection assay.

To give a numerical titer of the particles which are transduced in each case, it was used an infection assay with VGJd-kn as was described previously, resulting the following:

The parental strain C6706 and its derivative TCP mutant KHT52 were sensitive to the infection with VGJ®-Kn and, as indicator strains showed titres of 10M plaque forming units (PFU), while KHT46, a MSHA mutant, was fully resistant to the phage, less than 5 PFU/mL, after being infected with the same preparation of VGJ®-Kn.

Complementation of strain KHT46 with wild type mshA structural gene, restored phage sensitivity. These results confirm that a mutation that prevents the expression of MSHA pilus confers resistance to the VJG® infection.

Further assays to compare the capacity of HybP® and CTX® to infect Clasical and El Tor strains were done, using their kanamycin resistant variants. See the results in Table 1.

As it has been previously described, CTX®-Kn phage was obtained through the insertion of a kanamycin resistance cassette from the plasmid pUC4K (Amersham

Biosciences), in the unique restriction site, Nofl, of the replicative form of CTX®.

The HypP©® phage was obtained during an infection assay where cell free culture supernatant of 569b strain co-infected with CTX®-Kn and VGJ®-Kn was used to infect the receptor strain KHT52. The cells of this strain carrying kanamycin resistance, originally carried by CTX®-Kn and provided to HybP®, were purified and, they continued producing HybP® viral particles to the supernatant.

To check the efficiency of infection of the hybrid phage in Classical and El Tor vibrios, suspensions of CTX®-Kn and VGJ®-Kn of the same title (1-5x 10" particles/mL) were used to infect the receptor strains 569B (Classical) and C7258 (El

Tor). In both cases, the receptor strains were grown in optimal condition for TCP expression, the CTX® receptor. The assay was done as follows, 200 pL of pure phage preparation were mix with 20uL (about 108 cells) of a fresh culture of a receptor strain during 20 min at room temperature, plated on solid LB supplemented with kanamycin and incubated over nigh at room temperature.

® ®

The numbers of colonies carry the Kn-resistance gene in their genome is the result of phage infections and show the capacity of each phage to infect different strains in routine laboratory condition. Those results are exposed in Table 1.

Table 1. Titration of CTX¢-Kn and hybrid (HybP®-Kn) phages in 569B and C7258.

Phage

I ce | sexi | 0

As it is shown in Table 1 the hybrid phage transduces CT genes more efficiently than CTX®, the ordinary vehicle of these genes. These results point out the importance of the CTX® transmission mediated by VGJ® among Vibrio cholerae strains and stressed its relevance considering the ubiquity of MSHA, the functional receptor in these bacterial strains.

Example 3. Mobilization of CTX®, its mechanism and reversion to virulence.

Infection of V. cholerae O1 or 0139 strains that carry an active CTX® phage with

VGJO gives rise to the production of infective particles that bear the CTX® phage genome inserted in the genome of VGJ®. These particles of hybrid phages have been designated HybP®. The HybP® titers were evaluated by means of the use of a hybrid phage, which carries a kanamycin marker (HybP®-Kn), employing different strains as indicators. The resultant titers are shown in Table 1.

HybP®-Kn was purified starting from preparations derived of 569B (HybP®-Kn) strain and the single strand was sequenced to determine the junctions between CTX® and VGJ®. The cointegrate structure is graphically shown in the Figure 2 and the nucleotide sequences of the junctions among both sequences, what explains the mechanism by which VGJ® transduces CTX® toward other V. cholerae strains.

HybP®-Kn enters to V. cholerae using the same receptor that VGJ®, that is to say

MSHA.

®

V. cholerae 1333 strain is an attenuated clone described in the previous art, similar to the strains that were useful for obtaining the derivative of the present invention. This strain is a derivative of the pathogenic C6706 strain. As it shows in the

Figure 4, the inoculation of 10° colony-forming units of 1333 strain in suckling mouse does not have lethal effect, even when it is colonizing for the subsequent 15 days.

Several experiments to determine virulence, demonstrated the effect of the HybP®-Kn infection on the reversion to virulence. While a dose of 10° CFU of 1333 strain does not have a lethal effect, C6706 and 1333 (HybP®-Kn) strains have very similar lethality profiles, and don't allow survival of inoculated mouse beyond the fifth day (Figure 4).

Example 4. Constitutive expression of the VGJ® receptor, the MSHA fimbria, in different culture conditions.

To study the expression of MshA, the major subunit of MSHA fimbria, V. cholerae 1s C7258, C6706, and CA401 strains, were grown in different media. The media used were: LB pH 6.5 (NaCl, 10 g/l; bacteriological triptone, 10 g/l; yeast extract, 5 g/l), AKI (bacteriological peptone, 15 g/l; yeast extract, 4 g/l; NaCl, 0.5 g/l; NaHCO3, 3 g/l), TSB (pancreatic digestion of casein, 17 g/l; papaine digestion of soy seed, 3.0 g/l; NaCl, 5 g/l; dibasic phosphate of potassium, 2.5 g/l; glucose, 2.5 g/l), Dulbecco's (glucose, 4.5 g/l; HEPES, 25 mm; pyridoxine, HCI, HaHCO3), Protein Free Hybridoma Médium (synthetic formulation free of serum and proteins, suplemented with NaHCO3, 2.2 gf; glutamine, 5 mg/l; red phenol, 20 g/l) and Syncase (NaH2PO4, 5 g/l; KH2PO4, 5 g/l; casaminoacids, 10 g/l; sucrose, 5 g/l and NH4CI, 1.18 g/l). In all cases was inoculated one colony in 50 ml of culture broth and was grown in a rotary shaker during 16 hours at 2s 37°C, with the exception of the AKI condition in which the strains were grown first at 30°C in static form during 4 hours and later on rotator shaker at 37°C during 16 hours.

In each case, the bacterial biomass were harvested by centrifugation and used to prepare cellular lisates. Equivalent quantities of cellular lisates were analyzed by

Western Blot with the monoclonal antibody 2F12F1 for immunodetection of mshA. The 50 MSHA mutant strain KHT46 was used as negative control of the experiment. All the studied strains, except the KHT46 negative control strain, showed capacity to produce

MshA in all culture conditions tested. Equally, said strains cultured in the previous

® ® conditions have the capacity to hemagglutinate chicken erythrocytes (mannose sensitive), in the same titer or higher to 1:16 and are efficiently infected by VGJO-Kn, exhibiting titers higher than 10" particles per milliliter of culture. s Example 5. Obtaining of spontaneous mutants deficient in MSHA expression and evaluation of resistance to infection

Strain KHT46, a MSHA suppression mutant, derived from V. cholerae C6706 (O01, The Tor, Inaba), shows a refractory state to the infection with VGJ®, VGJ®-Kn and the hybrid HybP® phages. However, this is a pathogenic strain that is not property of the authors of the present application, neither of the juridical person who presented it,

The National Center for Scientific Research, in Havana City, Cuba.

To obtain the spontaneous mutants deficient in the expression of superficial

MSHA of the present application, was used a suppression mutant in the cholera toxin genes that during the process of obtainment resulted affected in their capacity to assemble MSHA in the cellular surface. Said mutants although are capable of producing the structural subunit of MSHA, do not assemble it in their surface and therefore do not have detectable titers of mannose sensitive hemagglutination, neither adsorb the activity of a specific monoclonal antibody against the MSHA in a competition ELISA.

Since this phenotype is notably stable, these mutants were subsequently genetically manipulated to introduce an insertional mutation in the hemagglutinin protease gene, following the procedure described in patent WO 99/35271 “V. cholerae vaccine candidates and the methods of their constructing” of Campos et al, and in the Robert's article, Vaccine, vol 14 No 16, 1517-22, 1996. The resultant mutants were named

JCGO01 and JCGO2, both of O1 serogrup, El Tor biotype, Ogawa serotype.

JCG01 and JCGO02 showed a refractory state to the infection with the VGJ®-Kn phage, a variant of the VGJ® phage that carries a resistance marker to kanamycin. A

VGJ®-Kn suspension that had a proven titer of ~ 10" units per mi, does not show capacity to infect said strains (non detectable titers, lower to 5 units for ml). This refractory state to the infection with VGJ®-Kn correspond with a very low titer of hemagglutination in the strains JCG01 and JCGO2 (1:2) regarding their parental (1:32) besides a total impairment in the MSHA dependent hemaglutination. Equally, whole cells of these mutants had null capacity to inhibit the interaction of the anti-MSHA

® ® monoclonal antibody (2F12F 1) to MshA fixed on the solid phase in a competition ELISA.

However, both strains produced the major structural subunit MshA, according to immunoblot experiments, indicating that the protein is not correctly assembling in the cellular surface although it is being produced. These mutants allowed proving the concept of this invention and passing to obtain suppression mutants.

Obtaining suppression mutants in the mshA gene starting from other cholera vaccine candidates. To obtain suppression mutants in the mshA structural gene, two segments of the genome of V. cholerae N16961, of ~ 1200 base pairs for each flank of the mshA structural gene were amplified by means of the polimerase chain reaction, using the following oligonucleotides: CNC-8125, ATG ATC GTG AAG TCG ACT ATG (21 mer); CNC-8126 CAG CAA CCG AGA ATT HERE ATC ACC ACG (27 mer); CNC- 8127, ATT CTC GGT TGC TGG AAC TGC TTG TG (26 mer); and CNC-8128, GCT

CTA GAG TAT TCA CGG TAT TCG (24 mer). The amplified fragments were cloned independently and assembled in vitro to generate the pAmshA clone. This clone contains these fragments in the same order and orientation that they are found in the bacterial chromosome; only the coding region of the mshA gene has been suppressed from the inner of the sequence. The fragment carrying the suppression was subcloned from the previous plasmid as a Sal I/’Xba | fragment in the suicide vector pCVD442 to obtain the plasmid pSAmshA.

The plasmid pSAmshA was used to suppress the chromosomal mshA gene in the V. cholerae vaccine strains by means of a traditional methodology of allelic replacement. For it, pSAmshA was introduced in the E. coli strain SM10Apir and mobilized toward V. cholerae by means of a procedure of bacterial conjugation. The resultant clones were selected for their resistance to the ampicillin antibiotic in plates of

LB medium supplemented with ampicillin (100 ug/ml). Most of these clones arise due to integration of the plasmid in the chromosome of the receptor vibrios by means of an event of homologue recombination between one of the flanking fragments to the chromosome mshA gene and that of the plasmid pSAmshA, originating a cointegrate between both. This event was verified by means of a Southern blot experiment, in which the total DNA of 10 clones was digested with the restriction enzyme Sma | and hybridized with a probe obtained from the plasmid pSAmshA (Sal I/Xba | insert). The clones of our interest are those that produce a band of 21 000 base pairs. A similar

® ® control of the parental strain in this experiment produced a band of 13 000 base pairs.

The adequate clones were conserved immediately in LB glycerol at -70°C. Then 3 of them were cultured in the absence of the antibiotic selective pressure to allow that an event of homologue recombination eliminated the genetic duplication existing. This can happen by means of suppression of the original genetic structure (intact mshA gene) and replacement by a mutated copy present in the plasmid (supressed mshA gene) as is shown in Figure 3. The clones in which the mutated gene replaced the intact gene were analyzed by Southern blot and identified by the presence of a band of 12 000 base pairs. Finally, the clones where the mshA gene was suppressed were selected and conserved appropriately as vaccine candidates (freezing at -80°C in LB supplemented with 20% glycerol). This procedure was performed with each clone where the mshA suppression mutant was constructed.

Serological characterization. After the introduction of each mutation in the vaccine strains described in this document, each derivative was checked for the correct expression of the lipopolysaccharide corresponding to the original serotype. For that, cells were collected from a fresh plate, resuspended in saline (NaCl, 0.9%) and immediately examined with an appropriate agglutination serum, specific for Ogawa,

Inaba or 01389 vibrios.

The major immune response generated by an anti-cholera vaccine, is against the

LPS, therefore the expression of the antigen corresponding to each one of the strains presented in this invention was confirmed by agglutination with specific antiserum.

Colonization assay in suckling mice. The colonization assay in suckling mice (Herrington et al., J. Exper. Med. 168: 1487-1492, 1988) was used to determine the colonizing ability of each strain. An inoculum of 10°-10° vibrios in a volume of 50 pl was administered by orogastric route to groups of at least 5 suckling mice. After 18-24 hours at 30°C the mice were sacrificed, the intestine was extracted and homogenized, and dilutions were plated in appropriate media for the growth of mutants.

Table 2. Colonizing capacity of the vaccine strains of the present invention. _ Strain___Inoculum __ Colonizing Genotype g Genotype

BLRO1 1.0x10 28x10 ACTX®, hap::.celA, AmshA

BLRO2 2.0x 10° 42x10* AVGJOX®D, hap::celA, lysA, AmshA

BLRO3 1.2 x 10° 8.0 x 10° ACTX®, hap::celA, metF, AmshA

EMGO1 3.0x10 80x10 ACTX®, hap::celA, AmshA

EMGO02 2.5x10° 3.0x 10° AVGJOX®D, hap::celA, lysA, AmshA

Co

PCT/CU2004 /000002

EMGO3 ~~ 4.0x10° 5.0 x 10° ACTX®, hap::celA, metF, AmshA

JCGO1 2.0x10 6.0 x 10” ACTXO, hap:.celA, MSHA

JCGO02 1.0 x 10° 6.0 x 10 ACTX®, hap::celA, MSHA

JCGO3 1.0x10 1.0x 10 ACTX®, hap::celA, AmshA

EVDO1 3.0x 10° 3.0x 10° AVGJOX®D, hap:celA, thyA, AmshA

KMDO1 1.0x 10° 7.0x 10° ACTX®, hap::celA, metF, AmshA

KMDO02 2.0x 10° 5.0x 10° ACTX®, hap::celA, lysA, AmshA

ESP06 1.7 x10° 6.0 x10° ACTX®, hap::celA, 4YC0934,

JCGO04 1.0x10 2.0x10 ACTX®, hap::celA, AmshA

ESPO1 1.0x 10° 50x 10° AVGJOX®, hap::.celA, metfF, AmshA

ESP02 6.0 x 10° 4.0x 10° ACTX®, hap:celA, lysA, AmshA

ESP04 8.0 x 10° 1.0x 10° ACTX®, hap::.celA, AVC0934,

RAFO1 3.1x10 5.0x10 ACTX®, hap::.celA, AmshA : EVDO2 2.8x 10° 3.1x 10° AVGJDXOD, hap::celA, thyA, AmshA

ESPO3 15x 10° 2.0 x 108 ACTX®, hap:.celA, metF, AmshA

KMDO03 2.3x 10° 3.4 x 10° ACTX®, hap::celA, lysA, AmshA

ESP05 2.1x10° 2.3x 10° ACTX®, hap::celA, AVC0934,

TLPO1 23x 10° 3.2x 10° ACTX®, hap::celA, AmshA

TLPO2 3.4x105 9.4 x 10° AVGJIOXD, hap:.celA, lysA, AmshA

TLPO3 2.7 x10° 8.8 x 10° ACTXD, hap::celA, metF, AmshA

All the strains showed adequate colonizing capacity to be used as live vaccine candidates. The colonization is needed to generate a strong immunological response because the local multiplication of the bacteria increases the duration of interaction with the mucosal immune system. In this case, although a perfect model for cholera does not exist, the suckling mice gives an adequate approach to what can be the subsequent colonization of each strain in humans.

Motility assay. The cells of a well isolated colony are loaded in the tip of a platinum loop from a master plate toward a plate for the motility detection (LB, agar 0.4%), introducing the tip of the loop 2-3 mm in the agar. The diameter of dispersion of each colony in the soft agar to 30°C is measured at 24 hours of incubation. A bacterial strain that reaches a diameter of 3 mm or less from the point of inoculation is considered as non-motile. A bacterial strain that grows in a diameter beyond 3 mm is considered as motile. All the strains included in this invention resulted to be motile.

Example 6. Methods to select and construct the vaccine candidates useful as startin strains to be modified by the procedure disclosed in the present invention.

Five pathogenic strains in our collection were selected as starting microorganism due to their lack of hybridization with VGJ® sequences. These strains are V. cholerae 21

AMENDED SHEET

A

C7258 (01, El Tor, Ogawa, Pert, 1991), C6706 (O1, El Tor, Inaba, Pera, 1991),

CRC266 (0139, La India, 1999), CA385 (Clasico, Ogawa) y CA401 (Clasico, Inaba).

The procedures disclosed in this example are not the subject of the present invention. They rather constitute a detailed description of the methods used to obtain attenuated strains that are the substrate to construct the mutants claimed in the present invention. These mutants being characterized in that they are refractory to infection by

VGJ® and the hybrid VGJO::CTX® are obtained by the methods described in the examples 4 and 5.

Below we describe the suicide plasmids used to introduce different sets of mutations into V. cholerae by allelic replacement before they are suitable to be modified by the methods of the present invention. The reader should note that the strains claimed in the present invention have in addition to the mutation that impairs the correct expression of MSHA fimbriae (a) a deletion mutation of the cholera enterotoxin genes or the entire CTX® prophage and (b) the hemaglutinin protease gene interrupted with the

Clostridium thermocellum endoglucanase A gene. They can also have additionally and optionally mutations in the genes (c) lysA, (d) metF, (e) VC0934 (coding for a glycosil transferase) and (f) thyA. (a) To construct atoxigenic strains by inactivation of the cholera enterotoxin genes or deletion of the CTX® prophage, the suicide plasmid used was pJAF (Benitez y cols, 1996, Archives of Medical Research, Vol 27, No 3, pp. 275-283). This plasmid was obtained from plasmid pBB6 (Baudry y cols, 1991, Infection and Immunity 60:428), which contains a 5,1 kb insert from V. cholerae 569B that encodes ace, zot, ctxAy ctxB.

Due to the absence of RS1 sequences 3' to the ctxAB operon in Classical vibrios, the

EcoR | site downstream to the ctxAB copy in this plasmid lies in the flanking DNA of undefined function. The plasmid pBB6 was modified by deletion of the Scal internal fragment to create plasmid pBSCTS5, which now contains a recombinant region deprived of the zot and ctxA functional genes. Then the Pstl of pPBSCT5 was mutated into EcoRI by insertion of an EcoRI linker to obtain pBSCT64 and the resultant EcoRI fragment was subcloned into the EcoRI site of pGP704 to obtain pAJF. (b) To construct strains affected in the expression of HA/P the suicide plasmid pGPH6 was used. This plasmid was constructed in different steps. First, plasmid pCH2 (Hase y Finkelstein, 1991, J. Bacteriology 173:3311-3317) that contains the hap gene in

¢ ® a 3,2 kb Hindlll fragment from V. cholerae 3083 was linealized by the Stul site, which is situated in the hap coding sequence. The 3.2 kb Hindlll-fragment containing the celA gene was excised from plasmid pCT104 (Cornet y cols, 1983, Biotechnology 1:589 - 594) and subcloned into the Stul site of pCH2 to obtain pAHC3. The insert containing of pAHC3, containing the hap gene insertionally inactivated with the celA gene, was subcloned as a Hindlll fragment to a pUC19 derivative that have the multiple cloning site flanked by Bglll sites to obtain plJHCI. The Bg! Il fragment of this plasmid was subcloned into pGP704 to originate pGPHS6, which contains a 6.4 kb fragment with the genetic hap::celA structure, where the hap gene is not functional. (c) y (d) When constructing mutants in the lysA or metfF genes, the suicide plasmids pCVIysAA1 or pCVMACIal were used. To construct these plasmids, the lysA y metF genes were PCR amplified from V. cholerae C7258, using a pair of oligonucleotides for each gene. The oligonucleotides were purchased from Centro de

Ingenieria Genética y Biotecnologia, Ciudad de La Habana, Cuba. The nucleotide sequesnces of the primers were: (lysA): (P 6488) 5'-GTA AAT CAC GCT ACT AAG-3' and (P 6487) 5'-AGA AAA ATG GAA ATGC-3' and (metF): (P 5872) 5-AGA GCA TGC

GGC ATG GC-3' and (P 5873) 5'-ATA CTG CAG CTC GTC GAA ATG GCG-3'. The amplicons were cloned into the plasmids pGEM®T (Promega) and plJ2925 (Janssen y cols, 1993, Gene 124:133-134), leading to the obtainment of the recombinant plasmids pGlysA3 y pMF29, which contain active copies of the lysA y metF genes, respectively.

The identity of each gene was checked by nucleotide sequencing.

The metF and IlysA genes cloned were mutated in vitro by deletion of the respective Clal (246 base pairs) and Pstl/Accl (106 base pairs) inner fragments, respectively. In the last of the cases the strategy was designed to keep the open reading frame leading to an inactive gene product to avoid exerting polar effects during and after construction of a /ysA mutant of V. cholerae. Each inactivated gen was cloned as a Bgl Il fragment in the suicide vector pCVD442 for the subsequent introduction into the cholera vaccine candidates of interest. The suicide plasmid containing the lysA alelle was termed pCV/ysAA1 and the one containing the metfF alelle was denominated pCVMACIal. (e) When constructing mutants of the VC0934 gene we constructed and used the suicide plasmid pCVDA34. In doing that, the VC0934 gene was PCR amplified using as

¢ ® template total DNA from strain N16961 and the primers: 5-GCA TGC GTC TAG TGA

TGA AGG-3' y 5-TCT AGA CTG TCT TAA TAC GC-3'. The amplicon was cloned into the plasmid pGEM5Zf T-vector to obtain plasmid pGEM34; a 270 base pair deletion was performed inside the VC0934 coding sequence using the restriction enzymes Narl/Bglll.

After flushing the ends with klenow and subsequent recircularization the plasmid obtained was named pG34. The resultant inactive gene was subcloned into the suicide vector pCVD442 digested with Sall and Sphl to obtain the plasmid pCVA34. This plasmid was used to make the allelic replacement of the wild type gene. (fy) When constructing mutants defective in thyA expression the suicide plasmid pEST was constructed and used. The steps to construct this plasmid comprised the cloning of the thyA gene from V. cholerae C7258 into pBR322 as an EcoRI-Hindlll cromosomal DNA fragment, to obtain pVT1 (Valle y cols, 2000, Infection and Immunity 68, No 11, pp6411-6418). A 300 base pairs internal fragment from the thyA gene, comprised between the Bg/ll and Mlul, sites was deleted from this plasmid to obtain pVMT1. This deletion removed the DNA fragment that codes for amino acids 7 to 105 of the encoded protein Thimidilate syntase. The mutated thyA gene was excised as an

EcoRI-Hindlil fragment, the extremes were blunted and then cloned into the Smal site of pUC19 in the same orientation as the p-galactosidase gene to obtain pVTS. The resultant gene was subcloned as a Sacl fragment from pVT9 to pCVD442 and the obtained plasmid was named pEST. This final construct was used to make the allelic replacement of the wild type gene in the strains of interest.

The described suicide vectors are a modular system that can be used to introduce secuencial mutation into V. cholerae vaccine candidates.

The allelic replacement with the genes encoded by these vectors is done following the sequence of steps denoted below:

In the first step the suicide vector, containing the allele of choice among those described, is transferred by conjugation from the E. coli donor SM10Apir to the V. cholerae recipient, this last being the subject of the planned modification. This event is done to produce a cointegrate resistant to ampicillin. The clones resultant from the conjugational event are thus selected in LB plates supplemented with ampicillin (100 ug/ml).

¢ ®

The procedure for this first stage is as follows. The donor strain, SM10Apir transformed with the sucide vector of interest, is grown in an LB plate (NaCl, 10 gf; bacteriological triptone, 10 g/l, and yeast extract, 5 g/l), supplemented with ampicillin (100 ug/ml), and the receptor strain, the V. cholerae strain to be modified, is grown in an

LB plate. The conditions for growth are 37°C overnight. A single colony of the donor and one from the receptor is streaked into a new LB plate. The donor strain is streaked firstly in one direction and the receptor (V. cholerae) secondly in the opposed orientation. This perpendicular and superimposed streaking warrant that both strain grow in close contact. In the next step the plates are incubated at 37°C for 12 hours, harvested in 5 ml of NaCl (0.9 %) y 200 pl of dilutions 102 10° 10* and 10° are disseminated in LB-ampicillin-polimixinB plates, to select the V. cholerae clones that were transformed with the suicide plasmid and counterselect the donor E. coli

SM10Apir. Ten such clones resulting from each process are preserved frozen at - 80°C in LB-glicerol at 20 % to be analyzed in the second step.

In the second step, a Southern blot hybridization is performed with a probe specific for the gene subjected to the mutational process; this is done to detect the structure of the correct cointegrate among the clones conserved in the previous step.

The clones in which the suicide plasmid integrated to the correct target by homologous recombination are identified by the presence of a particular cromosomal structure. This structure contains one copy of the wild type gene and one of the mutated allele separated only by plasmid vector sequences. This particular structure produces a specific hybridization pattern in Southern blot with the specific probe that allows its identification. The appropriate clones are conserved frozen at -80°C in LB glicerol.

This second step comprises the following substeps: Firstly, the total DNA of each clone obtained in the first step is isolated according to a traditional procedure (Ausubel y cols, Short protocols in Molecular Biology, third edition, 1992, unit 2.4, page 2-11, basic protocol). Total DNA from the progenitor strain is isolated as control. Then, the total

DNA of the ten clones the progenitor strain is digested with the appropriate restriction enzymes, to be mentioned subsequently in the document. One ug of DNA are digested from each clone and the mother strain and later electrophoresed in parallel lanes of an agarose gel. The DNA content of the gel is blotted into membranes in alkaline

¢ conditions (Ausubel y cols, Short protocols in Molecular Biology, third edition, 1992, unit 2.9 A, page 2-30, alternate protocol 1).

The blots are fixed by incubation at 80°C for 15 minutes. The free sites in the membrane are then blocked by prehybridization and subsequently probed with the 5s specific probe for each mutation.

What follows are the details of the restriction enzyme, the probe (digoxigenin- labelled using the method random primed method) and the size of the hibridization fragment that identify the desired structure for the cointegrate of each clone, according to the target gene: a) For suppression mutants of the CTX® phage genes, the total DNA of clones is digested with the restriction enzyme Hind Ill, and once in the membrane is hybridized with a probe obtained starting from the Pst | - EcoR | fragment of the pBB6 plasmid. The clones of interest are the ones that have the genetic structure that origin two bands in the Southern blot, one of 10 000 base pairs and another of 7 000 base pairs. As control the parental strain origins a single band of 17 000 base pairs in the same experiment of Southern blot. b) For suppression mutants of the hap gene, the total DNA of clones is digested with the restriction enzyme Xho |, and once in the membrane is hybridized with a probe obtained starting from the Hind Ill fragment of 3 200 base pairs presents in the pCH2 plasmid. The clones of interest are those that have the genetic structure that origins a single band in the Southern blot, of 16 000 base pairs. As control the parental strain generates a single band of 6 000 base pairs in the same experiment of Southern blot. c) For suppression mutants of lysA gene, the total DNA of clones is digested with the restriction enzyme Xho |, and once in the membrane is hybridized with a probe obtained from the Sph I/Sma | fragment of the pCVTlysA1 plasmid, contained the mutated gene lysA. The clones of interest are those that have the genetic structure that origins a single band in the Southern blot, of 12 500 base pairs. As control the parental strain generates a single band of 5 200 base pairs in the same experiment of Southern blot. d) For suppression mutants of metF gene, the total DNA of clones is digested with the restriction enzyme Nco |, and once in the membrane is hybridized with a

® probe obtained from the Bg! Il fragment of pCVM:iClal, contained the mutated metF gene. The clones of interest are those that have the genetic structure that origins a single band in the Southern blot, of 12 000 base pairs. As control the parental strain generates a single band of 5 000 base pairs in the same experiment of Southern blot. e) For suppression mutants of gene VC0934, the total DNA of clones is digested with the restriction enzyme Ava |, and once in the membrane is hybridized with a probe obtained from the Sal I/Sph | fragment of pCVDZ234, contained the mutated VC0934 gene. The clones of interest are those that have the genetic structure that origins two bands in the Southern blot, one of 1 600 or 1 900 and another of 8 200 or 7 900 base pairs. As control the parental strain generates a single band of 3 500 base pairs in the same experiment of Southern. f) For suppression mutants in thyA gene, the total DNA of clones is digested with the restriction enzyme Bstx |, and once in the membrane is hybridized with a probe obtained from the Sac | fragment of pEST1, contained the mutated thyA gene. The clones of interest are those that have the genetic structure that origins a single band in the Southern blot, of 9 600 base pairs. As control the parental strain generates a single band of 2 400 base pairs in the same experiment of

Southern blot.

In the third step of the procedure, 3 clones of interest, carrying a cointegrate with - one of the previous structures, are cultured in absence of the antibiotic selective pressure to allow the loss of the suicidal vector by means of homologue recombination and the amplification of resultants clones. In said clones the loss of the suicidal vector goes with the loss of one of the two copies of the gene, the mutated or the wild one, of the genetic endowment of the bacteria.

In a fourth step of the procedure, dilutions of the previous cultures are extended in plates to obtain isolated colonies, which are then replicated toward plates supplemented with ampicillin to evaluate which clones are sensitive to ampicillin. Said clones, sensitive to ampcillin, are conserved for freezing, as described previously.

In a fifth step, by means of a study of Southern blot with specific probes for each one of the genes of interest (describe in a, b, c, d, and, f) it is verified which clones retained in the chromosome the mutated copy of the allele of interest. These clones of

¢ ® interest are expanded to create a work bank and to carry out their later characterization, as well as the introduction of the modifications object of protection in the present invention application.

In the following paragraphs we detail the restriction enzyme, the probe and the sizes of the hybridization fragments that identify the desired structure in each of the mutants, according to each of the genes being the subject of modification: a) To analyze the mutants in the CTX® prophage, the total DNA is digested with the restriction endonuclease Hind lll. Once in the membrane it is hybridized with a probe derived from the Pst | - EcoR | fragment of plasmid pBB6. Are clones of interest such that do not produce hybridization bands in the Southern blot. b) For the mutants with the inactivated allele of hap, total DNA from the clones is digested with the restriction enzyme Xho | and once in the membrane it is hybridized with a probe derived from the 3 200 base pair Hind Ill fragment from plasmid pCH; that codes for the hap gen. The clones of interest are those that produce a single band in the Southern blot, of about 9 000 nucleotide pairs. c) For the mutants in the lysA gene total ADN is digested with the restriction enzyme

Xho |, and once in the membrane it is hybridized with a probe derived from the Sph

I/Sma | fragment isolated from the plasmid pCV4lysA, that contain the lysA mutated gene. The clones of interest are those having the genetic structure that produce a single band in Southern blot of about de 5 000 pairs of nucleotides. d) For the mutants with deletions in the metF gene, the total ADN of the clones is digested with the restriction enzyme Nco |, and once in the membrane it is hybridized with a probe derived from the Bgl Il fragment contained in plasmid pCVM4Clal, that contains the metF mutant gene. The clones of interest are those that have the genetic structure that leads to a single band of 4 700 base pairs in the

Southern blot. e) For the mutants in the VC0934 gene, total DNA of the clones is digested with the restriction enzyme Ava |, and the blots are hybridized with a probe obtained from the Sal I/Sph | fragment of pCVDA34, which contains the VC0934 mutant gene obtained in vitro. The clones of interest are those having the structure leading to a single band of 3 200 base pairs in the Southern blot.

¢ f) For the mutants in the thyA gene, total DNA of the clones is digested with the restriction enzyme Bstx |, and the blots are hybridized with a probe obtained from the Sac | fragment of pEST1, which contain the thyA gene. The clones of interest are those that have the genetic structure leading to a single band in the Southern blot of about 2 100 base pairs.

Example 7. Methods to preserve vaccine strains by means of lyophilization.

In following example microorganisms were cultured in LB broth at 37°C with an orbital shaking 150 and 250 rpm until reaching the logarithmic phase. Cells were harvested by centrifugation 5000 and 8000 rpm at 4°C during 10-20 minutes and then were mixed with the formulations that show good protection features of the microorganism, so that the cellular concentration was between 108 and 10° cells mi”. 2ml were dispensed for each 10R type flask. The lyophilization cycle comprised a deep freezing of the material, a primary drying keeping each product between -30°C and -39°C for space of 8 to 12 hours and a secondary drying at temperatures between 18°C and 25°C for not more than 12 hours. The viability loss was defined as the logarithmic difference of the

CFU/mL before and after the lyophilization or before and after the storage of the lyophilized material, which is always dissolved in a 1.33% sodium bicarbonate solution.

Formulation L+E+S

The BLRO1, JCGO03 and ESPO5 strains were processed by the previously described lyophilization process in a formulation of the type L (5.0%), E (2.0%) and S (2.0%). The freezing was performed at —60°C. During the primary drying, the temperature of the product was kept at =32°C for 10 hours and in the secondary drying the temperature was kept at 22°C for 12 hours. The dissolution of the lyophilized material in a 1.33% sodium bicarbonate solution was instant. The viability loss calculated immediately after the dissolution, with regard to the concentration of live cells before the lyophilization resulted to be 0.30, 0.43, and 0.60 logarithmic orders for BLR01, JCGO03 and ESPQ5, respectively.

Comparison of the L+P+S and L+E+S formulations with that of skim milk+peptone sorbitol.

The strain JCGO03 was lyophilized using two formulations: the type L (6.0%), P (2.0%) and S (2.0%), and the other type L (6.5%), E (1.8%) and S (1.6%). This strain was also

@ ® lyophilized in a formulation of 6.0% skim milk, 2.0% peptone and 2.0% sorbitol as a comparison formulation. The freezing was done at -60°C. During the primary drying, the temperature of the product was kept at -33°C for 12 hours and in the secondary drying the temperature was kept at 20°C for 14 hours. The dissolution of the lyophilized s material in a 1.33% sodium bicarbonate solution was instant when the lyophilization process took place in the formulations of the type L+P+S or L+E+S and slightly slower when was lyophilized in the comparison formulation. The viability loss calculated immediately after the dissolution, with regard to the concentration of live cells before the lyophilization resulted to be 0.48, 0.52 and 0.55 logarithmic orders for the L+P+S,

L+E+S and the comparison formulations, respectively, significantly similar.

Humidity and oxygen effects

The strain JCGO3 lyophilized in the three formulations mentioned in the previous paragraph, was exposed immediately after being lyophilized to the simultaneous action of humidity and oxygen. This was achieved, confining the samples during 3 days at 25°C in an atmosphere in sterile glass desiccators, under an 11% relative humidity (created by a saturated solution of lithium chloride). The viability loss in the L+P+S,

L+E+S and comparison formulations resulted to be 1.61, 1.10 and 3.43 logarithmic orders, respectively, what shows that the formulations object of this invention guarantee a bigger protection to humidity and oxygen than the comparison formulation.

Effect of the storage temperature.

The strains TLPO1, JCG01 and ESP05 were lyophilized in a formulation of the type L (5.5%), E(2.0%) and S(2.0%). The freezing was done at -58°C. During the primary drying, the temperature of the product was kept at -30°C for 12 hours and in the secondary drying the temperature was kept at 20°C for 14 hours. The dissolution in a 1.33% sodium bicarbonate solution was instant. The viability loss calculated immediately after the dissolution, with regard to the concentration of live cells before the lyophilization resulted to be 0.43, 0.55 and 0.44 logarithmic orders in TLPO1, JCGO1 and ESPO5, respectively. The lyophilized material was stored 1 year either at 8°C or - 20°C. The Table 3 shows the viability loss results obtained.

Table 3. Viability loss (1 year of storage).

ee

Strain 8°C -20°C === “Jeet CT T4020 089

ESP05 0.91 0.55

Example 8. Strains of the present invention and their characteristics

The strains of the present invention have been deposited in the Belgium Coordinated 5s Collection of Microorganisms (BCCM). Laboratorium voor Microbiologie -

Bacterienverzameling (LMG):

Vibrio cholerae JCG0O1 (LMG P-22149)

Vibrio cholerae JCG02 (LMG P-22150)

Vibrio cholerae JCG0O3 (LMG P-22151)

Vibrio cholerae KMDO01 (LMG P-22153)

Vibrio cholerae KMDO02 (LMG P-22154)

Vibrio cholerae KMDO03 (LMG P-22155)

Vibrio cholerae JCG04 (LMG P-22152)

Vibrio cholerae ESP01 (LMG P-22156)

Vibrio cholerae ESP02 (LMG P-22157)

Vibrio cholerae ESP03 (LMG P-22158)

Vibrio cholerae RAF01 (LMG P-22159)

Vibrio cholerae TLP0O1 (LMG P-22160)

Vibrio cholerae TLP02 (LMG P-22161)y

Vibrio cholerae TLPO3 (LMG P-22162)

They are described in Table 4.

Table 4. Vaccine strains of the present invention.

Strain Wild type Biotype/Serotype Relevante Genotype. parental strain

BLRO1 CA385 Classical/Ogawa ACTX®, hap::celA, AmshA

BLRO2 CA385 Classical/Ogawa ACTX®, hap:.celA, lysA, AmshA

BLRO3 ~~ CA385 Classical/Ogawa ACTX®, hap:celA, metF, AmshA

EMGO1 CA401 Classical/lnaba ACTX®, hap::celA, AmshA

EMGO02 CA401 Classical/lnaba ACTX®, hap::celA, lysA, AmshA

EMG03 ~~ CAd401_ Classical/lnaba _ ACTX®, hap::celA, metF, AmshA

JCGO1 C7258 El Tor/Ogawa ACTX®, hap:.celA, MSHA

JCG02 C7258 ElTor/Ogawa _ _ ACTXO®, hapicelA, MSHA

JCGO3 C7258 El Tor/Ogawa ACTX®, hap::.celA, AmshA

EVDO1 C7258 El Tor/Ogawa ACTX®, hap:.celA, thyA, AmshA

KMDO1 C7258 El Tor/Ogawa ACTX®, hap::celA, metF, AmshA

PCT/CU2004/000002

KMDO02 C7258 El Tor/fOgawa ACTX®, hap:.celA, lysA, AmshA

ESPO6 ~~ C7258 El Tor/Ogawa ACTX®, hap::.celA, AVC0934, AmshA

JCGO4 C7258 El Tor/Ogawa ACTX®, hap::celA, AmshA

ESPO1 C7258 El Tor/Ogawa ACTX®, hap::celA, metF, AmshA

ESPO2 C7258 El Tor/Ogawa ACTX®, hap::celA, lysA, AmshA

ESP04 ~~ C7258 El Tor/Ogawa ACTX®, hap::celA, 4VC0934, AmshA

RAF01 C6706 El Tor/Inaba ACTX®, hap::.celA, AmshA

EVDO2 C6706 El Tor/Inaba ACTX®, hap::celA, thyA, AmshA

ESPO3 C6706 El Tor/Inaba ACTX®, hap:.celA, metF, AmshA

KMDO03 C6706 El Tor/Inaba ACTX®, hap::celA, lysA, AmshA

ESPOS ~~ C6706 El Tor/Inaba ACTX®, hap::celA, AVC0934, AmshA

TLPO1 CRC266 0139 ACTX®, hap:.celA, AmshA

TLPO2 CRC266 0139 ACTX®, hap:.celA, lysA, AmshA

TLPO3 CRC266 0138 ACTX®, hap::celA, metF, AmshA

Brief description of the drawings.

Figure 1. Microphotography of VGJ® phage. Magnification x 32 000. VGJ® phage was purified from the supernatants of infected Vibrio cholerae 569B.

Figure 2. Diagram of the genome of hybrid phage HybP®-kn, which has a high potentiality for cholera toxin transmission.

Figure 3. Scheme of the genetic manipulation used to suppress mshA gene of V. cholerae vaccine candidates and the suicide vector used during the proceeding.

Figure 4. Suckling Mice survival inoculated with an attenuated strain and its derivative infected with HybP®-Kn that revert it to virulence.

Advantages

The present invention provide us with a methodology to protect live cholera vaccine candidates from the reacquisition of cholera toxin genes and others toxins from the CTX® bacteriophage mediated by VGJ® phage, and therefore from the conversion to virulence by this mechanism.

Equally provide us with the necessary information to assure that live cholera vaccine candidates will not spread CTX®, in the case that these vaccine candidates reacquire CTX®, by a specialized transduction with the VGJ® phage.

The present invention provide us the application of MSHA mutants as live cholera vaccine candidates, which exhibits an increase in their environmental safety due to resistance to the infection with CTX® mediated by VGJO®. 32

AMENDED SHEET

. PCT/CU2004/000002 ®

This invention provides us with a new characteristic to keep in mind during the design and construction of live cholera vaccine candidates to improve their environmental safety, that is to say that such vaccines are not able to spread the CTX® genes, mediated by VGJ®, in the case of reacquisition.

The above characteristic could be applied to the already made live cholera vaccine candidates, which have demonstrated an acceptable level of reactogenicity in volunteers studies, to reduce their potential environmental impact.

This invention also provide formulations to preserve by lyophilization all of the above-mentioned live cholera vaccine candidates and also improve their abilities to tolerate the remainder of oxygen and humidity in the container.

These formulations also guarantee the instant reconstitution of the lyophilized live cholera vaccine candidate powder in sodium bicarbonate buffer, making easier the manipulation, protecting the vaccines during this process and improving the organoleptic characteristic, specifically related with the visual aspect of lyophilized tablets and the reconstituted products.

One of the formulations provided here for conservation and lyophilization of live cholera vaccine candidates lack the bovine components usually added to many formulations to lyophilize human vaccines. 33

AMENDED SHEET

Claims (7)

. PCT/CU2004/000002 ® CLAIMS

1. Living attenuated strains of Vibrio cholerae useful to manufacture oral vaccines of improved environmental safety, the strains characterized in that they are:

a. Strains having at least one mutation comprising the inactivation of one gene essential for the biogenesis of MSHA, the receptor to phage VGJ® (SEC ID: No 1), in order to prevent the hybrid recombinant phage VGJO::CTX® (Fig. 2) from infecting the strains and reverting them to virulence.

b. Strains lacking sequences of the VGJ® phage (SEC ID: No 1) in their genomes to impair the formation of the hybrid recombinant phage VGJO::CTX® and prevent further spreading the cholera toxin genes of CTX via this new phage.

C. Strains presented in freeze dried formulations containing the substances: lactose, sorbitol and peptone or lactose, sorbitol and yeast extract, at a total concentration not higher than 10%.

2. The living attenuated strains according to claim 1, which are characterized in that they are: ) Strains having at least one mutation comprising the inactivation of one gene essential for the biogenesis of MSHA, the receptor to phage VGJ® (SEC ID: No 1), in order to prevent the hybrid recombinant phage VGJO::CTXP (Fig. 2) from infecting the strains and reverting them to virulence, the mutation being an spontaneous mutation. 34 AMENDED SHEET

PCT/CU2004/000002

®

. Strains lacking the VGJ® phage (SEC ID: No 1) in their genomes to impair the formation of the hybrid recombinant phage VGJ®::CTX® and prevent further spreading the cholera toxin genes of CTX® via this new phage.

. Strains presented in freeze dried formulations containing the substances: lactose, 6.0%; peptone, 2.0% and sorbitol, 2.0% or lactose, 5.0%; yeast extract, 2.0% and sorbitol, 2.0%.

3. The living attenuated strains according to claim 1, the strains being one or more of the following strains of Vibrio cholerae (serogroup O1, biotipe El Tor and serotipe Ogawa), deposited with the Belgium Coordinate Collection of Microorganisms (BCCM), Laboratorium Voor Microbiologie- Bacterienverzameling (LMG):

. Vibrio cholerae JCG01 (LMG P-22149)

. Vibrio cholerae JCG02 (LMG P-22150)

4. The living attenuated strains according to claim 1, the strains characterized in 15: that they are:

a. Strains having at least one mutation comprising the inactivation of one gene essential for the biogenesis of MSHA, the receptor to phage VGJO (SEC ID: No 1), in order to prevent the hybrid recombinant phage VGJO::CTX® (Fig. 2) from infecting the strains and reverting them to 200 virulence, the mutation being a deletion introduced in the mshA gene coding for the structural subunit of MSHA fimbria.

b. Strains lacking the VGJ® phage (SEC ID: No 1) in their genomes to impair the formation of the hybrid recombinant phage VGJ®::CTX® and prevent further spreading the cholera toxin genes of CTX® via this new phage. AMENDED SHEET

. PCT/CU2004/000002

®

C. Strains presented in freeze dried formulations containing the substances: lactose, 6.0%: peptone, 2.0% and sorbitol, 2.0% or lactose, 5.0%; yeast extract, 2.0% and sorbitol, 2.0%.

5. The living attenuated strains according to claims 1 and 4, the strains being one among the following deposited with the Belgium Coordinate Collection of Microorganisms (BCCM), Laboratorium Voor Microbiologie- Bacterienverzameling (LMG):

a. Vibrio cholerae JCGO3 (LMG P-22151), serogrupo O1, biotipo El Tor, serotipo Ogawa,

b. Vibrio cholerae KMDO1 (LMG P-22153), serogrupo O1, biotipo El Tor, serotipo Ogawa,

C. Vibrio cholerae KMDO2 (LMG P-22154), serogrupo O1, biotipo El Tor, serotipo Ogawa,

d. Vibrio cholerae KMDO3 (LMG P-22155), serogrupo O1, biotipo El Tor, serotipo Inaba,

e. Vibrio cholerae JCG04 (LMG P-22152), serogrupo O1, biotipo EI Tor, serotipo Ogawa,

f. Vibrio cholerae ESP01 (LMG P-22158), serogrupo O1, biotipo EI Tor, serotipo Ogawa,

a. Vibrio cholerae ESP02 (LMG P-22157), serogrupo O1, biotipo El Tor, serotipo Ogawa,

h. Vibrio cholerae ESP03 (LMG P-22158), serogrupo O1, biotipo El Tor, serotipo Inaba, 36 AMENDED SHEET

PCT/CU2004/000002 i. Vibrio cholerae RAFO1 (LMG P-22159), serogrupo O1, biotipo El Tor, serotipo Inaba,

i. Vibrio cholerae TLPO1 (LMG P-22160), serogrupo 0139,

k. Vibrio cholerae TLPO2 (LMG P-22161), serogrupo 0139, lL Vibrio cholerae TLPO3 (LMG P-22162), serogrupo 0139.

6. Strains according to any one of claims 1 to 5, substantially as herein described and illustrated.

7. New living, attenuated strains, substantially as herein described. 37 AMENDED SHEET