JP2003503066A - Delivery system - Google Patents

Abstract

  (57) [Summary] The present invention relates to delivery systems for heterologous antigens. In the present invention, a chromosomal locus within the rRNA operon (eg, the chromosomal locus of the 16S gene or the chromosomal locus of the 23S gene) has been identified as a useful site for integration of the nucleic acid into the chromosome of Vibrio cholerae bacteria. Was. In the present invention, a regulatory sequence particularly useful for directing high-level expression of a heterologous antigen in the Vibrio cholerae bacterium was identified as the OmpU promoter.

Description

Detailed Description of the Invention

The present invention relates to delivery systems for heterologous antigens. One aspect of the present invention is Vi
It relates to the identification of chromosomal loci useful for the integration of nucleic acids into the chromosome of brio cholerae. Another aspect of the invention relates to the use of the OmpU promoter to direct high level expression of heterologous antigens in Vibrio cholerae.

Vaccination as a deliberate attempt to protect humans against disease has a long history. However, it was only in the latter half of the 20th century that the technology that enabled the development of sophisticated vaccination techniques. With the advent of new procedures in molecular genetics, the number of techniques that can be used to design a vaccine is greatly expanding.

The general approach to vaccination has historically been to administer to human subjects biological material derived from the organism responsible for the particular disease. Such substances are usually attenuated in some way so that they do not actually cause disease. The humoral response provided by the immune system to attenuated foreign proteins primes the body against subsequent "real" infections, thus reducing the incidence of serious illness.

Recent advances in genetic engineering have facilitated the production of mutant proteins that can be screened for suitability as vaccine candidates. In view of the potential importance of successful vaccines, many groups have attempted to detoxify the virulence factors responsible for these diseases.

For example, in the case of enteropathogenic diseases, attenuated, non-toxic mutant enterotoxins have been developed that no longer induce pathogenic effects in the mammalian body but are structurally largely unaltered. Thus, when recognizing these proteins as foreign, the immune system directs specific antibodies against this attenuated toxin. These antibodies then become effective against wild-type toxin in the development of a true infection with live bacteria. Kaslow et al. (1992) reported the cholera toxin protein (CT) Asp9.
And His44 were mutated separately and the protein after residue 180 was truncated. These mutations attenuated the toxin's enzymatic activity. Similar studies have been conducted on heat-labile toxin protein (LT; Sixma et al. (1991) Nature 351 (
6325): 371-377; Tsuji et al. (1990), J. Am. Biol. Ch
em. 265 (36) 22520-22525; Harford et al. (1989).
), Eur J Biochem 183 (2) 311-316).

Detoxified variants of pertussis toxin have been reported to be useful both for direct intranasal vaccination and as mucosal adjuvants for other vaccines (Roberts et al. (1995) Infect Immun. 63
(6) Pages 2100-2108). A known detoxification mutation in the A subunit of Bordetella pertussis was found to have a similar effect in CT (Burnette et al., (1991) Infect Immun.
59 (11) 4266-4270).

Many attempts have utilized the ability to directly modify the function of bacterial or viral proteins to produce live strains with attenuated pathogenicity. Such "live" vaccines can only undergo limited replication in the host, or such "live" vaccines encode detoxified proteins. Generally, a broad spectrum of immunity can occur after a single inoculation; this immunity can persist for life. Such vaccination methods have the distinct advantage over multi-step vaccination regimens with purified attenuated proteins that are expensive and usually require readministration to ensure long-term efficacy. In particular, antigen purification processes for delivery to the body are expensive and can often result in protein denaturation.

There are also various problems associated with the use of attenuated “live” vaccines. Typically, these vaccines are virus-based, and generally plastic (
plastic). Thus, the replicating virus can revert to a more pathogenic form and cause an adverse reaction in the vaccinated patient or in humans contacted with this patient.

Attenuated bacteria have recently been investigated as candidates for antigen delivery. Strains of Salmonella typhi (CDV908) appear to be promising candidates for antigen delivery, and mucosal immunization with such strains
In addition to stimulating serum antibodies, it has the additional advantage of eliciting both mucosal and cell-mediated immune responses.

Genetic manipulation of such strains should allow simultaneous administration of multiple antigens,
This gives a broad spectrum of protection. Several groups are investigating this approach with the aim of producing a safe and effective multivalent oral diphtheria-tetanus-pertussis (DTP) vaccine. To date, by researchers, S
． Nontoxic Immunogenic Fragment C of Tetanus Toxin in Typhimurium
The successful expression and protective capacity of (FragC) has been demonstrated (Chatfi
eld et al., (1992) Vaccine 10 (1) pages 53-60; Fairw.
eather et al., (1990) Infect Immun 58 (5) 1323.
~ 1326; Galen et al., (1997) Vaccine 15 (7) 700.
~ 708).

The Vibrio cholerae bacterium has previously been found to be enteropathogenic E. coli (Butt).
Erton et al., 1995; Acheson et al., 1996) and Shigell.
The suitability of a sonnei (Viret et al., 1996; Favre et al., 1996) as a carrier for Shiga toxin was studied.

One problem with these live oral vaccines is that, to date, no good vector exists for effective high level expression of attenuated antigens in the human intestine.

[0013] Thus, bacterial and viral based diseases continue to be a major cause of morbidity and mortality, especially in the developing world. Poor health care and global poverty in these areas means that there is a serious need for cheap and effective vaccines that are not persistently effective and therefore do not require repeated doses.
Such vaccines also significantly increase the efficacy of preventive care and reduce health care costs in the developed world.

The accessible expression levels of heterologous antigens in bacterial “live” vaccine strains are clearly of great importance for optimizing the vaccination regimen and for ensuring effective vaccination. Here we have developed a system to facilitate the production of attenuated bacterial vaccines and improve the expression levels of heterologous antigens in these strains.

SUMMARY OF THE INVENTION The present invention provides a recombinant Vibrio cholerae bacterium comprising a nucleic acid encoding a heterologous protein or protein fragment inserted into a locus within the 16S gene or 23S gene in its chromosome. .

This system relies on the stable chromosomal integration of nucleic acids, which allows the expression of proteins heterologous to Vibrio cholerae at sites that are found not important for the survival of the bacterium. The phenomenon that has been shown to be a problem to date is that when a gene is randomly inserted into a bacterial chromosome, this often results in some degree of bacterial growth, either in its growth rate or its general viability. It is to cause damage. This often means that
It results in the widespread use of plasmid or bacteriophage vectors that are not suitable for stable high level expression of heterologous proteins.

Surprisingly, the rRNA operons (eg the 16S and 23S operons) have been found to be particularly suitable targets for chromosomal integration of foreign nucleic acid fragments. Insertion of foreign nucleic acid at these sites is linked to Vibrio chol.
It has been found to allow efficient high level expression of heterologous proteins without compromising the growth rate or viability characteristics of the erae bacterium.

This method of gene targeting involves replacing a portion of a wild-type rRNA gene with a disrupted copy of the gene, wherein in the disrupted copy of the gene, a nucleic acid encoding a heterologous protein. Has been inserted. This integration event can best be carried out by homologous recombination. The foreign gene has a sequence adjacent to that of the V. It is introduced into the Cholerae bacterium, which allows the recombination to occur. Techniques for homologous recombination are well known in the art and are described, for example, in Sambrook et al. (1989).

The advantage of using rRNA genes is that these genes are chole
It is present in more than one copy throughout the genome of rae, with the consequence that one copy disruption affects the viability of the strain and its ability to adhere, colonize, and grow in infected hosts. The point is not to. The advantage of using site-specific recombination for insertion is that the insertion site is well known and can be easily characterized.

Preferably, the nucleic acid encoding the heterologous antigen is the 23S rRNA in the bacterial chromosome.
Inserted in the operon.

According to a further aspect of the invention, the nucleic acid encoding the heterologous protein or heterologous protein fragment is an OmpU promoter (preferably V. choler.
Recombinant bacteria that are cloned under the control of the ae OmpU promoter) are provided. This promoter has been identified as an ideal promoter for introducing high level expression of foreign antigens in Vibrio cholerae. Because high level expression can be achieved without compromising the growth rate or viability of the strain.

V. In its native context in Cholerae, the OmpU promoter drives the expression of the 38 kDa outer membrane protein, which has been shown herein to be expressed at very high levels in cells. Preferably,
The OmpU promoter used is the natural promoter found in Vibrio cholerae, but it is recognized that improved potency of this promoter can be achieved by repeated rounds of mutation and selection. Such methods are known to those of skill in the art (eg, Ling and Robinson, 1
997, Anal Biochem 254: 157-178; Gorb et al., 1
997, J. Bacteriol. 179: 5398-5406).
.

According to a further aspect of the invention, a nucleic acid encoding a heterologous protein or protein fragment is cloned under the control of the OmpU promoter and inserted into a locus within the rRNA operon of the Vibrio cholerae chromosome. A replacement bacterium is provided. This combination of selective targeting and use of the OmpU promoter for gene insertion has been found to provide particularly high levels of heterologous protein expression without yet compromising the growth rate or general viability of the bacterial strain. Was issued. Preferably, the nucleic acid is Vibri
In the o cholerae chromosome, it is inserted into a locus within the 16S gene or the 23S gene, more preferably within the locus within the 23S gene.

“Heterologous protein or heterologous protein fragment” refers to V. chole
By any protein or fragment that is not endogenous to ra.
Thus, the protein can be from any source and is endogenous to the host subject to be immunized (eg, human tumor antigen) or adhesin (eg,
It can even be filamentous hemagglutinin (FHA), pertactin or pili. Appropriate proteins are immunogenic and thus elicit an immune response when expressed in a mammalian host which is intentionally infected with the Vibrio cholerae bacterium according to the invention. Therefore, of most relevance are proteins that are known to be particularly immunogenic. This immune response may be a cellular or humoral response.

Preferably, the heterologous protein produced by the present invention is derived from a known pathogen (eg virus and bacterium).

More than one heterologous protein or protein fragment may be targeted to the rRNA gene in its chromosome, and / or they are O
It is also recognized that it can be cloned under the control of the mpU promoter.
Thus, expression of multiple antigens in this manner may provide a broad spectrum of protection against many different diseases.

According to a further aspect of the invention, a recombinant bacterium according to any one of the above aspects of the invention, wherein the recombinant bacterium is effective in the host as an adjuvant and in the host as an antigen. Recombinant bacteria are provided that express at least one heterologous protein that is effective.

In particular, it will be appreciated that two, three, four or more heterologous proteins can be co-expressed in the recombinant bacterium of the invention. One or more of these proteins can act as an adjuvant, while the other proteins can act as antigens. For example, the LTK63 and CTK63 antigens (which include a serine to lysine substitution at position 63 of subunit A) and LTR72.
The antigen, which contains an alanine to arginine substitution at position 72 of subunit A, functions well as a mucosal adjuvant in animal models. Thus, heterologous proteins (eg LTK63 gene, CTK63 gene and / or L
TR72 gene) is V. at the rRNA gene target site in the cholerae chromosome,
It will be appreciated that it may be inserted, preferably operably linked to the OmpU promoter. The V. The cholerae chromosome already contains other rRNA operon insertion sites, one or more heterologous proteins (eg NAP and / or Ca).
The gene (s) encoding gA) may be included, also preferably operably linked to the OmpU promoter so as to maximize protein expression. In this manner, the recombinant "live" bacteria not only possess immunological capacity, but also act as effective adjuvants to increase their potency as a vaccine factor.

In many cases, the heterologous protein can be detrimental to the host if unmodified, so derivatives of the wild-type protein should be used. As used herein, the term “derivative” includes, for example, variants that include amino acid substitutions, insertions or deletions that do not significantly alter the immunogenicity of the protein. In particular, conservative amino acid substitutions generally have little effect on the activity of protein molecules and are particularly suitable. Vibrio chole
proteins that are endogenous to rae (eg, cholera toxin protein (CT
Derivatives of) are also considered suitable for use according to the invention.

Protein fragments are also suitable for expression in bacteria according to the invention. The advantage of these molecules is that they generally show significantly reduced toxicity but still retain their immunogenicity. Most suitable are fragments containing epitopes known to be the most immunogenic (eg Clostri).
It is a fragment C) of the degree of tetanus venom derived from aluminum tetani. The term "fragment" includes an antigenic peptide that contains or defines an antigenic epitope.

Heterologous proteins or heterologous protein fragments suitable for use in the present invention also include, for example: heat-labile toxin protein from a strain of E. coli, tetanus toxin, B. pertussis-derived tracheal colonization factor (tracheal colonization factor)
) And pertussis toxin, NAP, H. VacA and CagA from pylori
, E6 and E7 from papillomavirus, gD2 from herpesvirus,
HCV-derived E1 or E2 core antigens, HIV-derived gp120, meningococcal and gonococcal antigens, rotavirus-derived antigens, influenza virus-derived antigens and malaria parasite-derived antigens. Other suitable toxins are known to those of skill in the art.

Particularly suitable for the present invention as a heterologous antigen is the neutrophil-activating protein (NAP). NAP is a homodecamer of the 15 kDa subunit that promotes neutrophil activation and neutrophil adhesion to endothelial cells. This function was suggested to be unlikely to be related to its intracellular function, but its adhesion promotion (prodhesiv)
e) The activity can be neutralized by antisera. Since neutrophil activation and adhesion to endothelial cells constitute an inflammatory mechanism, and H. Since pylori is responsible for inflammation of the stomach, NAP may be responsible for inflammation in the early stages of gastric ulcer disease, where an abundant accumulation of neutrophils in the superficial mucosa of the stomach is observed. Pyroli factor,
Or indicate a cause. For this reason, NAP is an ideal candidate for use in live bacterial vaccines.

The heterologous protein expressed according to the present invention may be a toxin or toxin derivative. As used herein, the term "toxin" refers to any protein produced by a pathogen that is toxic to a host animal, whether synthetic or naturally derived (eg, bacterial or viral). Origin)). Preferably, toxins from bacterial pathogens are used according to the invention.

Toxins are particularly suitable for use according to the invention. Because any immune response elicited against the toxin not only eradicates the bacterium expressing the toxin with the recognized epitope, but the bacterium itself is killed by the immune system. Neutralize any copy of its toxin protein secreted by
te).

The enterobacterial toxin proteins have been studied in great detail and methods for producing detoxified variants (toxoids) of many known toxins from different pathogen species are known. As used herein, the term "toxoid" refers to a detoxified mutant toxin protein.

The term “detoxified” means that the mutant molecule exhibits reduced toxicity in the host as compared to the wild type protein. Toxicity need not be completely eliminated, but must be low enough to be used in an effective immunogenic composition without causing significant side effects in infected subjects.

Toxicity can be measured using one of several methods. Such methods include testing toxicity in mouse cells or CHO cells or assessing morphological changes induced in Y1 cells. Preferably, the toxicity is
It is evaluated by the rabbit ileal loop assay. Preferably, the toxicity of toxoid is reduced by a factor of 10,000 compared to its wild-type counterpart when measured in Y1 cells, or 10 minutes when measured by the ileal loop assay. It is less than 1.

Random or rationally directed mutations can be introduced into the amino acid sequence of toxin molecules to generate toxoid molecules using current conventional techniques of recombinant DNA methodology. Preferably, the mutation is introduced by site-directed mutagenesis (SDM). Currently, there are many techniques for SDM known to those skilled in the art, including using PCR (see, eg, Sambrook et al. (Molec.
ural clone, A laboratory manual. (1989
) Second edition. Cold Spring Harbor Laboratory P
oligonucleotide-directed mutagenesis, or using commercially available kits).

Many toxin coding genes suitable for use in this aspect of the invention are known in the art. E. reviewing the sequences of toxin A and toxin B subunits from various enterotoxic strains of E. coli, Domenighini et al., (19
95) Pay particular attention to the definitive reference by Mol microbiol 15 (6), 1165-1167.

For example, any modification in the amino acid sequence of any subunit of a toxin (eg, LT toxin and CT toxin) that has a detoxifying effect without significantly reducing immunogenicity (LT-herein). A and CT-A) are suitable for use in the present invention. The immunogenic detoxified protein may select substantially the same structural conformation as the wild-type naturally occurring toxin. It is immunologically active and can cross-react with antibodies to wild-type toxin.

For CT-A and LT-A, Val-53, Ser-63, Val-9
7, Ala-72, Tyr-104, or at a position corresponding to Pro106,
Alternatively, any insertion mutation, substitution mutation or deletion mutation at that position has the desired effect. Preferably the mutation is a substitution of a different amino acid. Preferred substitutions include Val-53-Asp, Ser-63-Lys, Ala-7.
2-Arg, Tyr-104-Lys, or Pro106-Ser.

If desired, the CT toxoid or LT toxoid may contain other mutations. Other mutations include, for example, Arg-7, Asp-9, Arg-11, His-.
44, Arg-54, Ser-61, His-70, His-107, Glu-.
Substitution with one or more of 110, Glu-112, Ser-114, Trp-127, Arg-146 or Arg-192 can be mentioned. These mutations were determined to be either completely non-toxic or have low residual toxicity as measured by the above assay.

Particularly suitable are the E. It is a mutant of Escherichia coli heat-labile enterotoxin (LT), and specifically, a mutant containing the substituted Ser-63-Lys (LTK63).

The amino acid residue (s) that replace the wild-type protein sequence may be naturally occurring or synthetic. Preferred substitutions are those that retain as much of the structural integrity of the protein as possible while altering the amphoteric and / or hydrophilic properties of the toxin. Such amino acids have the same steric effect as wild-type residues.

Preferably, for CT, the mutation used is Ser-63-Lys (herein CTK63), and for LT, the mutation used is Se.
r-63-Lys (LTK63 herein).

The selection of the bacterial Vibrio cholerae strain for use in the present invention includes any strain that can be readily cultured in vitro and depends on what purpose is desired. For example, if high levels of expression are required for protein purification purposes, high level production strains are used. More usually, the purpose is
To produce a live vaccine that is effective in humans to prevent infection from certain pathogens. In this scenario, attenuated Vibrio chol
The erae strain should be used.

Attenuated strains of Vibrio cholerae effectively but safely colonize the human intestine and produce antibodies with high bactericidal or viral activity. Such "live" strains retain attenuated pathogenicity and may either have limited growth in the host or may encode detoxified proteins. In general, a broad spectrum of immunity can occur after a single inoculation. This immunity can last a lifetime.

As used herein, the term “attenuation” means that the virulence of a bacterial strain is reduced to negligible levels in mammals. Such strains survive, have a normal life cycle, and secrete non-toxic mutated proteins or mutant protein fragments. Thus, the host does not suffer from any pathogenic condition associated with the wild-type (non-attenuated) strain, but the host's immune system is still exposed to foreign proteins expressed by bacterial factors. Preferably, bacterial strains suitable for use in the present invention are naturally attenuated bacteria.

In this context, a particularly suitable strain for use as a live vector for antigen presentation is the IEM101 strain, “Attenuated cholerae”.
Co-pending European Patent Application No. 98305 entitled "Strains"
It is the subject of No. 060.0. This strain reaches the small intestine in large numbers and colonizes. This bacterium is able to penetrate the mucus layer and attach to the mucosal epithelium. This indicates that they successfully elicit an immune response.

According to a further aspect of the invention, V. of the above aspects of the invention may be used with a pharmaceutically acceptable carrier. An immunogenic composition comprising a recombinant strain of Cholerae is provided.

Pharmaceutically acceptable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules (eg proteins, polysaccharides, polylactic acids, glycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (eg oil droplets or liposomes) and Inactive virus particles).
Such carriers are well known to those of ordinary skill in the art. Furthermore, these carriers may function as immune stimulators (adjuvants).

The composition may be used as a vaccine and thus may optionally include an adjuvant.

Suitable additional adjuvants for enhancing the efficacy of the immunogenic composition according to the invention include, but are not limited to: (1) of heat-labile toxin protein or pertussis toxin protein. Non-toxic mutants (eg Tsuj
i et al. (1990); Harford et al. (1989) and Roberts et al. (1995)); (2) oil-in-water emulsion formulations (other specific immunostimulatory factors such as muramyl peptides (see below). Or a bacterial cell wall component)), and these include, for example: (a) microfluidizer (
For example, Model 110Y Microfluidizer (Microflui
dics, Newton, MA) and formulated into submicron particles, MF59 (5% Squalene, 0.5% Tween 80, and 0.
PCT Publication No. WO 90/14837, including but not limited to 5% Span 85 (including but not limited to varying amounts of MTP-PE (see below) as needed). Formulation, (b) 10% Squalen
e, 0.4% Tween 80, 5% Pluronic block
-Blocked) polymer L121 and thr-MDP (see below) to produce a microfluidized or larger particle size emulsion in submicron emulsions. Either vortexed, and (c) 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components (monophosphoryl lipids).
A (MPL), trehalose dimycolic acid (TDM), and cell wall skeleton (CW)
Ribi ^™ adjuvant system (RAS), (Ribi Immunochoche) selected from the group consisting of S) and preferably comprising MPL + CWS (Detox ^™ )).
m, Hamilton, MT); (3) saponin adjuvant (eg, Sti
mulon ^™ (Cambridge Bioscience, Worcester
, MA) or particles produced therefrom (eg ISCOM (immunostimulatory complex) may be used)); (4) Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5) Cytokines (eg. , Interleukins (eg, IL-1, IL-2, IL-4, IL-5, IL-6, I
L-7, IL-12, etc.), interferon (eg, γ interferon)
, Macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.); and (6) other substances that act as immune stimulating factors and enhance the efficacy of this composition.

Examples of the muramyl peptide as described above include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normranyl-1-alanyl-d-isoglutamine (nor -MDP), N-acetylmuranyl-l-alanyl-d-isoglutaminyl-l-alanine-2- (1'-2'-
Examples include, but are not limited to, dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE) and the like.

According to a further aspect of the invention there is provided a process for formulating a vaccine composition, wherein the recombinant bacterium according to the above aspect of the invention is a pharmaceutically acceptable carrier,
A process is provided that includes combining the adjuvants, optionally together.

According to a still further aspect of the invention, a method of vaccinating a mammal against a disease, the preparation of a bacterium according to the above aspect of the invention expressing an antigen associated with the pathogen responsible for the disease. A method is provided that includes administering an article.

According to a further aspect of the invention there is provided a nucleic acid comprising sequences from the 5'and 3'regions of the rRNA operon, which flank the sequence encoding a protein or protein fragment that is heterologous to Vibrio cholerae. . Such nucleic acid may be recombinant Vibrio cho in accordance with the above aspects of the invention.
It is useful for the production of Lerae bacteria. Sequences derived from the rRNA operon allow for homologous recombination events to occur that result in the replacement of the endogenous rRNA operon for the disrupted version in which the nucleic acid encoding the heterologous protein or protein fragment is flanked by rRNA sequences. . Preferably r
The RNA operon is either the 16S gene or the 23S gene. In the most preferred embodiment of this aspect of the invention, the nucleic acid encoding the heterologous protein or protein fragment is cloned under the control of the OmpU promoter.

According to yet a further aspect of the invention there is provided a nucleic acid comprising an OmpU promoter operably linked to a nucleic acid encoding a protein or protein fragment that is heterologous to Vibrio cholerae. The term "operably linked" means that the promoter comprises the essential regulatory sequences necessary to drive the expression of a nucleic acid encoding a heterologous protein cloned downstream of the promoter.

According to a still further aspect of the invention, a nucleic acid comprising an OmpU promoter operably linked to a nucleic acid encoding a protein or protein fragment that is heterologous to Vibrio cholerae, the nucleic acid comprising:
Brio cholerae rRNA gene (preferably 16S or 23
A nucleic acid flanked by sequences from the 5'and 3'regions of the S gene) is provided.

The nucleic acids of these aspects of the invention can be incorporated into a vector such as a plasmid or bacteriophage. Any suitable vector can be used to transform the Vibrio cholerae bacterium, as will be apparent to those skilled in the art. Particularly suitable vectors include CVD422, which is incapable of replicating in host cells, used in this study.

According to a further aspect of the invention there is provided use of the rRNA operon as a location for inserting a nucleic acid encoding a heterologous protein or protein fragment into the chromosome of a Vibrio cholerae bacterium. Preferably rR
The NA operon is either the 16S gene or the 23S gene.

The present invention also provides the use of the OmpU promoter for the expression of heterologous antigens in Vibrio cholerae bacteria.

According to a further aspect of the invention, a chromosomal locus in the rRNA operon,
Preferably 16S or 23S in Vibrio cholerae bacteria
Provided is a method of expression of a heterologous protein or protein fragment in a Vibrio cholerae bacterium comprising the step of targeting a nucleic acid encoding a protein or protein fragment to an RNA gene.

The invention also provides a method of expression of a heterologous protein or heterologous protein fragment in a Vibrio cholerae bacterium, the method comprising cloning a nucleic acid encoding the heterologous protein or protein fragment under the control of an OmpU promoter. Includes.

According to a still further aspect of the invention there is provided a method of expression of a heterologous protein or heterologous protein fragment in a Vibrio cholerae bacterium, the method comprising: encoding a nucleic acid encoding a protein or protein fragment into a rRNA operon in a Vibrio cholerae bacterium. Targeting to a chromosomal locus (preferably 16S or 23S RNA gene) in, and expressing a nucleic acid encoding a heterologous protein or protein fragment thereof under the control of the OmpU promoter.

According to a further aspect of the invention there is provided a method of vaccinating a mammal against a disease, which method according to the above aspects of the invention comprises a heterologous protein associated with the pathogen responsible for the disease. Or administering a preparation of recombinant bacteria expressing a heterologous protein fragment.

The host to be infected by the bacterium of the present invention is a human; c
It is the only organism susceptible to infection by Holerae.

All documents mentioned in this specification are incorporated herein by reference.

Various aspects and embodiments of the invention are described in greater detail herein by way of example. It is understood that modifications of detail may be made without departing from the scope of the invention.

EXAMPLES The practice of the present invention will employ, unless otherwise specified, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are of skill in the art. It is within the range. Such techniques are explained fully in the literature. For example, Mo
regular Cloning; A Laboratory Manual,
Second Edition (Sambrook, 1989); DNA C
longing, Volumes I and ii (Glover, 1985)
); Oligonucleotide Synthesis (Gait ed., 19
84); Nucleic Acid Hybridization (Hames)
& Higgins, 1984); Transcription and Tr
anslation (Hames & Higgins 1984); Anima
l Cell Culture (Freshney, 1986); Immob
ilized Cells and Enzymes (IRL Press, 1
986); A Practical Guide to Molecular
Cloning (Perbal, 1984); the Methods i.
n Enzymology series (Academic Press, Inc.
), Especially 154 and 155; Gene Transfer Vector.
s for Mammalian Cells (Miller & Calos ed.,
1987, Cold Spring Harbor Laboratory);
Immunochemical Methods in Cell and M
olecular Biology (Edited by Mayer & Walker, 1987)
; Protein Purification: Principles and
Practice (Scopes, 1987); Handbool of E
xperimental Immunology, Volumes I-IV (W
Eir & Blackwell, 1986).

Example 1 Ribosomal RNA (rRNA of Vibrio cholerae
) Amplification and cloning of genes encoding 16S and 23S) Ribosomal RNA 16S and 23S genes (rRNA 16S and rR
NA 23S) is a V.N. There are 9 copies along the cholera chromosome. The inventors have identified them as chromosomal loci for inserting genes encoding heterologous antigens. Using a database from GeneBank,
The nucleotide sequences of the 16S rRNA and 23S rRNA genes were identified using accession numbers X74696 and U10956, respectively. Two fragments were generated by PCR containing the respective 5'and 3'ends of the two genes, respectively. For the 16S rRNA gene, a fragment containing nucleotides 1 to 434 was amplified using the following oligonucleotides: 5'-GCTCTAGAATTGAAGAGTTTTGATCCTGGCTCA.
G-3 '(forward) (including XbaI restriction site) and 5'-CGCGGATC
CGTTAACGTACTTTTACAACCCGAAGGCC-3 '(reverse direction)
(Including BamHI and HpaI restriction sites). A fragment containing 1116-1455 nucleotides was amplified using the following oligonucleotides: 5
'-CGGGATCCGGGGTACCAACTGCAGCCTTGTTTGC
CAGCACGT-3 '(forward) (including BamHI, KpnI and PstI restriction sites) and 5'-CCGGAATTCTCCCGAAGGTTAAAC.
TACTGCTTC-3 '(reverse direction) (including EcoRI restriction site). 23S
For the rRNA gene, a fragment containing nucleotides 33-370 was amplified using the following oligonucleotides: 5'-GCTCTAGAGGGC.
AGTCAGAGGGCGATGAG-3 '(forward) (including XbaI restriction site) and 5'-CGGGATCCCAAGCTTCCGCCCTACTCGAT
TTCAG-3 '(reverse direction) (including BamH1 and HindIII restriction sites). The fragment containing nucleotides 708 to 1077 was amplified using the following oligonucleotide: 5'-CGCGGATCCCCAGCTGCAGCA.
ACTGGAGGACCGAACC-3 ′ (forward direction) (BamHI, PvuII
, And PstI restriction sites) and 5′-CCGGAATTCTTTCT
TTAAATGATGGCTGCTTCTAAG-3 '(reverse direction) (EcoRI
Including restriction sites).

The DNA fragment containing the 5'and 3'regions of the rRNA 16S gene was cloned together into the Blue Script KS vector using the following strategy: This 434 bp amplified fragment was cloned using XbaI and BamHI. Digested, ligated with this amplified 339 bp fragment digested with BamHI and EcoRI, and cloned into XbaI-EcoRI digested Bluescript KS to generate BS-16S.

The 5 ′ and 3 ′ regions of rRNA 23S were put together in BlueScript KS
It was cloned into the vector using the following strategy: This 337 bp
Was amplified with XbaI and BamHI, ligated with the amplified 368 bp fragment digested with BamHI and EcoRI, and cloned into XbaI-EcoRI digested BluescrptKS to generate BS-23S.

Example 2: Vibri knocked out in the rRNA 23S gene
Strategy used to construct o strain) The CVD422 suicide plasmid was used to knock out the 23S gene by inserting the kanamycin resistance gene into the Vibrio chromosome. CV
D422 contains the β-lactamase gene and the SacB gene, which are useful markers for selection. The kanamycin resistance gene was isolated as an approximately 1.2 kb BamHI fragment from the pUC4K plasmid and cloned into BamHI digested BS-23S to generate a recombinant BS-23SKm ^r plasmid. The BS-23SKm ^r plasmid was used as a restriction enzyme for SacI and Ec.
The oRV digested, approximately 2.0 kb fragment was cloned into SacI-SmaI digested CVD422 to generate CVD-23SKm ^r . This plasmid was transformed into E. E. coli SM10 (pir donor strain) was transformed and this recombinant strain was used for conjugation of the IEM101 recipient strain (polymyxin B, gentamicin resistance). The first recombination event was selected by plating the bacteria and recovered from the conjugate on LB agar containing 100 μg / ml ampicillin, 0.75 μg / ml polymyxin B and 0.75 μg / ml gentamicin. The second recombination event (where suicide vector (CVD422, Am
p ^r , Sucrose ^s ) was deleted from the chromosome) was selected by growing the bacteria at 28 ° C. on medium containing 10% sucrose. Sucrose resistant bacteria were transformed into L. cerevisiae supplemented with 15 μg / ml kanamycin. B. Re-plated on agar. A recombinant IEM101 strain (Amp ^s , Suc ^r , Km ^r ) having a kanamycin resistance gene integrated in the rRNA 23S gene on the chromosome was designated as IEM-23.
It was named SKm ^r .

Example 3: IEM10 knocked out in the rRNA 23S gene
Characterization of 1 strain) One copy of ribosomal gene 23S interferes with IEM101 strain and IEM23S
It was evaluated whether the two strains of Km ^r affect bacterial survival and growth. To do this, IEM101 and four IEM23Km ^r recombinant strains (1a,
1b, 2a and 2b) in an overnight culture at 37 ° C. in 20 ml LB medium.
: Grow with 50 dilutions starting. Samples were collected at the beginning of growth (T ₀ ) and 3 and 6 hours after growth. Optical density at 600 nm (OD
． ₆₀₀ ), the culture was diluted, and the bacterial dilution was plated on LB agar.

The results of this experiment were plotted in the graph shown in FIG. With these results,
It is shown that knocking out one copy of the rRNA gene does not affect the growth rate.

The chromosomal site where the kanamycin resistance gene was inserted in the four IEM23SKm ^r recombinant strains was characterized by Southern blot analysis. To do this,
Chromosomal DNA from IEM101 and recombinant IEM23SKm ^r 1a, 1b, 2a and 2b strains were digested with PvuII restriction enzyme and loaded on a 0.8% agarose gel. After transferring to nitrocellulose membrane, chromosomal DNA,
It was probed with a labeled 520 bp XhoI / HindIII fragment of the Km ^r gene. The results of this experiment showed that the IEM23SKm ^r 1a and 1b strains had the same hybridization pattern, and this pattern was I
It is shown to be different from the pattern of the EM23SKm ^r 2a and 2b strains (Figure 2). This means that IEM23SKm ^r 1a and 1b, and IEM2
Insertion of the kanamycin resistance gene in 3SKm ^r 2a and 2b resulted in rRN
It shows that it occurred in two different copies of the A23S gene.

Example 4 Identification of OmpU Outer Membrane Proteins as Highly Expressed Proteins in V. cholerae Total Cell Extracts The OmpU promoter was identified as follows: IEM101 strain in 50 ml L. B. Growing at 37 ° C starting with a 1:50 dilution of the overnight culture in medium. A 1 ml sample was collected every 30 minutes for 6.5 hours. Sample O. D. _{A 600} was evaluated and the relative curve is reported in Figure 3a. The bacterial sample was centrifuged and the pellet resuspended in PBS to give O.C. D. ₆₀₀
= 28 was obtained. 10 μl of each sample was loaded on a 10% polyacrylamide gel.

The results reported in FIG. 3b showed that the protein of approximately 38 kDa was more highly expressed relative to other proteins present in total cell extract. This protein is expressed at high levels preferentially in late log phase and
After hours the bacterial culture O. D. _{When the 600} was 2, the maximum was reached.

To identify proteins, samples corresponding to 6 hours of growth were treated with 10% SD
S-PAGE loaded and Sequi-blot PVDF membrane (Biora).
d, Hercules, California). Bec with on-line phenylthiohydantoin amino acid analyzer (system gold)
By automated sequence analysis according to the manufacturer on a kman sequencer (LF3000),
The amino terminus was determined. Using the deduced amino-terminal sequence, NCBI Basic
Screen the BLAST protein database and chole
It was identified as the amino terminal portion of the rae OmpU outer membrane protein (residues 22-41).

Example 5: Amplification of OmpU outer membrane protein promoter region and pGEM
Cloning in 3 vector) The nucleotide sequence for the OmpU gene was obtained from the GeneBank database using accession number U73751. The promoter and upstream regulatory region were identified by sequence analysis and the fragment derived from nucleotides 600-824 (immediately before the ATG codon) was amplified. The following oligonucleotides:

[0082]

[Chemical 1] (Including restriction sites BamHI, HpaI and NruI) and oligonucleotides

[0083]

[Chemical 2] The restriction sites BamHI, HpaI, EcoRV and XhoI were used for amplification. The amplified fragment of approximately 280p was digested with BamHI. This fragment was digested with BamHI to the pGEM3 vector (Promega).
, WI. USA) to generate the pGEM-omp plasmid.

Example 6: Helicobacter under control of OmpU promoter
Cloning of gene encoding NAP protein derived from pylori) The gene encoding NAP protein was cloned into plasmid pSM214G (Rob
erto Petracca, IRIS, Chiron S. et al. p. A. , Sie
(provided by Na.), the following oligonucleotides:

[0085]

[Chemical 3] (Including the restriction sites PstI and XhoI) and

[0086]

[Chemical 4] Amplification was carried out (including the restriction sites KpnI and EcoRV). This amplified fragment of approximately 470 bp containing the coding sequence for NAP was digested with the enzymes XhoI and EcoRV and XhoI / EcoRV digested pGEM-om.
p-ompNAP plasmid was generated by cloning into p.

(Example 7: Construction of IEM-ompNAP recombinant strain) The NAP gene under the control of the OmpU promoter was replaced with the rRNA of IEM101.
To insert the OmpU-NAP DNA sequence into the 23S chromosomal sequence,
It was cloned within the 5'and 3'regions of the gene. To do this, p-o
The mpNAP plasmid was digested with BamHI and BamHI digested p
Clone into BS-23S to generate BS-23 SompNAP plasmid. This plasmid was digested with SacI-SalI and obtained 1.5k
fragment of b (adjacent to the 5'and 3'ends of the rRNA 23S gene,
Containing the NAP gene under the control of the OmpU promoter), SacI-SalI
Cloned into the CVD422 vector digested with
AP plasmid was generated. This plasmid was introduced by transforming into SM10 (pir donor strain) and this recombinant strain was used for conjugation of the IEM-23SKm ^r recipient strain (gentamicin, polymyxin and kanamycin resistance).

After conjugation, this recombinant strain (resulting from a single recombination event, and on the chromosomal rRNA
(Including the entire recombinant plasmid integrated into the 23S locus), 100 μg
/ Ml ampicillin, 0.75 μg / ml gentamicin and 0.75 μg / ml
L. supplemented with ml polymyxin and 15 μg / ml kanamycin. B. Selected by plating on agar.

To select for loss of plasmid sequences and thus for the second recombination event, Amp, Gm, Pol, Kan resistant recombinant colonies were grown overnight at 28 ° C. in medium containing 10% sucrose. . To select for bacteria in which the gene encoding kanamycin resistance has been replaced by the gene encoding NAP, sucrose resistant colonies were transformed with L. B. Agar, and L. a. Supplemented with 15 μg / ml kanamycin. B. Plated on both agars. Kanamycin-sensitive colonies (containing the OmpU-NAP gene integrated in the 23S gene) were transferred to IEM23S-omp.
It was named NAP and used for further analysis.

Example 8 Expression of NAP Protein by IEM-ompNAP Strain IEM23S-ompNAP strain was tested for viability, proliferation and N in a time course experiment.
AP expression was analyzed and the IEM101 wild type strain was used as a control.

To do this, overnight cultures of both strains were diluted 1:25 in BL medium and incubated at 37 ° C. for 6 hours. A 1 ml sample was collected every 30 minutes. Bacterial viability was analyzed by plating various sample dilutions on LB. The level of NAP expression was obtained by sonicating bacterial pellets from 0.5 ml cultures (4 7 impulses every 30 seconds) with anti-NAP rabbit polyclonal antisera. Evaluated by Western blot analysis of 10 μl min.

The results of this experiment are reported in Figures 4 and 5. As shown in FIG.
The growth rate was the same in the two strains (IEM101 and IEM-ompNAP). This indicates that NAP gene insertion under the control of OmpU promoter does not affect the viability of the strain. Further, as shown in FIG.
The level of AP expression was very high during the first 2 hours of growth and decreased at 5 hours,
It increased over the next 6 hours.

Vibrio cholerae has previously been shown to be capable of producing NAP protein in its decameric form (see co-pending International Patent Application PCT / IB99 / 00695). NAP is H.264. It is also known to be produced as a decamer in Pylori (Doyce and Evans (1995
) Infect Immun, 63: 2213-2220). To confirm whether the NAP expressed by Vibrio could form decamers even when the expression was regulated by the OmpU promoter, the soluble fraction was subjected to Western blotting under non-reducing and non-denaturing conditions. analyzed.

To do this, the bacterial pellet obtained from 50 ml of overnight culture was
Resuspended in 1 PBS and sonicated. 10 ml of sonicated material was non-reduced (no dithiothreitol) and non-denatured (no dithiothreitol) in 8% SDS-PAGE
Loaded under both conditions (without heating) and analyzed by Western blot. The results reported in FIG. 6 show that under these conditions the NAP antigen migrates as a 150 kD protein (corresponding to the decamer form) on SDS polyacrylamide gels.

Example 9: Construction of IEMnirNAP strain E. coli induced by anaerobic conditions. N under the control of the E. coli nirB promoter
Recombinant IEMnirNAP strain expressing AP antigen was obtained as described.
The nirB promoter is represented by the following oligonucleotide: 5'-CGCGGA
TCCGTTAACTCGCGAGAATTCAGGTAAATT-3 '(forward) (including BamHi, HpaI, NruI and EcoRI restriction sites) and 5'-CGCGGATCCCGTTAACGACATCCTCCGAGCAGAA
P from pnir100 plasmid using AGTCTCCTGT-3 ′ (reverse direction) (including BamHI, HpaI, EcoRV and XhoI restriction sites)
Amplified by CR. The amplified 140 bp DNA fragment was used as B
It was digested with amHI and cloned into pGEM3 vector to obtain pGEMn.
An ir plasmid was obtained. This plasmid was digested with XhoI and EcoRV,
It was then ligated to the XhoI / EcoRV fragment containing the NAP gene (described previously) to generate the pnirNAP plasmid. This plasmid is Ba
digested with mHI and containing the NAP gene under the control of the nirB promoter,
The resulting 610 bp DNA fragment was digested with BamHI BS-
Ligation with 23S (described previously) generated the BS-23SnirNAP plasmid. This plasmid was digested with SacI and SalI and rR
This generated 1500 bp DNA fragment containing the nirNAP gene flanked by the 5'and 3'ends of the NA23S gene was cloned into SacI and Sa
Ligation with the I1 digested CVD442 vector generated CVD23S-nirNAP. This plasmid was transformed into the SM10λpir strain and introduced into the IEM23SKm ^r strain by conjugation. Recombinant strains resulting from homologous recombination were isolated using the above selection. NirB integrated into 23S rRNA gene
This kanamycin-sensitive strain containing the -NAP gene was designated as IEM23S-ni.
It was named rNAP.

Example 10: Comparison of NAP levels when expression is driven by two different promoters: OmpU and nirB. Previous studies have shown that the nirB promoter is expressed in the V.I. It was shown to be a strong promoter in cholerae. We have two strains IEMomp
The activity of the nirB promoter was compared to that of the OmpU promoter by analyzing the amount of NAP produced by NAP and IEMnirNAP. These two strains contain the gene encoding NAP under the control of the OmpU promoter or the nirB promoter, respectively. These strains were cultured overnight at 37 ° C. with aeration. Dilute these cultures 50-fold,
The IEMompNAP strain was grown at 37 ° C. for 2 hours under aerobic conditions,
The IEMnirNAP strain was grown at 37 ° C. for 4 hours under low aeration. These cultures were centrifuged and the bacterial pellet was resuspended in PBS to obtain approximately 10 ¹⁰ cells / ml. Add 15 μl of each preparation to 15% SD
Loaded on S-polyacrylamide gels and analyzed for the presence of NAP by Western blot analysis using an anti-NAP rabbit monoclonal antiserum. These results, as reported in FIG. 7, indicate that the expression level of NAP antigen was significantly higher when the expression was driven by the OmpU promoter compared to the nirB promoter.

Example 10 Immunization of Mice BalbC mice (8 mice in each group) were evaluated on days 0, 28 to evaluate the anti-NAP immune response induced by the IEMompNAP and IEMnirNAP strains, respectively. Eyes, days 42 and 56 were immunized intranasally with 10 ⁸ live bacteria. Control animals were immunized with 10 μg of purified NAP alone or with 10 μg of purified NAP in combination with 1 μg of CTK63 as an adjuvant (in these cases each group contained 5 mice). Animals
Blood was drawn on days 0, 27, 41, 55 and completed on day 70. In addition, on day 70, bile was collected and nasal washes were performed.

Example 11: ELISA for measuring anti-NAP antibody The anti-NAP Ig antibody response in the serum of mice after each immunization, or the IgA antibody response in nasal washes and bile after these four immunizations was determined. , ELISA. The ELISA test was performed as follows. In each well of a 96-well plate,
Add 100 ng of purified NAP (50 μl / well) and plate at 4 ° C.
Incubate overnight. The wells were then plated with PBS, 0.05% Tween.
Wash 3 times with n-20 (PBT) and saturate with 100 μl / well 1% BSA in PBS for 1 hour at 37 ° C. 100 μl of 1:50 diluted serum or bile and 100 μl of undiluted nasal wash were added and serially diluted. The plates were incubated at 37 ° C for 2 hours and washed as above. All I
For detection of g, the wells were added with 50 μl of 1: 1000 dilution of rabbit anti-mouse I
g (horseradish peroxidase (HRP) conjugated) at 37 ° C. for 1 and a half hours. For detection of IgA, add wells with 50 μl of 1:10.
With biotin-conjugated goat anti-mouse IgA (α chain specific) at 00 dilution, 37 ° C
Incubate for 1 hour and a half and wash with PBT, 50 μl 1: 100
0 dilution of HRP-conjugated streptavidin was added to each well and the plates were incubated at 37 ° C for 1 hour. Antigen-bound antibody was visualized by adding o-phenylenediamine (OPD) as a substrate. After 15 minutes, the reaction was blocked by the addition of 50 μl / well of 12.5% H ₂ SO ₄ at a final concentration of 1 M and the absorbance was read at 490 nm. EL in serum
The ISA titer was arbitrarily determined as the reciprocal of the final dilution that produced an OD _490nm greater than 0.3 over pre-immune serum, while for mucosal lavage and bile IgA titers pre-immune serum was less than 0. Expressed as the reciprocal of the final dilution that produced an OD _490nm greater than 2. These values were normalized using positive control sera on each plate.

As shown in FIG. 8, Ig anti-NAP antibodies were only detectable in mice immunized with the IEMompNAP strain or with purified NAP in the presence of CTK63 as an adjuvant. The Ig immune response is IEMnirNA
It was not detected in the serum of mice immunized with P strain or purified NAP alone. Interestingly, the level of anti-NAP immune response induced when the NAP antigen was delivered in vivo by the recombinant IEM101 strain was higher than that when the purified protein was administered in the presence of mucosal adjuvant. It was

Furthermore, after the fourth immunization, IgA immune responses were only observed in the sera of mice immunized with the IEMompNAP strain or in the sera of mice immunized with purified NAP in combination with CTK63 (FIG. 9), whereas No IgA immune response was detectable in mice immunized with IEMnirNAP or purified NAP.
IgA immune responses were also detected in bile and nasal washes of each group of mice (FIGS. 10a and 10b), and in this case, this level was significantly higher when mice were immunized with IEMompNAP or NAP and CTK63. It was

DISCUSSION In the present study, we found that IEM101 was found to have high levels of H. cerevisiae in soluble and decamer forms. It was possible to express the pylori NAP protein and, interestingly, showed that a detectable amount of NAP was also present in the culture supernatant.
In addition, we have found that V. The Cholerae OmpU promoter was identified as a useful promoter for high level expression of foreign antigens, and we have determined the activity of this promoter in E. coli as previously described. E. coli nirB promoter.

The NAP gene is expressed in r of IEM101 under the control of these two different promoters.
The RNA integrates into the 23S chromosomal locus, and their results indicate that both the nirB and OmpU promoters operate in IEM101, but the amount of protein is higher when expression is driven by the OmpU promoter. Since we can achieve high level expression without compromising the growth rate or viability of the strain, we are ideal for inducing high level expression of foreign antigens in Vibrio. It can be concluded that the promoter has been identified.

Furthermore, surprisingly, the rRNA operon was cloned by the V. It has been found to be a particularly suitable target for chromosomal integration of foreign genes in cholerae.
In fact, insertion of a foreign nucleic acid sequence at this site (resulting in the presence of r in the chromosome)
(With partial deletion of one of the nine copies of RNA). cholera
It enables the efficient high level expression of heterologous antigens without compromising the growth rate or viability characteristics of e.

The immunological response of the recombinant strain IEMompNAP was evaluated in a mouse model of intranasal immunization. This IEMompNAp strain induces the highest antibody titers against serum Ig and mucosal IgA. The stimulation of the immune response by the NAP protein when expressed at high levels in vivo by the IEMompNAP strain is better than the immune response induced by purified NAP antigen in combination with CTK63 as an adjuvant. This finding indicates that the amount of expressed protein affects the antibody response. In fact, the IEMompNAP strain is more immunogenic than the IEMnirNAP strain, which expresses antigen in lower amounts.

In conclusion, live attenuated IEM101 V.V. The cholerae strain is considered a good carrier for antigen delivery at mucosal surfaces, which can stimulate high titers of systemic Ig and secretory IgA. The IEMompNAP recombinant strain is a V. cho
Lera and H .; Represents an ideal vaccine candidate against pylori.

[Brief description of drawings]

FIG. 1 Knocking out a copy of the 23S rRNA gene does not affect the bacterial growth rate.

FIG. 2. Insertion of the kanamycin resistance gene in IEM23SKm ^r 1a and 1b, and IEM23SKm ^r 2a and 2b resulted in 2 of the rRNA 23S gene.
It occurs in two different copies.

FIG. 3 a) Growth curve of IEM101 Vibrio cholerae strain. b) IE
Protein expression at various points in M101 growth.

FIG. 4 Comparison of growth rates for the two strains IEM101 and IEM-ompNAP.

[Figure 5]   Expression levels of NAP in strain IEM-ompNAP.

FIG. 6: NAP was identified in the decamer form in V. Western blot showing expression in Cholerae.

FIG. 7: Comparison of the levels of NAP whose expression is driven by the OmpU or nirB promoters. Western blot of whole cell extracts loaded on 15% SDS PAGE. Lane 1, purified NAP. Lane 2, IEMompNAP. Lane 3,
IEMnirNAP.

FIG. 8. Serum Ig anti-NAP titer. Purified NA in combination with purified NAP and purified CTK63
Mean Ig anti-NAP titers in sera of mice immunized with P, IEMompNAP, and IEMnirNAP live bacteria.

FIG. 9. Serum IgA anti-NAP titer. Purified NAP, purified NAP in combination with CTK63
Mean Ig anti-NAP titers in the sera of mice immunized with live, IEMompNAP, and IEMDMIPnirNAP bacteria.

FIG. 10. Mucosal IgA anti-NAP. a) Purified NAP, purified NAP and CTK63, IEM
I in the bile of mice immunized with ompNAP and IEMnirNAP strains
Individual titers of gA anti-NAP. b) Purified NAP, purified NAP and purified CTK63
, Individual titers of IgA anti-NAP in nasal washes of mice immunized with IEMompNAP and IEMnirNAP. Black bars in a and b indicate average titers.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 31/12 C12R 1:63 C12N 1/21 C12N 15/00 ZNAA // (C12N 1/21 A C12R 1 : 63) (C12N 15/09 C12R 1:63)

Claims (32)

[Claims] 1. A recombinant Vibrio cholerae bacterium comprising a nucleic acid encoding a heterologous protein or a heterologous protein fragment inserted at a locus within the rRNA operon of the bacterium's chromosome. 2. A recombinant Vibrio cholerae bacterium which comprises Om
A recombinant bacterium comprising a nucleic acid encoding a heterologous protein or a heterologous protein fragment cloned under the control of the pU promoter. 3. A recombinant Vibrio cholerae bacterium which comprises Om
It has been cloned under the control of the pU promoter and is r of the bacterial chromosome.
A recombinant bacterium comprising a nucleic acid encoding a heterologous protein or a heterologous protein fragment inserted at a locus within the RNA operon. 4. The recombinant bacterium according to claim 1 or 3, wherein the rRNA operon is either 16S gene or 23S gene. 5. The nucleic acid comprises 23 of the Vibrio cholerae chromosome.
The recombinant bacterium according to claim 4, which is inserted into a locus within the 3S gene. 6. The recombinant bacterium according to any one of claims 1 to 5, wherein the Vibrio cholerae bacterium is an attenuated bacterium. 7. The recombinant bacterium of claim 6, wherein the Vibrio cholerae bacterium is naturally attenuated. 8. The Vibrio cholerae bacterium is IEM101.
The recombinant bacterium according to claim 7, which is a strain of bacterium. 9. The heterologous protein or heterologous protein fragment comprises:
The recombinant bacterium according to any one of claims 1 to 8, which is a membrane protein or a part of a membrane protein. 10. A recombinant bacterium according to any one of claims 1 to 9, wherein the heterologous protein or heterologous protein fragment is or is a part thereof: NAP. Proteins, human tumor antigens, or adhesion factors such as fibrous hemagglutinin (FHA), pertactin or pili,
Fragments C and E of tetanus toxin from Clostridium tetani
． E. coli strain-labile heat-labile toxin protein and its genetically detoxified derivative, Neisseria meningitidis antigen, Neisseria gonorrhoeae antigen, influenza antigen, malaria antigen, tetanus toxin, B. a tracheal colony forming factor or pertussis toxin from Pertussis, H. pertussis.
VacA and CagA from pylori, E6 and E7 from papillomavirus, gD2 from herpesvirus, E1 or E2 core protein from HCV, gp120 from HIV, LT-A and CT-A,
LT mutant LTK63 or LT mutant LTR72, CT mutant CTK63, or other variants thereof. 11. A recombinant bacterium according to any one of claims 1 to 10, wherein the bacterium comprises nucleic acids encoding two or more heterologous proteins, at least one of the proteins as an adjuvant. A recombinant bacterium capable of acting, and at least one heterologous protein capable of acting as an antigen. 12. An immunogenic composition comprising a recombinant bacterium according to any one of claims 1 to 11 together with a pharmaceutically acceptable carrier. 13. A vaccine composition comprising the recombinant bacterium according to any one of claims 1 to 11 together with a pharmaceutically acceptable carrier. 14. The vaccine composition of claim 13, further comprising an adjuvant. 15. A process for the formulation of the vaccine composition according to claim 13, wherein the recombinant bacterium according to any one of claims 1 to 11 is used as a pharmaceutically acceptable carrier. A process that includes the steps of combining. 16. A process for the formulation of a vaccine composition according to claim 14, comprising the step of combining the recombinant bacterium according to any one of claims 1 to 11 with an adjuvant. process. 17. A recombinant bacterium according to any one of claims 1 to 11 for use as a medicament. 18. A recombinant bacterium according to any one of claims 1 to 11 for use as an adjuvant. 19. Use of a recombinant bacterium according to any one of claims 1 to 11 in a vaccine composition. 20. A method for preventing or treating a disease in a subject, which comprises an immunologically effective dose of the composition of claim 12 or 13.
A method comprising the step of administering to said subject. 21. A nucleic acid comprising a sequence encoding a protein or protein fragment that is heterologous to Vibrio cholerae, wherein the nucleic acid is inserted between sequences derived from the 5'and 3'regions of the rRNA operon. Nucleic acid, wherein the rRNA sequence is adjacent to a sequence encoding the heterologous protein. 22. A nucleic acid comprising an OmpU promoter operably linked to a nucleic acid encoding a protein or protein fragment that is heterologous to Vibrio cholerae. 23. A nucleic acid comprising an OmpU promoter operably linked to a nucleic acid sequence encoding a protein or protein fragment heterologous to Vibrio cholerae, the nucleic acid comprising:
A nucleic acid flanked by sequences from the 5'and 3'regions of the oleae rRNA gene. 24. The nucleic acid according to any one of claims 21 to 23, wherein the rRNA operon is either the 16S gene or the 23S gene. 25. A vector comprising the nucleic acid according to any one of claims 21 to 24. 26. Use of the rRNA operon as a location for insertion of a nucleic acid encoding a heterologous protein or a heterologous protein fragment into the chromosome of a Vibrio cholerae bacterium. 27. Use of the OmpU promoter for the expression of a heterologous antigen in a Vibrio cholerae bacterium. 28. A method of expressing a heterologous protein or a heterologous protein fragment in a Vibrio cholerae bacterium, wherein the nucleic acid encoding the protein or protein fragment is the Vibrio chore.
A method comprising targeting to a chromosomal locus in the rRNA operon of the lerae bacterium. 29. A method of expressing a heterologous protein or heterologous protein fragment in a Vibrio cholerae bacterium, the method comprising cloning a nucleic acid encoding the heterologous protein or heterologous protein fragment under the control of an OmpU promoter. 30. A method of expressing a heterologous protein or a heterologous protein fragment in a Vibrio cholerae bacterium, wherein the nucleic acid encoding the protein or protein fragment is said Vibrio chore.
A method comprising targeting to a chromosomal locus in the rleae bacterial rRNA operon and expressing a nucleic acid encoding the heterologous protein or heterologous protein fragment under the control of the OmpU promoter. 31. The use according to claim 26, or the method according to claim 28 or claim 30, wherein the rRNA operon is either the 16S gene or the 23S gene. 32. A method of vaccinating a mammal against a disease comprising:
12. A method comprising administering a recombinant bacterial preparation according to any one of claims 1 to 11 which expresses a heterologous protein or a heterologous protein fragment associated with the pathogen responsible for the disease.