AU2003208135B2 - Novel vibrio cholerae vaccine candidates and the methods of constructing - Google Patents

Description

-S.

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Centro Nacional De Investigaciones Cientificas Actual Inventor(s): Javier Campos Gomez, Rafael Alfredo Fando Calzada, Boris Luis Rodriguez Gonzalez, Talena Yamile Ledon Perez, Edgar Valle Diaz, Anisia Juana Silva Cabrera, Jorge Antonio Benitez Robles Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: NOVEL VIBRIO CHOLERAE VACCINE CANDIDATES AND THE METHODS OF

CONSTRUCTING

Our Ref: 698193 POF Code: 463671/464064 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): -1- SOo6w NOVEL VIBRIO CHOLERAE VACCINE CANDIDATES AND THE METHODS OF

CONSTRUCTING.

This is a divisional application divided out of Application No. 19591/99, tihe entire contents of which are incorporated herein by reference.

Technical sector The field of invention is that of Biotechnology and more specifically the generation of Vibrio cholerae vaccines and the methods of constructing them by using genetic engineering tools.

Background of the invention.

A brief explanation on the terminology used through the text of the invention is listed below.

By ctxT virus is meant the particle of protein-coated DNA, produced by certain Vibrio cholerae strains, which is capable of transducing its DNA, comprising cholera toxin genes, to other Vibrio cholerae strains.

By cholera toxin is meant the protein responsible for the clinical :iymptoms of cholerae when produced by the bacteria.

By ctxD-encoded toxin genes are meant, in addition to cholera toxin genes, zot and ace genes, which code for the "zonula occludens toxin" and for tho "accessory cholera enterotoxin", respectively.

For non-toxigenic strains of Vibrio cholerae it most be understood any strain devoid of the genes coding for the above toxins, which are as well, useful as vaccines but still produce an undesired reactogenic syndrome.

The term safe vaccine or safe strain refers to such strain lacking the residual reactogenicity of non-toxigenic strains of Vibrio cholerae.

By hemagglutinin/protease is meant the protein manifesting dual function, being one of them the ability to agglutinate erythrocytes from certain species and the other the property to degrade proteins such as mucin.

The term ce/A refers to the nucleotide sequence coding for the endcglucanase A protein. This protein naturally occurs in Clostridium thermocellum strains and has a p(1- 4) glucan-glucane hydrolytic activity able to degrade cellulose and its derivatives.

By-Thymidylate synthase is meant the protein capable of catalyzing "he reductive methylation of -deoxyuracil monophosphate (dUMP) by NS-N'-methylenetetrahydrofolate to yield 2,5 deoxythymidyne phosphate (dTMP) and dihydrofolate.

Substantially pure DNA is DNA that is free from both of the coding sequences immediately contiguous by the 5' or the 3' end of thyA coding sequence, in the naturally 2 occurring genome of the microorganism from which the DNA of the invention is derived.

The term thereof includes, for example, a recombinant DNA which is incoroorated on a vector strain, cell line or plasmid, or which exists as a separate molecule cDNA, restriction or PCR fragment). It also includes recombinant DNA molecules which are part of a hybrid gene encoding additional sequences.

Homologous sequences refers to DNA or protein sequences which share similar or identical residues being nucleotides or amino acids, respectively, in identical positions of two or more given strings. The greater the number of identical/similar residues in certain position, the greater the percent of identity/similarity between two them.

Clinical cholera is an acute diarrheal disease that results from an oral infection with the bacterium Vibrio choerae. After more than 100 years of researc- on cholera there remains the need for an effective and safe vaccine. Humankind 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 predominance of Vibrios belonging to El Tor Biotype. Recently, beginning in January cf 1991, this pandemic has extended to South America causing greater than 25 000 cases and over 2000 deaths in Peru, Ecuador, Colombia, and Chile. By November 1992, a new serogroup emerged in India and Bangladesh, the 0139, showing a groat epidemic potential that became a new cause of concern throughout the developing world. These recent experiences reinforce the need for effective cholera vaccines against disease caused by V. cholerae of serogroups 01 (El Tor) and 0139.

Because convalescence to cholera is followed by an state of immunity lasting at least 3 years, much of the efforts in Vibrio cholerae vaccinology have boen made to produce live, attenuated cholera vaccines, that closely mimic the disease in its immunization properties after oral administration, but do not result reactcgenic to the individuals ingesting them. Vaccines of this type involve deletion mutations of all toxin genes encoded by the ctxO Vibrio-phage. See patents of Kaper, J. et al.; WO 91,18979 and Mekalanos, WO 95,18633.

The first vaccine to be assayed against cholera dates from 1885-1892. It was a traditional vaccine that comprised administration by parenteral route of "attenuated" vibrios. It resulted limited in efficacy and unacceptably reactogenic (Finkelstein R. A., International Symposium on Cholera on the America Continent. Sao Paulo, SP, Brasil, 3 1992). Oral vaccination was tried first in 1892, using attenuated Vibrio cho;erae strains.

The results of this attempt were misinterpreted and the strategy immediately abandoned. Oral vaccination was later rescued in 1970-1980 at the Cente- for Vaccine Development of Maryland, USA, by using chemically mutagenized vibrios as immunizing agents. Reversion to virulence of these mutants impeded further spread of the strategy (Levine et al., Infect and Imm, N 0 2, 1984; Finkelstein et al., patent US 4,328,209) and prompted the researchers to generate genetically defined rnon-toxigenic mutants unable to revert. Although these mutants have shown to confer solid immunological protection against disease (Kaper J. B. and Levine M. Patents US 06,472,276 and 581,406), the essential drawback for their use is the high level of adverse reactions they produce in vaccinees (Levine et al., Infect. and Imm. Vol 56,

N

0 1, 1988). According to these data the major issue to be overcome when producing an effective cholera vaccine is safety. Additionally, researches worldwide are currently concerned on the horizontal transfer of genetic information among bacteria, thus it is necessary to pay attention to this aspect when designing live bacterial vaccines, with the aim to ameliorate the environmental impact during vaccination. It is al..o necessary to achieve good levels of stability and immunogenicity.

A dead cholerae vaccine consisting of whole cells supplemented with the B subunit of cholera toxin is available (Holmgren et al., Current topics in Microbiology and Immunology, Vol. 146, 1989). Such vaccine is safe and effective but requires multiple doses to generate an immune response equivalent to that of a cholera infection and consequently is very expensive.

Another alternative for cholera vaccination is the recent licensed CVI)103-HgR, a live cholera vaccine belonging to the classical biotype. It is safe, effective and cheap; however its protective efficacy against the current circulating El Tor and 0' 39 vibrios is not as good as against Classical vibrios (See patent USA, 5399494).

Other live vaccine candidates have been described in patent W0 95/18633.

Such mutants represent all serotypes of the current pandemic, including the, 0139. They are safe, their production is cheap, and have been shown to be preliminary effective; however they are not as extensively tested as CVD103-HgR. All these candidates are protothrophic bacteria able to survive natural conditions of the 3nvironment.

Additionally, although a procedure to obtain defined mutants is descibed in the document, the proposed candidates constitute non-motile spontaneous mutants. It has been proposed by the inventors that the non-motile nature of these vaccin.e candidates limits their ability to reach the enterocyte surface and avoid the elicitation of the reactogenic response characteristic of their parentals.

Robert et al., Vaccine, Vol. 14, N 0 16, 1517-22, 1996, demonstrated the factibility of using the hemaggiutinin/protease locus for the insertion of heterologous tags, without detriment of the colonizing capacity of vibrios. Colonization of the human small bowel by Vibrio cholerae is essential to induce a strong localized immune response of secreted IgA in the intestinal mucosa and to produce a long lasting immunity against cholera (Taylor et al., The Journal of Infectious Diseases, 1994, 170: 1518-23).

It has been wisely sustained by Dr. Mekalanos, that uptake of bacter a by Peyer's patches is a consequence of the colonization process that does not lead to reactogenicity and is considered an essential step in the localized immuie response pathway. In contrast, the interaction of bacteria with enterocytes results in adverse reactions unacceptable for vaccine purposes (Mekalanos J. et al., Bull. Inst, Pasteur, 93: 255-262,1995). According to this criteria mutations that interfere with the capacity of vibrios to reach the enterocytes are desired features of cholera vaccines.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Summary of the Invention According to a first aspect, the present invention provides a method to remove or reduce to an acceptable level residual reactogenicity from non-toxigenic mutants of Vibrio cholerae intended to be used for vaccination against cholera comprising inactivation of the hemagglutinin/protease gene (hap), by deletion, insertion or any other defined and irreversible genetic manipulation.

According to a second aspect, the present invention provides a medicinal preparation suitable for administration to humans for inducing immunological protection against cholera comprising as an essential ingredient one or more Vibrio cholerae rr r vaccine strains derived from non-toxigenic parental, the strain or strains having an acceptable level of residual reactogenicity due to the presence of a dysfunctional S..o ohemagglutinin/protease gene (hap), resulting from deletion, insertion or any other defined and irreversible genetic manipulation.

According to a third aspect, the present invention provides the use, for reducing 20 the level residual reactogenicity of a medicinal preparation suitable for administration to Shumans for inducing immunological protection against cholera, of one or more Vibrio S'cholerae vaccine strains derived from non-toxigenic parental, the strain or strains having a dysfunctional hemagglutinin/protease gene (hap), resulting from deletion, insertion or S any other defined and irreversible genetic manipulation.

According to a fourth aspect, the present invention provides a method of producing a vaccine strain suitable for administration to humans for inducing immunological protection against cholera wherein residual reactogenicity is removed or reduced to an acceptable level from non-toxigenic mutants of Vibrio cholerae by inactivation of the hemagglutinin/protease gene (hap), by deletion, insertion or any other defined and irreversible genetic manipulation and further wherein a defined and irreversible mutation is introduced into its thyA gene coding for Thymidilate synthase whereby proliferation in natural ecosystem is limited.

According to a fifth aspect, the present invention provides a medicinal preparation with improved environmental biosafety suitable for administration to humans for inducing immunological protection against cholera comprising as an essential ingredient one or more Vibrio cholerae vaccine strains derived non-toxigenic parental, the strain or strains having an acceptable level of residual reactogenicity due to the presence of a dysfunctional hemagglutinin/protease gene (hap) resulting from deletion, insertion or any other defined and irreversible genetic manipulation and further having a dysfunctional genetically modified allele of a thyA gene instead of its natural one, resulting from deletion, insertion or any other defined and irreversible genetic manipulation, whereby proliferation in natural ecosystems is limited.

According to a sixth aspect, the present invention provides the use, for reducing the level residual reactogenicity of a medicinal preparation suitable for administration to humans for inducing immunological protection against cholera, of one or more Vibrio cholerae vaccine strains derived from non-toxigenic parental, the strain or strains having a dysfunctional hemagglutinin/protease gene (hap), resulting from deletion, insertion or r any other defined and irreversible genetic manipulation and further having a dysfunctional genetically modified allele of a thyA gene instead of its natural one, resulting from deletion, insertion or any other defined and irreversible genetic manipulation, whereby proliferation in natural ecosystems is limited.

According to a seventh aspect, the present invention provides a mutant of strain 638 as defined in the second aspect which has been further manipulated to introduce a deletion within its thyA gene between the Bgl II and Mlu I restriction sites.

According to an eighth aspect, the present invention provides a mutant of strain r "1333 as defined in the second aspect which has been further manipulated to introduce a deletion within its thyA gene between the Bgl II and Mlu I restriction sites.

According ot a ninth aspect, the present invention provides a mutant of strain L911 as defined in the second aspect which has been further manipulated to introduce a deletion within its thyA gene between the Bgl II and Mlu I restriction sites.

According to a tenth aspect, the present invention provides a method to produce a dead Vibrio cholerae vaccine comprising using as a source a strain of Vibrio cholerae as defined in the fifth aspect.

According to an eleventh aspect, the present invention provides pure DNA of the thy A gene comprising the nucleotide sequence coding for the natural Thymidilate.

5b Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

Description of the Invention Vibrio cholerae mutants, despite having deletions in the cholera toxin genes (atoxigenic mutants), still produce unacceptable levels of reactogenicity in humans, which has precluded the use of several live attenuated mutants for vaccination. This invention issues a method to abolish the residual reactogenicity of non-toxigenic mutants of Vibrio Cholerae by inactivation of the hemagglutinin/protease gene (hap), which is useful to obtain a safer, genetically defined, and stable mutant of Vibrio cholerae useful as a live, oral vaccine for inducing immunological protection against cholera in humans.

Such mutant derives from a non-toxigenic strain of Vibrio cholerae by disruption of the hemagglutinin/protease gene (hap) with the marker gene celA. In preferred r 15 embodiments of this document the vaccine strain belongs to either of the two serotypes f of the El Tor Biotype or to the 0139 serotype of Vibrio cholerae. Preferably this strain c- t- .derives from a non-toxigenic mutant of Vibrio cholerae, obtained by means of genetic engineering tools and is tagged with the marker gene celA within the hap locus. In the .most preferred fashion the strain is 638, 1333, or L911.

20 This invention additionally includes a method to enhance the environmental c biosafety of any strain of Vibrio cholerae intended to be used as a live or as the source 'for large scale production of dead vaccines. The method described herein involves ,I cloning and genetically manipulating the thyA gene to perform an internal deletion, and Lthe exchange of the wild type copy in the chromosome by the mutated allele created "in vitro".

Among preferred embodiments of this invention results any vaccine strain of Vibrio cholerae belonging to. existent biotypes and serotypes or any emergent serotype against which current vaccines are not effective and which has been obtained by means of genetic manipulations in the genes coding for HA/P and Thymidilate Synthase, to render a double mutant with improved environmental biosafety features. More preferably the mutant is a derivative of 81, 413 or SG251a, and in the most preferable way the strain is 638T or the thyA~ derivatives of 1333 and L911. As well this contained version of vaccine strains is useful as an entity for antigen delivery to the mucosal immune system.

We have found such mutants to be low reactogenic in laboratory and/or clinical tests, although yet able to elicit strong immune responses when adminislered by oral route. As a result these hemagglutinin/protease defective mutants and their further derivatives share the desirable properties for a vaccine against cholera in humans.

Additionally, the invention provides substantially pure DNA encod ng the thyA gene for Vibrio cholerae Thymidilate Synthase. By thyA DNA is meant the DNA sequence shown in figure 1, SEQ. ID. No 1, and fragments, deletions, disruptions and homologous sequences thereof.

The construction of vaccine strains described herein involves replacement of the chromosomal gene coding for the wild type Hemagglutinin/Protease by a ce/A disrupted allele in non-toxigenic mutants of Vibrio cholerae having deletec all genes encompassing the ctx4 prophage. These mutants are genetically well defined and very stable in their proteolytic defect, showing no reversion detectable in 10 9 cells. Said mutants are equally stable in their cellulolytic activity conferred by the ce/A chromosomal marker, even after passage by the intestine of mice or humars.

Construction of further derivatives with enhanced environmenlal biosafety L features involves introducing a genetic deletion in the thyA gene of the mutant, to obtain a thymine/thymidine auxotrophic derivative. The resultant triple mutant is genetically well defined and stable. As a consequence of this mutation the strain is provided with a resistance marker to the antibiotic trymethoprim, which is conditioned to the presence of thymine/thymidine. Such marker is unlikely to transform other bacteria by horizontal transfer due to its recessive nature.

This invention issues non-toxigenic, genetically defined, stable, and safe mutants of Vibrio cholerae which are useful as a live, oral vaccines for inducing immunological protection against cholera in humans. When designirg our nonreactogenic cholerae vaccine candidates we have stuck to the idea of reactogenicity as a consequence of interaction between Vibrio cholerae and enterocytes. We reasoned that inactivation of the major secreted protease, responsible for mucin degradation, would render a strain of Vibrio cholerae inefficient in penetration of the tnick mucous layer of the enterocytes, but unchanged in the ability to reach the surface of the M cell and elicit a strong immune response. The major secreted, mucin degrading protease in strain C7258, C6706 and SG25-1 was found to be the soluble hemagglutinin/protease, a putative virulence factor described by Finkelstein, et al. Journal of Bactcriology, Vol.

173, No. 11, pp. 3311-3317, 1991. Paralleling the inactivation of the hemagglutinin/protease gene, a DNA fragment coding for the endoglucanase A of Clostridium thermocellum was inserted in the Vibrio cholerae chromosome. This mutation combined with the deletion of cholerae toxin genes of the c:tx) genome resulted in tagged vaccine candidates with excellent properties as immurizing agents for vaccinating humans against cholera.

As an example, rather than as an interest to be limiting; the non-toxigenic mutants of Vibrio cholerae useful for constructing safe mutants defective in the expression of hemagglutinin/protease for vaccine purposes, are describec in table la.

Non-toxigenic mutants are specifically characterized by the absence of al ctxt coding sequences in their chromosome, and by the presence of a single RS1 element, the nucleotide sequence of which was confirm by DNA sequencing. The methods of producing non-toxigenic mutants of Vibrio cholerae are well described elsewhere (Archives of Medical Research, 27, No. 3, pp. 275-283).

Table la. Starting strains for constructing HA/P defective mutants.

Vaccine candidate Biotype/Serotype Genotype 81 El Tor/Ogawa Actx$ 413 El Tor/Inaba Actx4 SG25-1" 0139 Actx$ nAl straiiL andU me meinoas or making are described in Archives of Medical Research. Vo. 27, No. 3, pp.

275-283, 1996. 81 and 413 derive from C7258 and C6706, respectively; both of which are clinical isolates from Pert, 1991. SG25-la is a derivative of the 0139 isolate SG25-1 from Calcutta, India, 1993.

In a similar way, table lb provides the hemagglutinin/protease mutants useful for constructing thyA defective derivatives with better features of biosafety.

Table 1 b. Hemagglutinin/Protease mutants useful for constructing thyA mutants.

Vaccine candidate Relevant properties 638 81 hap::ceA 1333 413 hap::ce/A L911. SG251a hap::celA Characterization of non-toxigenic vaccines with additional mutations intended to enhance their biosafety Serological characterization.

After any new mutation was introduced in vaccine strains described herein the derivative was demonstrated to retain the expected serotype. Cells were harvested from a plate, suspended in saline and immediately tested with Difco typing serum specific for Inaba, Ogawa or 0139.

The major immune response elicited by vaccinees is directed against the! LPS of the bacterium. All strains being issued in the present invention retained expression of the expected O-antigen as confirmed by serological tests. Additionally LPS profiles remained unchanged in polyacrylamide gel electrophoresis and Western blot Infant mouse colonization assay.

The infant mouse colonization assay (Herrington et al, J. Epe Med 168:1487:1492, 1988) was used to asses the colonization properties of each mutant An inoculum of 0 s-106 vibrios in a final volume of 50 pl was administered intragasthically to groups of at least five mice. After 18-24 hours at 30'C, mice were sacrificed, the intestine was dissected, homogenized and plated on bacteriological media containing appropriate supplements to support growth of mutants. Colonies that grew after overnight incubation were tested for additional markers.

The ability of doubly and triply mutated (Actx}, HAP)f(thyA') strains cif Vibrno cholerae to colonize the intestine of suckling mice can be observed in table- 2 Strains .638, 1333 and 638T are well colonizers of the small bowell of mice. Colcnization of gastrointestinal tract of infant mice is widely accepted to correlate well with colcnization of the human gut, which is necessary to prime the mucosal immune systerr and to induce a strong secretory IgA response. Although L911 colonizes less eficiently 'han the rest it is still able to colonize.

it should be noted that'638T is a thyA mutant of 638. The mutation in-rocluced in this strain creates a thymine or thymidine dependence that reduces the abililh of this strain to multiply in natural environments where free pyrimidines are usu.ally absent from.

Table 2. Colonizing capacities of hap::celA vaccine strains.

Strain Input Output Biotype/Serotype Relevant genotype 638 Ix10' 2.8x10 El Tor/Ogawa ActxY,hap::ce'A 1333 2x1 0" 4.2xt10 El TorInaba Actx4,hap::ce'A L911 1.2x106 8 x10 0139 Actx),hap::ce'A

I

638T 1 .7x10 6x10 5 El Tor/Ogawa Actx<,hap::ce'A. thyA- Detection of protease activity.

Milk-LB plates were used to detect proteolytic activities in supernatants of TS8grown vibrios. For quantitation of protease activity the azocasein method was adapted from Ginther CL., Antimicrob. Agents Chemother. 15, 522-526, 1979. Briefly 1.1 ml of buffer (CaCl 2 imM; Tris 0.2M, pH 7.2; Azocasein were mixed with 200 pl of culture supernatant and incubated for 1hr at 37'C. The unreacted substrate was precipitated with 83 Ip of TCA 40% for 10 min. followed by 10 min. centrifugation at 12000 rpm. The colored product remaining in solution was neutralized with NaOH and read at 450 nm. One unit of enzymatic activity was defined as the quantity of enzyme producing a net increase of one in the optical density of the sample in one hour of reaction.

The mutation introduced in the hemagglutinin/protease gene of strains disclosed herein, accounts for a reduction in 60-80% of the proteolytic activity as observed in mutants when compared to their non-toxigenic parents, Detection of vibrios expressing the endoglucanase A marker.

For CelA activity detection, vibrios were grown in LB plates for 24 hours, overlayed with CMC-indicator agar and incubated for 4 hours at 60'C. Endoglucanase A positive colonies were visualized after Congo Red staining and washing, as red colonies surrounded by a transparent halo in the red background of the plate. CMCindicator agar was composed of 0.7% agarose, 0.5% CM-cellulose in phosphate-citrate buffer pH 6.3 and staining solution was 1% Congo Red in water.

The ce/A marker used to unequivocally distinguish the vaccine, is stably expressed and inherited in Vibrio cholerae. The appearance of tagged vi rios can be observed in figure 2.

Scoring for a thymidine auxotrohpy in vaccine strains generated by specific mutagenesis of thvA qene.

M9-salts supplemented with glucose as the carbon source and thymine/thymidine (200 pg/ml) were used to detect growth of thyA defective mutants impaired to grow in M9-salts glucose medium. LB plates supplemented with 200 pg/ml of thymidine were used to check for trimethropim resistance in the tfyA mutants generated. The stability of thymidine auxotrophy was analyzed by replica-plating colonies from M9-glucose-thymidine to M9-Glucose plates.

Strains 638, 1333, and L911 are prototrophic bacteria able to grow in mineral salts employing glucose as the sole carbon source. Strain 638T require; addition of thymidine or thymine for growth in minimal medium. When one of these supplements is present 638T is resistant to up to 200 ig/ml of trimethoprim.

Assay for motility.

Cells from single, well isolated colonies were picked from a master plaie aided with the tip of a platinum needle and inoculated by insertion (2-3 mm) into a motility agar plate (LB, agar The diameter each colony spreaded through soft agar was recorded at 24 hours of incubation at 30*C. The criteria for motility was as fo'lows A bacterial strain spreading 3 mm or less from the point of application was :ornsidered non-motile. A bacterial strain spreading >3 mm beyond the point of inoculat on was considered motile.

All strains under the scope of this patent, but L911 were found to be moti e. L911 is a derivative of SG251, which in our hands, resulted non-motile when -assayed in motility agar.

Assay for flaoella Motile and non-motile bacteria were assayed for the presence of flagella by electron microscopy. Briefly, Vibrio cholerae was grown for 4 hours at on colonization factor antigen agar (CFA, casaminoacids, 1 Yeast extract 0 Magnesium Sulphate, 0.05%; Manganesium Chloride, 0.005%), harvested and washed in saline (NaCI, Bacteria were negatively stained with 1% uranyl acetate for 3 min. and analyzed by transmission electron microscopy.

The flagellate nature of bacteria was also demonstrated for all vaccine strains as above, with greater emphasis on non-motile 0139 strain L911 (figure 2) All strain described herein were shown to be flagellated, including the non-motile L911 Assays for TCP Mutant strains were examined for the presence of toxin corregulated ;:ilus on bacterial surface by immunogold electron microscopy. Cells were cultured as for the assay of flagella. Fresh harvested suspensions of vibrios were (10 gp) depositad on a .carbon coated nickel grid, fixed for 1 min. by exposing the grid to a 60 watt,; lamp and the excess of liquid removed on a filter pad. The grid was inverted in a drop of a TCPspecific sera diluted in saline, 1% BSA, 0.Q5% tween-80, incubated during 15 min. for reaction and washed in saline-BSA-Tween. Washed grids were incubated with he gold labeled conjugate diluted in the same buffer, let to stand for 15 min. and washed 3 times with saline-BSA-Tween. After washing, samples were stained during 1 min wirh a 1 ammonium molibdate solution.

The most important features of cholera vaccines are safety and antifienicity. By genetic manipulation, V. cholerae strains are rendered safe, but care should )e :aken to retain their antigenicity unchanged. Vibrio cholerae strains disclosed in thepr set patent application were evaluated for the expression of most prominent antigen; in the bacterium. TCP was visualized on bacterial surface by immunogolj electron microscopy. Alternatively, expression of this key colonization factor was detected by Western analysis. All strains analyzed 638, 1333, L911 and 638T p oduced normal levels bf TCP protein and assembled it into large appendages on bactenal surface (figure 3).

Live Vaccine strains Strains 638, 1333, L911 and 638T described herein, can be used to achieve adequate immunological protection against cholerae in humans, provided the high rates of seroconversion they elicit and the low levels of adverse reactions th'ey )roduce when orally administered to human volunteers. Depending upon relevant local epidemiology a single strain or combinations of them could be used for immunization Culturability of vaccine strain Vaccine strains were cultured in standard laboratory medium. Suplements of thymidine (200 pg/ml) were added when necessary for growth, but no supplements were included in the inoculum.

Dosage A single' oral dose of live vaccine was administered to people in bicarbonate buffer after harvesting bacteria from a fresh plate in aseptic saline (NaCI Useful inoculum are 10 7 -10 9 cells per immunizing dose. Alternatively more than one dose, separated 7-28 days apart, can be given to each subject. Preferably vib.ios can be lyophilized in a formulation that preserves viability and mixed with bicarbonate prior to inoculation.

Clinical testing of 638.

Strain 638 has been tested in human trials involving 42 volunteers aged 8-40 A summary of clinical findings after ingestion of strain 638 and placebo is shown n Table 3. All clinical manifestations observed were mild and of short duration. No s:atistical significance could be demonstrated between the inoculated and placebo proulps with the present data. Gurgling and abdominal cramps were the reactions more frequently reported by volunteers irrespective of dose. Four volunteers developed mild diarrhea i (grade Three of these volunteers received the high dose and one the rredium dose.

One volunteer who received the high dose, had 5 loose stools (72 h after inoculation) and a total output of 680 g. Two volunteers had 2 loose stools 28 and 72 h after inoculation with total outputs of 220 and 500 g, respectively. The other voljnteer had a single diarrheal output of 300 g 73 h after inoculation.

Bacteriological isolation of vaccine strain.

As indicated in Table 4, strain 638 was recovered in 37 out of 42 volunteers inoculated (88 For the higher dose, excretion of the vaccine strain terd to peak at 72 h after inoculation. Three out of the 4 cases of diarrhea occurred at this time. Of the 37 volunteers that excreted vibrios, 12 excreted on at least 4 days, 19 on at least 3 days, and 28 on 2 days. The number of volunteers excreting strain 638 aid the mean number of vibrios/ g stool declined in the lower dose. Vibrios isolated from the stool of volunteers produced endoglucanase A indicating that the celA reporter gene was stably maintained during growth in the human intestine. We conclude that strain 638 is a good colonizer of the human small bowel.

Immune response to vaccine strain.

Strain 638 elicited a significant and consistent immune response in terms of serum vibriocidal antibodies, serum anti-Ogawa LPS IgG or IgA, and Ogawa LPS-specific IgA ASC (Tables 5 y Although reciprocal GMT peaked 14 days after inoculation, seroconversion was attained on day 7 and titers remained high till day 28: Seroconversion rates, peak reciprocal GMT, and ELISA titers were dose-dependent.

However, even at the lowest dose, strain 638 elicited a significant vibriocdal antibody response compared to placebo. A significant proportion of the volunteers which experimented seroconversion developed relatively high 1024) vibriocidal titers (Table The high percentage of responders in the ASC evaluation (Table 5) reflects an effective stimulation of mucosal immunity, mainly slgA, by strain 638 in correspondence with the elevated anti-LPS IgA titers encountered 14 days after inocjlation. One volunteer who ingested placebo seroconverted for anti-LPS IgG. This volunteer had very low pre-inoculation anti-LPS serum IgG which increased to the cutoff value at day .7 and remained constant thereafter. Another volunteer who ingested placebo reached the cutoff value of ASC. Similarly, this volunteer had a very low pre-inoculation number of LPS-specific ASC. We conclude that strain 638 elicits a significant immune response.

Table 3. Frequency of occurrence of adverse reactions after ingestion of candidate vaccine strain 638.

El Tor Ogawa Confidence Probability Symptom Group of volunteers R.R. n e interval 4 (Fisher) Inoculated 1 Placebo Diarrhea 4 38 1 13 1.33 0.16-10.96 0.6329 Abdominal cramps 13 29 2 12 2.17 0.56-8.44 0.1944 Gurgling 14 28 3 11 1.56 0.52-4.63 0.3143 Heartburn 6 36 2 12 1.00 0.23-4.40 0.6850 Headache 7 35 0 14 0.1163 Vomiting 1 41 0 14 0.7500 Volunteers with diarrhea Mean diarrheal stool weight (range) 425 g (220-680) Mean number of diarrheal stools per ill volunteer (range) 2 Notes: 'N 42, N 14, 3 Relative Risk, 4 (95 Table 4. Recovery of Vibrio cholerae strain 638 from the stools of volunteers.

Volunteers excreting vaccine strain/:otal Group of Volunteers Time after inoculation Total Mean CFU/g stool 24 48 72 96 120 High dose (1-2 x 10) 7/29 10/29 16/29 15/29 10/29 28/29 4.4 x Medium dose (2 x 108) 616 5/6 4/6 4/6 3/6 616 5.5 x 10 6 Low dose (4 x 10 7 1/7 2/7 2/7 2/7 2/7 317 2.7 x 10 Table 5. Anti-LPS igA ASC response in peripheral blood of volunteers following ingestion of Vibrio cholerae strain 638.

Dose Positives Mean ASC per 10' PBMC (range) High 27/29 (93.1) 485 (0-4750; Medium 6/6 (100) 377 (40-1285) Low. 6/7 (85.7) 5 (0-65) Placebo 1/14(7.1) 371 (0-2040' Table 6. Serum antibody responses in volunteers orally administered Vibrio cholerae El Tor Ogawa strain 638 Group of volunteers Response High dose Medium dose Low dose Vibriocidal antihodins Seroconversion rate 1 GMT (range): Pre-inoculation Post-inoculation peak [14 days] Responders with titers 1. 1024 Vibriocidal antibodies 24/29 (82) 47 (0-160) 873 (0-20480) 10/24 5/6(83) 32 (0-40) 639 (0-2560) 4/5 5/7(71) 33 (0-40) 389 (40-2560) 4/5 Placebo 0/14 (3) 37 (0-320) 46 (0-320) 0 Anti-Oaawa LPS InC, Seroconversion rate 2 Anti-Oaawa LPS InG 23/29 (79) Log reciprocal titer 3

SD:

Pre-inoculation 0.1; Post-inoculation 1.8 Peak [14 days] 4/6 (67) 0.12 0.29 1.59 1.49 3/7 (44) 0 1.07 1.36 1/14(7) 0.03 ±012 0.19 0 57 2±0.25 6±1.12' Anti-Onawa I PR InA Seroconversion rate 26/29 (90) tog reciprocal titer 3

SD:

.Pre-inoculation 0.1 t 0.37 Post-inoculation peak 2.43 1.0 .peak (14 days] Ani-nwaLR 6/6(100) 0 2.68 0.48 57 (71) 0.27 0.37 0/14 (3) 0.13 ±0.39 1.96 ±1.25 0.19 0.53 Notes: 1 Number of volunteers with fourfold increase in titer/total, 2 Number of volunteers with a twofold increase in titer/total, 3 logarithm of the reciprocal arithmetic mean titer Abbreviations: GMT, geometric mean titer; SD, standard deviation of the mean IV Examples The examples described herein were conceived to illustrate rather than to limit the invention.

Constructing safer vaccine candidates from non-toxigenic parentals.

For constructing Hemagglutinin/Protease defective derivatives from non-toxigenic mutants described in table la, each parental strain was equally processed. F rst suicide vector pGPH6 (figure 4) containing the HA/P gene (hap) inactivated by insertion of reporter gene ce/A was transferred from E. coli SMlOXpir to the non-toxigenic mutant to produce an ampicillin resistant co-integrate. Second, Southern hybridization demonstrated ,the co-integrate to contain the insertionally inactivated hao gene (hap::celA) and its wild type allele (hap) separated by vector DNA. Third, the above ampicillin-resistant co-integrate was allowed to segregate in antibiotic-free rredum and ampicillin-sensitive colonies selected. Ampicillin-sensitive colonies designated 638.

1333 and L911, were characterized by Southern analysis and shown to contain the hap::celA mutant allele.

Constructing 638T. a Thvmidilate Synthase defective derivative of Vibrio cho,erae strain 638.

For constructing mutants of Vibrio cholerae which are defective in the exp ression of' Thymidilate Synfhase, its.coding gene thyA was first cloned and sequerced. ThyA gene from Vibrio cholerae was first cloned by complementing a spontaneous trimethoprim-resistant thymidine-requiring mutant of.strain 81, with a genom c library of strain C7258 constructed in pBR322. One clone was selected, purified and the insert brought to pUC19 which served as template to facilitate sequencing with universal primers. Nucleotide sequence of thyA gene and its predicted polypeptide of Thymidilate Synthase (TSasa) protein are shown in SEQ ID NO 1.

To construct a TSasa defective vaccine strain, an internal restrictio-n fragment was deleted "in vitro" from the thyA open reading frame. Deletion comprised nucleotides between Mlul and BgIl restriction sites and relmved DNA sequences coding fcr amino acid 7 to amino acid 105 of ThyA protein. The resultant gene construct was mpaired in its ability to complement the thyA defect of spontaneous mutant Vibrio cholerae 815.

This fragment was cloned into the unique Sad restriction site of pCV0442 to obtain pEST (figure This plasmid was transferred from E. coli SMICpir Vibrio cholerae strain 638 and an ampicillin-resistant co-integrate was selected Southern J 16 analysis of the co-integrate demonstrated pEST was specifically integratea within Ihe thyA gene. The ampicillin-resistant co-integrate was allowed to segregate in antibioticfree medium supplemented with thymidine. Sucrose-resistant colonies were selected in the presence of thymidine. A thymidine-requiring, ampicillin-sensitive colony designated 638T, was characterized by Southern analysis and demonstrated to contain the dysfunctional thyA allele. pEST was used to construct 638T and will also be useful for constructing 1333T, and L911T or any additional defined thyA defective derivative other V. cholerae strain.

A brief description of the drawings follows.

Figure 1. Detection of cholerae vibrios tagged with ce/A in plate assays employing CMC-indicator agar.

Figure 2. Detection of flagella on Vibrio cholerae mutant L911 of the 0139 se ogroup Figure 3. Detection of TCP on bacterial surface of 638T.

Figure 4. Schematic representation of suicide vector pGPH6 used to construct hap.:ce/A mutants of V. cholerae.

Figure 5. Schematic representation of suicide vector of pEST used to construct thyA mutants.

Advantages This invention provides us with an approach to create safer mutanis of Vibno cholerae by inactivation of the hemagglutinin/protease gene of non-toxigen c mutants.

Such derivatives are useful as vaccines.

Safety and immunogenicity of strains 638, 1333 and L911 are similar to that presented in patent WO 95/18633, Additionally they are representative cf all Vibrio cholerae strains circulating during the current pandemic and of the iew 0139 serogroup. Said strains are also tagged with a distinguishable marker to facilitate environmental sampling of the vaccine. All mutations introduced during con truction of the vaccines described herein are well defined as gene deletions or as geno insertions of known nature.

This invention also provides a method to improve the environmental biosafety of live cholera strains to be used as oral vaccines; This improvement is attained by creating a defined mutation in the gene thyA which is also described herein.

vaccines described herein are well defined as gene deletions or as gene insertions of known nature.

This invention also provides a method to improve the environmenta biosafety of live cholera strains to be used as oral vaccines. This improvement is attained by creating a defined mutation in the gene thyA which is also described herein Vaccine strain 638T is featured by its enhanced environmental biosEfety. It has a thymidine auxotrophy that limits its proliferation in natural ecosystems, where free pyrimidines are scarce if existent.

Trimethoprim resistance conferred to Vibrio cholerae vaccine candidate 638T by its mutant thyA gene is unlikely to be transmitted into other bacteria due to its recessive nature. Additionally, resistance to trimethoprim is conditioned to the presence of thymine or thymidine in a culture.

Reacquisition of cholerae toxin genes or other DNA by means of horizontal gene transfer is superfluous in these strains since they do not proliferate out from the laboratory.

V. DPofits Under the terms of the Budapest Treaty on the internatlonal Recognition of the Deposit of Microorganlsms for the Purpose of Patent Procedure, deposits of Vibrio choletae strains described herein have been made with the DSMZ-Deuteche Sammiung von Mikrooganiarmen und Zellkulturen Gmbh (German Colleation of Microorganlsm and Cell Cultures), Braunachweig, Federal Republic of Germany as follows: Vibrio oholerse 1333 OSM 12767 Vibrio chaleree LO11 DSM 12758 Vibrfo choalrse 638 DSM 12758 Vbrio Ocholerse 838T DSM 12760 All of the deposits were made on April 7, 1999.

a 18 Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

Claims (13)

1. A method to remove or reduce to an acceptable level residual reactogenicity from O non-toxigenic mutants of Vibrio cholerae intended to be used for vaccination against 5 cholera wherein the hemagglutinin/protease gene (hap) is inactivated by deletion, insertion or any other defined and irreversible genetic manipulation.

The method of claim 1 wherein the inactivation of the hemagglutinin/protease gene O Sis performed by disruption of the hap gene with the marker gene ce/A.

3. A medicinal preparation suitable for administration to humans for inducing (N immunological protection against cholera comprising as an essential ingredient one or more of the following Vibrio cholerae vaccine strains: Vibrio cholerae 1333 and Vibrio cholerae L911.

4. The preparation of claim 3 comprising one or more of the following vaccine strains: Vibrio cholerae 1333 being a mutation of strain 413, Biotype/Serotype El Tor/Inaba, Genotype Actx( with a dysfunctional hemagglutinin/protease gene (hap) resulting from the insertion in the hap gene of the marker gene celA; (ii) Vibrio cholerae L911 being a mutation of strain SG25-1a, Biotype/Serotype 0139, Genotype Actx( with a dysfunctional hemagglutinin/protease gene (hap) resulting from the insertion in the hap gene of the marker gene celA.

5. A preparation according to claim 3 or 4 suitable for oral administration.

6. A preparation according to claim 5 prepared from a lyophilized formulation of a Vibrio cholerae strain as defined in any preceding claim.

7. A preparation according to claim 5 or 6 wherein the strain or strains are admixed with a buffer suitable for oral administration.

8. A preparation according to claim 7 comprising bicarbonate buffer. IO

9. The use of the inactivation of the hemagglutinin/protease gene (hap), in one or more Vibrio cholerae vaccine strains derived from non-toxigenic parentals by deletion, O insertion or any other defined and irreversible genetic manipulation for reducing the 'C 5 level residual reactogenicity of a medicinal preparation suitable for administration to humans and for inducing immunological protection against cholera.

10. A method of producing a vaccine strain suitable for administration to humans for 0 inducing immunological protection against cholera characterized in that residual reactogenicity is removed or reduced to an acceptable level from non-toxigenic Smutants of Vibrio cholerae by inactivation of the hemagglutinin/protease gene (hap), NC by deletion, insertion or any other defined and irreversible genetic manipulation and wherein a defined and irreversible mutation is introduced into its thyA gene coding for Thymidilate synthase whereby proliferation in natural ecosystem is limited.

11. The method of claim 10 wherein inactivation of the hemagglutinin/protease gene is performed by disruption of the hap gene with the marker gene celA and irreversible mutation in thyA consists in the deletion of the Mlu I-Bgl II internal fragment from its coding sequence (SEQ ID No 1).

12. A medicinal preparation with improved environmental biosafety suitable for administration to humans for inducing immunological protection against cholera comprising as an essential ingredient one or more Vibrio cholerae vaccine strains derived non-toxigenic parental, the strain or strains having: an acceptable level of residual reactogenicity due to the presence of a dysfunctional hemagglutinin/protease gene (hap) resulting from deletion, insertion or any other defined and irreversible genetic manipulation; and (ii) a dysfunctional genetically modified allele of a thyA gene instead of its natural one, resulting from deletion, insertion or any other defined and irreversible genetic manipulation, whereby proliferation in natural ecosystems is limited.

13. The preparation of claim 12 comprising the vaccine strain: Vibrio cholerae 638T.