CA2244800A1 - Immunogenic detoxified mutants of cholera toxin - Google Patents

Abstract

An immunogenic detoxified protein comprising the amino acid sequence of subunit A of a cholera toxin (CT-A) or a fragment thereof in which at least one amino acid is substituted with another amino acid characterized in that, in purified form, the immunogenic detoxified protein exhibits a residual toxicity greater than 10000 fold lower than its naturally occurring counterpart. In the described embodiment, the amino acid at, or in a position corresponding to Pro-106 is replaced with another amino acid. The immunogenic detoxified protein is useful as vaccine for Vibrio cholerae and is produced by recombinant DNA means by site-directed mutagenesis.

Description

W O 97/29771 PCT~B97/00183 IMMUNOGENIC DETOXIFIED MUTANTS OF CHOLERA TOXIN

~ Field of the invention ~ 5 The present invention relates to immunogenic detoxified proteins of cholera toxin (CT) wherein at least one amino acid is su~stituted with another amino acid with the result that, in purified form, the immunogenic detoxified protein exhibits a residual toxicity greater than 10000 fold lower than its naturally occuring counterpart and to their use in vaccines which are useful for the prevention or treatment of cholera and as mucosal adjuvants for other immunogenic proteins. The detoxified i~munogenic proteins can be suitably produced using recombinant DNA techniques by site-directed mutagenesis of DNA encoding the wild type toxins.

Backqround to the Invention Cholera is a con~agious disease widely distributed in the world, in particular in the Third World, where, in certainareas, it is endemic. The serious disorders which develop in the intestinal system prove fatal in a high percentage of the recorded cases of the disease.

The etiological agent of cholera is the Gram-negative microorganism Vibrlo cholerae (V. cholerae) . This colonises the intestinal tract of individuals who have come into contact with it through ingestion of contaminated food or water, and multiplies to very high concentrations. The principal symptom is severe diarrhoea as a result of which the patient can lose as much as 10-15 litres of liquids per day via the faeces. As a result of the severe dehydration and loss of electrolytes, the patient does not withstand the infection in 50-60% of cases, and dies. The diarrhoea caused by V. cholerae is due to the secretion of cholera toxin, CT, which acts by stimulating the activity of the adenylate cyclase enzyme so as to induce disturbances at cell level.

W O97/29771 PCT~B97/00183 Although cholera can be effectively cured by controlled and intense rehydration, the distribution of a vaccine is desirable with a view to complete control and future eradication of the disease.
At the present time, there exists a vaccination against cholera, consisting of parenteral administration of killed bacteria. Although some countries insist on vaccination against the disease, there are serious doubts as to its real usefulness, given that the current cellular vaccine protects against the consequences of the infection in only 50% of the cases and that the protection is also extremely limited in duration, to less than 6 months.

In Bangladesh, an experimental trial is in progress ~1990-92) of an oral vaccine consisting of ~illed bacteria with the addition of subunit B of cholera toxin, which is known to be highly immunogenic. This product succeeds in inducing lasting protection, without special side effects (Holmgren J., Clemens J., Sack DA., Sanchez J. and Svennerholm AM;
"Oral Immunization against cholera" Curr. Top. Microbiol.
Immunol. (1988), 146, 197-204).

The CT toxin comprises a single A subunit (or protomer A) responsible for the enzymic activity of the toxin (herein CT-A) and five identical B subunits (or protomer B) which are involved in the binding of the toxin to the intestinal epithelial cells (herein CT-B).

The A subunit penetrates the cell membrane and causes activation of adenylate cyclase by NAD-dependent ADP-ribosylation of a GTP-binding protein which controls the activity of the enzyme. The clinical effect of this is to cause massive fluid loss into the intestine.
Considerable research has been conducted on cholera toxin.
Its sequence is known and has been described (Mekalanos J.J.
et al Nature 306, page 551 (1983)).

In view of the potential clinical significance of vaccines ~ ~ against cholera there is a continuing and great interest in producing a detoxified toxin capable of immunising against cholera. The techniques of genetic engineering allow specific mutations to be introduced into the genes encoding the toxins and the production of the mutated toxins using now conventional techniques of gene expression and protein purification.

Kaslow, H.R. et al (Abstract B291 of the 92nd General Meeting of the American Society for Microbiology, 26-30th May 1992) describe mutating Asp-9 and ~is-44 and truncating after amino acid 180 in CT-A which all essentially eliminate activity. Mutating Arg-9 is said to markedly attenuate activity. Mutating other amino acid sites had little effect on toxicity.

Burnette, W.N. et al (Inf. and Immun. 59fll), 4266-4270, (1991)) describe site-specific mutagenesis of cT-A to produce an Arg-7-Lys mutation paralleling that of a known detoxifying mutation in the A subunit of the Bordetella pertussis toxin. The mutation resulted in the complete abolition of detectable ADP-ribosyltransferase actLvity.
.

International patent application WO 92/19265 (Burnette, Kaslow and Amgen Inc.) describes mutations of CT-A at Arg-7, Asp-9, Arg-ll, His-44, His-70 and Glu-112.

A mutations at Glu-110 has also been described in the literature (Lobet, Inf. Immun., 2870, l991; Lai, Biochem.
Biophys. Res. Comm. 341 1983; Okamoto J. Bacteriol. 2208, 1988).

It is known that the development of toxicity of the A
subunits of CT requires proteolytic cleavage of A1 and A2 subunits at around amino acid Arg-ls2 (Grant et a7 Inf. &
Immun. (1994) 62(10) 4270-4278).

W O97/29771 PCT~B97/00183 Immunogenic detoxified proteins comprising the amino acid ~ ~ sequence of subunit A of a cholera toxin or a fragment thereof, wherein one or more amino acids at, or in positions corresponding to Val-53, Ser-63, Val-97, Tyr-104 or Pro-106 are replaced with another amino acid are disclosed in WO
93/13202 (Biocine Sclavo SpA). Optionally the amino acid sequence may include other mutations such as, for example, substitutions at one or more of Arg-7, Asp-9, Arg-11, His-44, Arg-54, Ser-61, His-70, His-107, Glu-110, Glu-112, Ser-114, Trp-127, Arg-146 or Arg-192. These mutations are described as ~eing completely detoxified as measured by the assays described.

A double mutant comprising the amino acid sequence of CT-A
or a fragment thereof is described in UK patent application 9513371.6 (filed 30th June 1995) wherein the amino acids at, or in positions corresponding to, Ser-63 and Arg-192 are replaced with another amino acid.

Detoxified mutants of pertussis toxin have been reported to be useful both for direct intranasal vaccination and as a mucosal adjuvant for other vaccines (Roberts et al Inf. &
Immun. (1995) 63(6) 2100-2108). Published International patent application WO 95/17211 (Biocine SpA) describes the use of detoxified mutants of CT as mucosal adjuvants.

SummarY of the invention We have discovered that a residual low level of toxicity provides an improved altered CT for use in a vaccine and/or a mucosal adjuvant.

Although detoxified in the sense of having a much lower toxicity than the wild-type protein, these proteins retain traces of enzymatic activity. The mutation causes a decrease of in vivo toxicity of at least lOOOO fold and this makes the protein very useful for human use. In addition to the immunological and adjuvant properties typical of mutants W O 97/29771 PCTnB97/00183 devoid of enzymatic activity, the mutant protein surprisingly exhibits an increase in adjuvant activity.

According the present invention, there is provided an immunogenic detoxified protein comprising the amino acid sequence of subunit A of a cholera toxin (CT-A) or a fragment thereof wherein at least one amino acid is substituted with another amino acid characterised in that, in purified form, the immunogenic detoxified protein exhibits a residual toxicity greater than 10000 fold lower than its naturally occuring counterpart.

The immunogenic detoxified protein according to the present invention exhibits a carefully selected balance of toxicity m~;mising the immunogenicity and/or adjuvant effects of the protein whilst maintaining a sufficiently low toxicity to be tolerated by the immunised individual.

Preferably the amino acid at, or in a position corresponding to, Pro-106 is replaced with another amino acid.

That this particular preferred embodiment would exhibit the claimed features was not predictable ~rom the prior art which generally teaches that absolute removal of toxicity was the goal for the development of mutant toxin vaccines and adjuvants and in particular teaches that this particular mutated toxin would have no toxicity (see WO 93/13202).

In this specification, references to CT encompass the various naturally occurring strain variants as well other variants encompassing changes from the sequences disclosed herein which do not affect the immunogenicity of the assembled toxoid.

The amino acid sequences for CT are definitively described in Domenighini et al Molecular Microbiology (1995) 15(6) 1165-1167.

CA 02244800 l998-07-3l W O97/29771 PCT~B97/00183 The amino acid substituted for the wild type amino acid may ~ ~ be a naturally occurring amino acid or may be a modified or synthetic amino acid, provided that the mutant retains the necessary immunogenic properties and exhibits a greater than 10 000 fold reduction in toxicity relative to the naturally occuring counterpart. The substitution may involve deletion or addition of one or more amino acids.

Substitutions which alter the amphotericity and hydrophilicity whilst retaining the steric effect of the substituting amino acid as far as possible are generally preferred.

As used herein, the term l'detoxified" means that the immunogenic composition exhibits greater than 10 000 fold reduction in t~xicity relative to its naturally occurring toxin counterpart. The reduction in toxicity should be sufficiently low for the protein to be used in an immunogenic composition in an immunologically effective amount as a vaccine with causing significant side effects.

As used herein, the term "residual toxicity" means that the detoxified immunogenic protein retains a measurable toxicity. More particularly the level of toxicity is o p t i m i s e d b y b a l a n c i n g i n c r e a s e d immunogenicity/adiuvanticity against the toxic effects of administration in a benefit/side effect trade-o~f.

The residual toxicity of the immunogenic composition is greater than 10 000 fold reduced relative to its natural occurring counterpart, preferably greater than 30 000 fold and most preferably greater than 50 000 fold.

The toxicity may be measured in mouse CH0 cells or preferably by the rabbit ileal loop assay or by evaluation of the morphological changes induced in Y1 cells.

Most preferably the toxicity of the immunogenic composition CA 02244800 l998-07-3l W O 97/29771 PCTnB97/00183 is reduced relative to its natural occurring counterpart by ~ about 30 000 fold as measured by the evaluation of the morphological changes induced in Y1 cells or 10 000 fold as measured by the rabbit ileal loop assay.
The term "toxoid" as used herein means a genetically detoxified toxin.

The immunogenic protein may be a CT subunit A toxoid, but is preferably an assembled toxin molecule comprising a mutated CT-A subunit and five B subunits of CT. The B subunit may be a naturally occurring subunit or may i~self be mutated.

The immunogenic protein is preferably a naturally occurring CT-A suitably modified as described above. However, conservative amino acid changes may be made which do not affect the immunogenicity or the toxicity of immunogenic protein and preferably do not affect the ability of the immunogenic protein to form complete toxin with B subunit protein. Also, the immunogenic protein may be a fragment of CT-A provided that the fragment is immunogenic and non toxic and contains at least one of the conserved regions containing one of the mutations according to the invention.

Preferably Pro-106 is replaced with Ser-106.

According to a second aspect of the invention, there is provided an immunogenic composition for use as a vaccine comprising an immunogenic detoxified protein of the first aspect of the invention and a pharmaceutically acceptable carrier.

The invention also provides a vaccine composition comprising an immunogenic detoxified protein according to the first aspect of the invention and a pharmaceutically acceptable carrier. The vaccine composition may further comprise an adjuvant. Alternatively, the vaccine composition may comprise a second antigen capable of raising an W O97/29771 PCT~B97/00183 immunological response to another disease. In such an ~ ~ alternative composition, the immunogenic detoxified protein acts as a mucosal adjuvant.

According to a third aspect of the invention, there is provided a method of vaccinating a mammal against Vi~rio cholerae comprising administering an immunologically effective amount of an immunogenic detoxified protein according to the first aspect of the invention. Optionally, the immunogenic detoxified protein of the invention may act as an adjuvant for a second immunogenic protein administered separately, sequentially or with the immunogenic detoxified protein of the invention.

The immunogenic detoxified proteins of the invention may be synthesised chemically using conventional peptide synthesis techniques, but are preferably produced by recombinant DNA
means.

According to a fourth aspect of the invention there is provided a DNA sequence encoding an immunogenic detoxified protein according to the first aspect of the invention.

Preferably the DNA se~uence contains a DNA sequence encoding a complete CT comprising DNA encoding both the detoxified subunit A and subunit B in a polycistronic unit.
Alternatively, the DNA may encode only the detoxified subunit A.

According to a fifth aspect of the invention, there is provided a vector carrying a DNA according to the fourth aspect of the invention.

According to a sixth aspect of the invention, there is provided a host cell line transformed with the vector according to the fifth aspect of the invention.

The host cell may be any host capable of producing CT or but W O 97/29771 PCT~B97/00183 is preferably a bacterium, most suitably E. coli or V. cholerae suitably engineered to produce the desired immunogenic detoxified protein.

In a further embodiment of the sixth aspect of the invention, the host cell may itself provide a protective species, for example a V.cholerae strain mutated to a phenotype lacking wild type CT and carrying and expressing an immunogenic detoxified protein of the first aspect of the invention.

In a further embodiment of the sixth aspect of the invention the host cell is capable of expre~sing a chromosomal CT-A
gene according to the first aspect of the invention.
According to a seventh aspect of the invention, there is provided a process for the production o~ an immunogenic detoxified protein according to the first aspect of the invention comprising cu-turing a host cell according to the sixth aspect of the invention.

According to a eighth aspect of the invention there is provided a process for the production of DNA according to the fourth aspect of the invention comprising the steps of subjecting a DNA encoding a CT-A or a fragment thereof to site-directed mutagenesis.

According to a ninth aspect of the invention there is provided a process for the formulation of a vaccine comprising bringing an immunogenic detoxified protein according to the first aspect of the invention into association with a pharmaceutically acceptable carrier and optionally with an adjuvant.

Industrial ApPlicabilitY

The immunogenic detoxified protein of the invention constitutes the active component of a vaccine composition WO97/29771 PCT~B97/00183 use~ul for the prevention and treatment of cholera ..
infections. The immunogenic detoxified protein may also ~e used in a vaccine composition as a mucosal adjuvant. The compositions are thus applicable for use in the pharmaceutical industry.

~e~ailed Descri~tion of Embodiments of the Invention The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook, et al., MOLECULAR CLONING; A LABORATORY MANUAL, SECOND EDITION
(19B9); DNA CLONING, VOLUMES I AND II (D.N Glover ed.
1985~; OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed, 1984);
NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. Higgins eds.
1984); TRANSCRIPTION AND TRANSLATION (B.D. Hames & S.J.
Higgins eds. 1984); ANIMAL CELL CULTURE (R.I. Freshney ed.
1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B.
Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE
TRANSFER VECTORS FOR MAMMALIAN CELLS (~.H. Miller and M.P.
Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY
(Academic Press, London), Scopes, (1987), PROTEIN
PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.), and HANDBOOK OF EXPERIMENTAL IM-MUNOLOGY, VOLUMES I-IV (D.M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification. All publications ,patents, and patent applications cited herein are incorporated by reference.

W O 97129771 PCT~B97/00183 In particular, the following amino acid abbreviations are - - used:

Alanine A Ala Arginine R Arg 5 Asparagine N Asn Aspartic Acid D Asp Cysteine C Cys Glycine G Gly Glutamic Acid E Glu Glutamine Q Gln ~istidine H His Isoleucine I Ile Leucine ~ Leu Lysine K Lys 10 Methionine M Met Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T Thr Tryptophan w Trp Tyrosine Y Tyr Valine V Val As mentioned a~ove examples of the immunogenic detoxified protein that can be used in the present invention include polypeptides with minor amino acid variations from the natural amino acid sequence of the protein other than at the sites of mutation specifically mentioned.
A significant advantage of producing the immunogenic detoxified protein by recombinant DNA techniques rather than by isolating and purifying a protein from natural sources is that equivalent quantities of the protein can be produced by using less starting material than would be required for isolating the protein from a natural source. Producing the protein by recombinant techniques also permits the protein to be isolated in the absence of some molecules normally present in cells. Indeed, protein compositions entirely free of any trace of human protein contaminants can readily be produced because the only human protein produced by the ~ recombinant non-human host is the recombinant protein at issue. Potential viral agents from natural sources and viral components pathogenic to humans are also avoided.
Also, genetically detoxified toxin are less likely to revert to a toxic from than more traditional, chemically detoxified toxins.

W O97/29771 PCT~B97tOO183 Pharmaceutically acceptable carriers include any carrier = that does not itself induce the production of antibodies harm~ul to the individual receiving the composition.
Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes) and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
1~ Additionally, these carriers may function as immunostimulating agents (adjuvants).

Preferred adjuvants to enhance e~fectiveness of the compo-sition include, but are not limited to: aluminum salts (alum) such as alumini~m hydroxide, aluminium phosphate, aluminium sul~ate etc., oil emulsion formulations, with or without other specific immunostimulating agents such as muramyl peptides or bacterial cell wall components, such as for example (1) MF59 (Published International patent application W0-A-90/14837, containing S% Squalene, 0.5%
Tween~ 80, 0.5% Span~ 85 (optionally containing various amounts of MTP-PE (see below), although not required) formulated into submicron particles using a microfluidizer such as Model llOY micro~luidizer (Microfluidics, Newton, MA
~2164), (2) SAF, containing 10~ squalene, 0.4% Tween 80, 5%
pluronic-blocked polymer L121, and thr-MDP (see ~elow) either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (3) RIBI~
adjuvant system (RAS) (Ribi Immunochem, Hamilton, MT) containing 2~ S~ualene, 0.2% Tween~ 80 and one or more bacterial cell wall components from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM~, and cell wall skeleton (cWS) pre~erably MPLtCWS (Detox~), muramyl peptides such as N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-iso-glutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(l'-2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE) etc., and -W O97/29771 PCT~B97/00183 cytokines, such as interleukins (IL-1, IL-2 etc) macrophage - - colony stimulating factor (M-CSF), tumour necrosis factor (TNF) etc. Additionally, saponin adjuvants, such as Stimulon~ (Cambridge Bioscience, Worcester, MA) may be used or particles generated therefrom such as ISCOMS
(immunostimulating complexes). Furthermore, Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA) may be used. Alum and MF59 are preferred.

The immunogenic detoxified protein of the invention may used as an adjuvant for a second antigen in an immunologically active composition for the treatment or vaccination of the human or animal body.

The immunogenic compositions (e.g. the antigen, pharmaceutically acceptable carrier and adjuvant) typically will contain diluents, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions;
solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect as discussed above under pharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologically effective amount of the antigenic polypeptides, as well as any other of the above-mentioned components, as needed. By "immunologically effective amount", it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic CA 02244800 l998-07-3l W O 97/29771 PCT~B97/00183 group of individual to be treated (e.g., nonhuman primate, primate, etc.), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other rel-evant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

The immunogenic compositions are conventionally administered parenterally, e.g. by injection either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include oral and pulmonary formulations, suppositories and transdermal applications.
lS Dosage treatment may be a single dose schedule or a multiple dose schedule. The vaccine may be administered in conjunc-tion with other immunoregulatory agents.

The term "recombinant polynucleotide" as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation~ is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or (3) does not occur in nature.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for example, labels which are known in the art, methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., CA 02244800 l998-07-3l W O97/29771 PCT~B97/00183 phosphorothioates, phosphorodithioates, etc.), those - containing pendant moieties, such as, for example proteins ~ (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modi~ied linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide.
A "replicon" is any genetic element, e.g., a plasmid, a chromosome, a virus, a cosmid, etc. that behaves as an autonomous unit of polynucleotide replication within a cell;
i.e., capable of replication under its own control. This lS may include selectable markers.

A "vector" is a replicon in which another polynucleotide segment is attached, so as to bring about the replication and/or expression of the attached segment.
"Control sequence" refers to polynucleotide sequences which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence; in eukaryotes, generally, such control sequences include promoters and transcription termination se~uence. The term "control sequences" is intended to include, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

3s "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to ~unction in their intended manner. A control sequence "operably linked" to a coding sequence is ligated W O97/29771 PCTnB97/00183 1~

in such a way that expression of the coding sequence is - - achieved under conditions compatible with the control sequences.

An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding se~uence or a total coding sequence.

A "coding sequence" is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, cDNA, and recombinant polynucleotide sequences.

"PCR" re~ers to the techni~ue of polymerase chain reaction as described in Saiki, et al., Nature 324:163 (1986); and Scharf et al., Science (1986) 233:1076-1078; and U.S.
4,683,195; and U.S. 4,683,202.

As used herein, x is ''heterologousll with respect to y if x is not naturally associated with y in the identical manner;
i.e., x is not associated with y in nature or x is not associated with y in the same manner as is found in nature.

"Homology" refers to the degree of similarity between x and y. The correspondence between the sequence from one form to another can be determined by techniques ~nown in the art.
For example, they can be determined by a direct comparison of the sequence information of the polynucleotide.
Alternatively, homology can be determined by hybridization of the polynucleotides under conditions which form stable duplexes between homologous regions (for example, those which would be used prior to S1 digestion), followed by digestion with single-stranded specific nuclease(s), fol-W O97/29771 PCT~B97/00183 lowed by size determination of the digested fragments.
.
As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like.
Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (includin~, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

A polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is im-munologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.

The protein may be used for producing antibodies, either monoclonal or polyclonal, specific to the protein. The methods for producing these antibodies are known in the art.

"Recombinant host cells", "host cells," "cells," "cell cultures," and other such terms denote, for example, microorganisms, insect cells, and mammalian cells, that can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not CA 02244800 l998-07-3l WO 97/29771 PCT~B97/00183 necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. Examples for mammalian host cells include Chinese hamster ovary (CHO~
and monkey kidney (COS) cells.

Specifically, as used herein, "cell line," refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. The term "cell lines" also includes immortalized cells. Preferably, cell lines include nonhybrid cell lines or hybridomas to only two cell types.

As used herein, the term "microorganism" includes prokaryotic and eukaryotic microbial species such as bacteria and fungi, the latter including yeast and filamentous fungi.
.

"Transformation", as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

By "genomic" is meant a collection or library of DNA
molecules which are derived from restriction fragments that have been cloned in vectors. This may include all or part of the genetic material of an organism.

By "cDNA" is meant a complementary DNA se~uence that W O 97/29771 PCT~B97/00183 hybridizes to a complementary strand of DNA.

By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term ~'purified" as used herein preferably means at least 75% by weight, more prefera~ly at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present).

once the appropriate coding sequence is isolated, it can be expressed in a variety of different expression systems; for example those used with ~mal ian cells, baculoviruses, bacteria, and yeast.

i. Mammalian Systems Mammalian expression systems are known in the art. A
mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, usually located 25-30 base pairs (bp) upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II
to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element, usually located within 100 to 200 bp upstream of the TATA
box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation [Sambrook et al. (1989) "Expression of Cloned Genes in Mammalian Cells." In Molecular Cloninq: A
LaboratorY Manual, 2nd ed.l.

W O97/29771 PCTnB97/00183 Mammalian viral genes are often highly expressed and have a ~ broad host range; therefore sequences encoding mammalian viral genes provide particularly useful promoter sequences.
Examples include the SV40 early promoter, mouse m~m~ry tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. In addition, sequences derived from non-viral genes, such as the murine metallotheionein gene, also provide useful promoter sequences. Expression may be either constitutive or regulated (induci~le), depending on the promoter can be induced with glucocorticoid in hormone-responsive cells.

The presence of an enhancer element (enhancer), combined with the promoter elements described above, will usually increase expression levels. An enhancer is a regulatory DNA
se~uence that can stimulate transcription up to 1000-fold when linked to homologous or heterologous promoters, with synthesis beginning at the normal RNA start site. Enhancers are also active when they are placed upstream or downstream from the transcription initiation site, in either normal or flipped orientation, or at a distance of more than 1000 nucleotides from the promoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989) Molecular Bioloqy of the Cell, 2nd ed.~. Enhancer elements derived from viruses may be particularly useful, because they usually have a ~roader host range. Examples include the SV40 early gene enhancer [Dijkema et al (1985) EMB0 J. 4:761] and the enhancer/promoters derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b) Proc.
Natl. Acad. Sci. 79:6777~ and from human cytomegalovirus [Boshart et al. (1985) Cell 41:521]. Additionally, some enhancers are regulatable and become active only in the presence of an inducer, such as a hormone or metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215;
Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. A promoter se~uence may be directly linked with the W O 97~9771 PCT~B97/00183 DNA molecule, in which case the first amino acid at the N-- - terminus of the recombinant protein will always be a ~ methionine, which is encoded by the ATG start codon. If desired, the N-terminus may be cleaved from the protein by ~ 5 in vitro incubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimeric DNA
molecules that encodè a fusion protein comprised of a leader se~uence fragment that provides for secretion of the foreign protein in mammalian cells. Prefera~ly, there are processing sites encoded between the leader fragment and the ~oreign gene that can be cleaved either in v1vo or ln vitro.
The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The adenovirus triparite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
Usually, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRMA is formed by site-specific post-transcriptional cleavage and polya-denylation [Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988) "Termination and 3' end processing of eukaryotic RNA. In TranscriPtion and splicinq (ed. B.D.
Hames and D.M. Glover); Proudfoot (1989) Trends Biochem.
Sci. 14:105]. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples of transcription - terminater/polyadenylation signals include those derived from SV40 [Sambrook et al (1989) "Expression of cloned genes in cultured mammalian cells." In Molecular Cloninq: A
LaboratorY Manual].

WO97/29771 PCT~B97/00183 Some genes may }~e expressed more efficiently when introns (also called intervening sequences) are present. Several cDNAs, however, have been efficiently expressed from vectors that lack splicing signals (also called splice donor and 5 acceptor sites) [see e.g., Gothing and Sambrook (1981) Nature ~93:620]. ~ntrons are intervening noncoding sequences within a coding sequence that contain splice donor and acceptor sites. They are removed by a process called "splicing," following polyadenylation of the primary 10 transcript [Nevins (1983) Annu. Rev. Biochem. 52:441; Green (1986) Annu. Rev. Genet. 20:671; Padgett et al. (1986) Annu.
Rev. Biochem. 55:1119; Krainer and Maniatis (1988) "RNA
splicing." In TranscriPtion and sPlicinq (ed. B.D. Hames and D.M. Glover)].
Usually, the above described components, comprising a promoter, polyadenylation signal, and transcription termination sequence are put together into expression constructs. Enhancers, introns with functional splice donor 20 and acceptor sites, and leader sequences may also be included in an expression construct, if desired. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as mammalian cells or bacteria.
25 Mammalian replication systems include those derived from animal viruses, which require trans-acting factors to replicate. For example, plasmids containing the replication systems of papovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] or polyomavirus, replicate to extremely high copy 30 number in the presence of the appropriate viral T antigen.
Additional examples of mammalian replicons include those derived from bovine papillomavirus and Epstein-Barr virus.
Additionally, the replicon may have two replication systems, thus allowing it to be maintained, for example, in mammalian 35 cells for expression and in a procaryotic host for cloning and amplification. Examples of such mammalian-bacteria shuttle vectors include pMT2 [Kaufman et al. (1989) Mol.
Cell. Biol. 9:946 and pHEB0 [Shimizu et al. (1986) Mol.

W O97/Z9771 PCTnB97/00183 Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to be transformed. Methods for introduction of heterologous polynucleotides into mammalian cells are known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (A~CC), including but not limited to, Chinese hamster ovary (CH0) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a number of other cell lines.

ii. Baculovirus Systems The polynucleotide encoding the protein can also be inserted into a suitable insect expression vector, and is operably linked to the control elements within that vector. Vector construction employs techniques which are known in the art.

Generally, the components of the expression system include a transfer vector, usually a bacterial plasmid, which contains both a fragment of the baculovirus genome, and a convenient restriction site for insertion of the heterologous gene or genes to be expressed; a wild type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows for the homologous recombination of the heterologous gene in to the baculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfer vector, the vector and the wild type viral - - genome are transfected into an insect host cell where the vector and viral genome are allowed to recombine. The packaged recombinant virus is expressed and recombinant pla~ues are identified and purified. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac" kit). These techniques are generally known to those skilled in the art and fully described in Summers and Smith, Texas Aqricultu~al Ex~eriment Station Bulletin No. 1555 (1987) (hereinafter "Summers and Smith").

Prior to inserting the DNA sequence encoding the protein into the baculovirus genome, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are usually assembled into an intermediate transplacement construct (transfer vector). This construct may contain a single gene and operably linked regulatory elements;
multiple genes, each with its owned set of operably linked regulatory elements; or multiple genes, regulated by the same set of regulatory elements. Intermediate transplacement constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as a bacterium. The replicon will have a replication system, thus allowing it to be maintained in a suitable host for cloning and amplification.
Currently, the most commonly used trans~er vector for introducing foreign genes into AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed. These include, for example, pVL985 (which alters the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 basepairs downstream from the ATT; see Luckow and Summers, ViroloqY (1989) 17:31.

CA 02244800 l998-07-3l WO 97/29771 PCT~B97/00183 The plasmid usually also contains the polyhedrin ~ polyadenylation signal (Miller et al. (1988) Ann. Rev.
Microbiol., 42:177) and a procaryotic ampicillin-resistance (am~) gene and origin of replication for selection and propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA seguence capable of binding a baculovirus RNA polymerase and initiating the downstream (5' to 3') transcription of a coding sequence (e~g. structural gene) into mRNA. A
promoter will have a transcription initiation region which is usually p}aced proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A baculovirus transfer vector may also have a second domain called an enhancer, which, if present, is usually distal to the structural gene. Expression may be either regulated or constitutive.
Structural genes, abundantly transcribed at late times in a viral infection cycle, provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedron protein, Friesen et al., 2S (1986) "The Regulation of Baculovirus Gene E~pression," in:
The Molecular BioloqY of Baculoviruses (ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the gene encoding the plO protein, Vlak et al., (1988), J. Gen.
Virol. 69:765.
DNA encoding suitable signal sequences can be derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively, since the signals for mammalian cell posttranslational modifications (such as signal peptide cleavage, proteolytic cleavage, and phosphorylation) appear to be recognized by insect cells, and the signals required for secretion and nuclear W O 97129771 PCT~B97/00183 accumulation also appear to be conserved between the - - invertebrate cells and vertebrate cells, leaders of non-insect origin, such as those derived from genes encoding human cY-interferon, Maeda et a~., (1985), Nature 315:592;
human gastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell. Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci. USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; and human glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also be used to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressed intracellularly or, if it is expressed with the proper regulatory sequences, it can be secreted. Good intracellular expression of nonfused foreign proteins usually requires heterologous genes that ideally have a short leader sequence containing suitable translation initiation signals preceding an ATG start signal. If desired, methionine at the N-terminus may be cleaved from the mature protein by in vitro incubation with cyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are not naturally secreted can be secreted from the insect cell by creating chimeric DNA molecules that encode a fusion protein comprised of a leader sequence fragment that provides for secretion of the foreign protein in insects.
The leader sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the translocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding the expression product precursor of the protein, an insect cell host is co-transformed with the heterologous DNA of the transfer vector and the genomic DNA of wild type baculovirus -- usually by co-transfection. The promoter and transcription termination sequence of the construct will CA 02244800 l998-07-3l w o97ng771 PCT~B97/00183 usually comprise a 2-5kb section of the baculovirus genome.
~ ~ Methods for introducing heterologous ~NA into the desired site in the baculovirus virus are known in the art. (See Summers and Smith su~ra; Ju et al. (1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow and Summers (1989)). For example, the insertion can be into a gene such as the polyhedrin gene, by homologous double crossover recombination; insertion can also be into a restriction enzyme site engineered into the desired baculovirus gene.
Miller et al., (1989), BioessaYs 4:91.The DNA sequence, when cloned in place of the polyhedrin gene in the expression vector, is flanked both 5' and 3' by polyhedrin-specific sequences and is positioned downstream of the polyhedrin promoter.
~he newly formed baculovirus expression vector is subsequently packaged into an infectious recombinant baculovirus. Homologous recombination occurs at low frequency (between about 1% and about 5%); thus, the majority of the virus produced after cotransfection is still wild-type virus. Therefore, a method is necessary to identify recombinant viruses. An advantage of the expression system is a visual screen allowing recombinant viruses to be distinguished. The polyhedrin protein, which is produced by the native virus, is produced at very high - levels in the nuclei of infected cells at late times after viral infection. Accumulated polyhedrin protein forms occlusion bodies that also contain em~edded particles.
These occlusion bodies, up to 15 ~m in size, are highly refractile, giving them a bright shiny appearance that is readily visualized under the light microscope. Cells infected with recombinant viruses lack occlusion bodies. ~o distinguish recombinant virus from wild-type virus, the transfection supernatant is plaqued onto a monolayer of insect cells by techniques known to those skilled in the art. Namely, the plaques are screened under the light ~icroscope for the presence (indicative of wild-type virus) or absence (indicative of recombinant virus) of occlusion WO 97/29771 PCT~B97/0~183 bodies. "Current Protocols in Microbiology" Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990); Summers and Smith, ~supra; Miller et al. (1989).

Recom~inant baculovirus expression vectors have been dèveloped for infection into several insect cells. ~or example, recombinant baculoviruses have been developed for, inter alia: Aedes aeqYPti , Autoqrapha californica, ~ombyx _~Ei~ Drosophila melanoqaster, Spodo~tera fruai~erda, and Tricho~lusia ni (~CT Pub. No. W0 89/046699; Carbonell et al., (1985~ J. Virol. 56:153; Wright (1986) Nature 321:718;
Smith et al., (1983) Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) In Vitro Cell. Dev. Biol.
25:22~).
Cells and cell culture media are commercially available for both direct and fusion expression of heterologous polypeptides in a baculovirus/expression system; cell culture technology is generally known to those skilled in the art. See e.q., Summers and Smith supra.

The modified insect cel}s may then be grown in an appropriate nutrient medium, which allows for stable maintenance of the plasmid(s) present in the modified insect host. Where the expression product gene is under inducible control, the host may be grown to high density, and expression induced. Alternatively, where expression is constitutive, the product will be continuously expressed into the medium and the nutrient medium must be continuously circulated, while removing the product of interest and augmenting depleted nutrients. The product may be purified by such techniques as chromatography, e.g., HPLC, affinity chromatography, ion exchange chromatography, etc.;
electrophoresis; density gradient centrifugation; solvent extraction, or the like. As appropriate, the product may ~e further purified, as required, so as to remove substantially any insect proteins which are also secreted in the medium or result from lysis of insect cells, so as to provide a product which is at least substantially free of host debris, - ~ e.g., proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derived from the transformants are incubated under conditions which allow expression of the recombinant protein encoding sequence. These conditions will vary, dependent upon the host cell selected. However, the conditions are readily ascertaina~le to those of ordinary skill in the art, based upon what is known in ~he art.

iii. Bacterial Systems Bacterial expression techniques are known in the art. A
bacterial promoter is any DNA sequence capable of binding bacterial RNA polymerase and initiating the downstream (3") transcription of a coding sequence (e.g. structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. A bacterial promoter may also have a second domain called an operator, that may overlap an ad~acent RNA polymerase binding site at which RNA
synthesis begins. The operator permits negative regulated (inducible) transcription, as a gene repressor protein may bind the operator and thereby inhibit transcription of a specific gene. Constitutive expression may occur in the absence of negative regulatory elements, such as the operator. In addition, positive regulation may be achieved by a gene activator protein binding sequence, which, if present is usually proximal (5') to the RNA polymerase binding sequence. An example of a gene activator protein is the catabolite activator protein (C~P), which helps initiate transcription of the lac operon in Escherichia col i fE.
coli~ [Raibaud et al. (1984) Annu. Rev. Genet. 18:173].
Regulated expressiOn may therefore be either positive or negative, thereby either enhancing or reducing W O97/29771 PCT~B97/OU183 transcription.

Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) [Chang et al. (1977) Nature 198:1056], and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (tr~) tGoeddel et al. (1980) Nuc. Acids Res.
8:4057; Yelverton et al . (1981) Nucl. Acids Res. 9:731; U.S.
Patent No. 4,738,921; EPO Publ. Nos. 036 776 and 121 775].
The g-laotamase (bla) promoter system [Weissmann (1981) "The cloning of interferon and other mistakes." In Interferon 3 (ed. I. Gresser)], bacteriophage lambda PL [Shimatake et al.
(1981) Nature 292:128] and T5 [U.S. Patent No. 4,689,406]
promoter systems also provide useful promoter sequences.

In addition, synthetic promoters which do not occur in nature also function as bacterial promoters. For example, transcription activation sequences of one bacterial or bacteriophage promoter may be joined with the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Patent No. 4,551,433]. For example, the tac promoter is a hybrid tr~ promoter comprised of both tr~ promoter and lac operon sequences that is regulated by the lac repressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA
polymerase and initiate transcription. A naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. The bacteriophase T7 RNA polymerase/promoter system is an example of a coupled promoter system [Studier et al. (1986) J. MQ1 . Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad.
Sci. 82:1074]. In addition, a hybrid promoter can also be W O 97/29771 PCT~B97/00183 comprised of a bacteriophage promoter and an E. coli ~ ~ operator region (EP0 Publ. No. 267 851).

In addition to a functioning promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes an initiation codon (ATG) and a sequence 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [Shine et al. (1975) Nature 254:34~.
The SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' and of E. coli 16S rRNA ~Steitz et al. (1979) "Genetic signals and nucleotide sequences in messenger RNA."
In Biological Requlation and Development: Gene Ex~ression (ed. R.F. Goldberger)]. To express eukaryotic genes and prokaryotic genes with weak ribosome-binding site [Sambrook et al. (1989) "Expression of cloned genes in Escherichia coli." In Molecular Cloninq: A ~aboratorY Manual].
A DNA molecule may be expressed intracellularly. A promoter sequence may be directly linked with the DNA molecule, in which case the first amino acid at the N terminus will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by n vitro incubation with cyanogen bromide or by either n vivo on n vitro incubation with a bacterial methionine N-terminal peptidase (EP0 Publ.
No. 219 237).
Fusion proteins provide an alternative to direct expression.
Usually, a DNA sequence encoding the N-terminal portion of an endogenous bacterial protein, or other stable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the bacteriophage lambda cell gene can be linked at the 5' terminus of a foreign gene and expressed in bacteria. The resulting fusion protein CA 02244800 l998-07-3l W O97/29771 PCT~B97/00183 preferably retains a site ~or a processing enzyme (factor ~ Xa) to cleave the bacteriophage protein from the foreign gene [Nagai et al. (1984) Nature 309:810]. Fusion proteins can also be made with sequences from the lacZ [Jia et al.
(1987) Gene 60:1973, trPE [Allen et al. (1987) J.
Biotechnol. 5:93; Makoff et al . (1989) J. Gen. Microbiol.
135:11], and Chev [EPO Publ. No. 324 647] genes. The DNA
sequence at the iunction of the two amino acid sequences may or may not encode a cleavable site. Another example is a ubiquitin fusion protein. Such a fusion protein is made with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin specific processing-protease) to cleave the ubiquitin from the foreign protein.
Through this method, native foreign protein can be isolated [Miller et al. (1989) Bio/Technoloqy 7:698].

Alternatively, foreign proteins can also be secreted from the cell by creating chimeric DNA molecules that encode a fusion protein comprised of a signal peptide sequence fragment that provides for secretion of the foreign protein in bacteria [U.S. Patent No. 4,336,336]. The signal sequence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell. The protein is either secreted into Z5 the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria). Preferably there are processing sites, which can be cleaved either in vivo or n vitro encoded between the signal peptide fragment and the foreign gene.

DNA encoding suitable signal sequences can be derived from genes for secreted bacterial proteins, such as the E. coli outer membrane protein gene (omPA) [Masui et al. (1983), in:
~xPerimental ManiPulatiOn of Gene ExPression; Ghrayeb et al.
(1984) EM~O ~ 3:24373 and the E. coli alkaline phosphatase signal sequence (PhoA) [Oka et al. (~985) Proc. Natl. Acad.
Sci. 82:7212]. As an additional example, the signal WO97/29771 PCT~B97/00183 sequence of the alpha-amylase gene from various Bacillus ~ ~ strains can ~e used to secrete heterologous proteins from B.
subtilis [Palva et al. (1982) Proc. Natl. Acad. Sci. USA
79: 5582; E~O Publ. No. 244 042].

Usually, transcription termination sequences recognized by bacteria are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Transcription termination sequences frequently include DNA sequences of about 50 nucleotides capable of forming stem loop structures that aid in terminating transcription. Examples include transcription termination sequences derived ~rom genes with strong promoters, such as the t~p gene in E. coli as well as other biosynthetic genes.

Usually, the above described components, comprising a promoter, signal sequence (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in a procaryotic host either for expression or for cloning and amplification. In addition, a repllcon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about lO to about l50. A host containing a high copy number plasmid will preferably contain at least about lO, and more preferably at least about 20 plasmids. Either a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host.

Alternatively, the expression constructs can be integrated CA 02244800 l998-07-3l W O97/29771 PCT~B97/00183 into the bacterial genome with an integrating vector.
~ Integrating vectors usually contain at least one sequence homologous to the bacterial chromosome that allows the vector to integrate. Integrations appear to result from recombinations between homologous DNA in the vector and the bacterial chromosome. For example, integrating vectors constructed with DNA from various Bacillus strains integrate into the Bacillus chromosome (EP0 Publ. No. 127 328).
Integrating vectors may also be comprised of bacteriophage or transposon sequences.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of bacterial strains that have been transformed.
Selectable markers can be expressed in the bacterial host and may include genes which render bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline ~Davies et al. (1978) Annu. Rev.Microbiol. 32:469~. Selectable markers may also include biosynthetic genes, such as those in the histidine, tryptophan, and leucine biosynthetic pathways.

Alternatively, some of the above described components can be put together in transformation vectors. Transformation vectors are usually comprised of a selectable market that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomal replicons or integrating vectors, have beendeveloped for transformation into many bacteria. For example, expression vectors have been developed for, inter alia, the following bacteria: Bacillus subtilis tPalva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EPo Publ.
Nos. 036 259 and 063 953; PCT Publ. No. W0 84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128;
Amann et al. (1985) Gene 40:183; Studier et al. (1986) J.
Mol. ~iol. 189:113; EP0 Publ. Nos. 036 776, 136 829 and 136 -W O 97/29771 PCT~B97/~0183 907], Streptococcus cremoris tPowell et al. (1988) A~pl.
~ ~ Environ. Microbiol. 54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ. Microbiol. 54:655],~
Streptomyces lividans [U.S. Patent No. 4,745,0563.
~ 5 Methods of introducing exogenous DNA into bacterial hosts are well-known in the art, and usually include either the transformation of bacteria treated with CaC12 or other agents, such as divalent cations and DMS0. DNA can also be introduced into bacterial cells ~y electroporation.
Transformation procedures usually vary with the bacterial species to be transformed. See e.g., [Masson et al. (1989) FEMS Microbiol. Lett. 60:273; Palva et al. (1982) Proc.
~atl. Acad. sci. USA 79:5582; EPO Publ. Nos. 036 259 and 063 953; PCT Publ~ No. W0 84/04541, Bacillus], tMiller et al.
(1988) Proc. Natl. Acad. Sci. 85:856; Wang et al. (1990~ J.
Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110; Dower et al. ~1988) Nucleic Acids Res. 16:6127; Kushner t1978) "An improved method for transformation of Escherichia coli with ColEl-derived plasmids. In Genetic Enqineerinq: Proceedinqs of the International Svm~osium on Genetic Enqineerinq (eds. H.W.
Boyer and S. Nicosia); Mandel et al. (1970) J. Mol. Biol.
53:159; Taketo (1988) Biochim. Bio~hvs. Acta 949:318;
Escherichia]/ ~Chassy et al. (1987) FEMS Microbiol. Lett.
44:173 ~actobacillus]; ~Fiedler et al. (1988) Anal. Biochem ~70:38, Pseudomonas]; tAugustin et al. (1990) FEMS
Microbiol. Lett. 66:203, Staphylococcus], [Barany et al (1980) J. Bacteriol. 144:698; Harlander (1987) "Transformation of Streptococcus lactis by electroporation, in: Stre~tococcal Genetics (ed. J. Ferretti and R. curtiss III); Perry et al. (1981) Infec. Immun. 32:1295; Powell et al. (1988) APP1. Environ. Microbiol. 54:655; Somkuti et al.
(1987) Proc. 4th Evr. Cona. Biotechnoloqy 1:412, Streptococcus].

iv. Yeast Expression W O97129771 PCT~B97/00183 Yeast expression systems are also known to one of ordinary ~ ~ skill in the art. A yeast promoter is any DNA sequence capable of binding yeast RNA polymerase and initiating the downstream (3') transcription of a coding sequence (e.g.
structural gene) into mRNA. A promoter will have a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region usually includes an RNA
polymerase ~inding site ~the "TATA Box") and a transcription initiation site. A yeast promoter may also have a second domain called an upstream activator sequence (UAS), which, if present, is usually distal to the structural gene. The UAS permits regulated (inducible) expression. Constitutive expression occurs in the absence of a UAS. Regulated expression may be either positive or negative, thereby either enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, therefore sequences encoding enzymes in the metabolic pathway provide particu~arly useful promoter sequences. Examples include alcohol dehydrogenase (ADH) (EPO Publ. No. 284 044), enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase (PyK) (EPO Publ. No. 329 203); The yeast PHO5 gene, encoding acid phosphatase, also provides useful promoter sequences tMyanohara et al. (1983) Proc. Natl. Acad. Sci.
USA 80:1].
In addition, synthetic promoters which do not occur in nature also function as yeast promoters. For example, UAS
sequences of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region (U.S. Patent Nos.
4,876,197 and 4,880,734). Other examples of hybrid W O97/29771 PCT~B97/00183 promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GA~10, OR PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK (EPO Publ. No. 164 556~. Furthermore, a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription. Examples of such promoters include, inter alia, [Cohen et al. (1980) Proc. Natl. Acad. Sci. USA
77:1078; Henikoff et al. (1981) Nature 283:835; Hollenberg çt al. (1981) Curr. To~ics Microbiol. Immunol. 96:119;
Hollenberg et al. (1979) "The Expression of Bacterial Antibiotic Resistance Genes i the Yeast Saccharomyces cerevisiae," in: Plasmids of Medical Environmental and Commercial Im~ortance (eds. K>N> Timmis and A. Puhler);
Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al.
(1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast.
A promoter sequence may be directly linked with the DNA
molecule, in which case the first amino acid at the N-terminus of the recombinant protein will always be a methionine, which is encoded by the ATG start codon. If desired, methionine at the N-terminus may be cleaved from the protein by n vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, as well as in mammalian, baculovirus, and bacterial expression systems. Usually, a DNA sequence encoding the N-terminal portion of an endogenous yeast protein, or otherstable protein, is fused to the 5' end of heterologous coding sequences. Upon expression, this construct will provide a fusion of the two amino acid sequences. For example, the yeast or human superoxide dismutase (SOD) gene, can be linked at the 5' terminus of a foreign gene and expressed in yeast. The DNA sequence at the junction of the two amino acid sequences may or may not encode a cleavable site. See e.g., EPO Publ. No. 196 056. Another example is W O 97/29771 PCT~B97/00183 a ubiquitin fusion protein. Such a fusion protein is made ~ ' with the ubiquitin region that preferably retains a site for a processing enzyme (e.g. ubiquitin-specific processing protease) to cleave the u~iquitin from the foreign protein.
Through this method, therefore, native foreign protein can be isolated (see, e.g., PCT Publ. No. WO 88/024066).

Alternatively, foreign proteins can also be secreted from the cell into the growth media by creating chimPric DNA
molecules that encode a fusion protein comprised of a leader sequence fragment that provide for secretion in yeast of the foreign protein. Preferably, there are processing sites encoded between the leader fragment and the foreign gene that can be cleaved either in vivo or in vitro. The leader se~uence fragment usually encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene (EPO Publ. No. 012 873; JPO Publ. No.
62,096,086~ and the A-factor gene (U.S. Patent No.
4,588,684). AlternatiVely, leaders of non-yeast origin, such as an interferon leader, exist that also provide for secretion in yeast (EPO Publ. No. 060 057).

A preferred class of secretion leaders are those that employ a fragment of the yeast alpha-factor gene, which contains both a "pre" signal sequence, and a "pro" region. The types of alpha-factor fragments that can be employed include the full-length pre-pro alpha factor leader (about 83 amino acid residues) as well as truncated alpha-factor leaders (usually about 25 to about 50 amino acid residues) (U.S. Patent Nos.
4,546,083 and 4,870,008; EPO Publ. No. 324 274). Additional leaders employing an alpha-factor leader fragment that provides for secretion include hybrid alpha-factor leaders made with a presequence of a first yeast, but a pro-region from a second yeast alphafactor. (See e.g., PCT Publ. No.

W O 97/29771 PCT~B97/00183 WO 89/02463.) Usually, transcription termination sequences recognized by yeast are regulatory regions located 3' to the translation stop codon, and thus together with the promoter flank the coding sequence. These sequences direct the transcription of an mRNA which can be translated into the polypeptide encoded by the DNA. Examples o~ transcription ~erminator sequence and other yeast-recognized termination sequences, such as those coding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader (if desired), coding sequence of interest, and transcription termination sequence, are put together into expression constructs. Expression constructs are often maintained in a replicon, such as an extrachromosomal element (e.g., plasmids) capable of stable maintenance in a host, such as yeast or bacteria. The replicon may have two replication systems, thus allowing it to be maintained, for example, in yeast for expression and in a procaryotic host for cloning and amplification. Examples of such yeast-bacteria shuttle vectors include YEp24 [Botstein et al.
(1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl.
Acad. Sci USA 81:4642-4646], and YRpl7 [Stinchcomb et al.
(1982) J. Mol. Biol. 158:157~. In addition, a replicon may be either a high or low copy number plasmid. A high copy number plasmid will generally have a copy number ranging from about 5 to about 200, and usually about 10 to about 150. A host containing a high copy number plasmid will preferably have at least about 10, and more preferably at least about 20. Enter a high or low copy number vector may be selected, depending upon the effect of the vector and the foreign protein on the host. See e.g., Brake et al., su~ra.
r Alternatively, the expression constructs can be integrated into the yeast genome with an integrating vector.
Integrating vectors usually contain at least one sequence homologous to a yeast chromosome that allows the vector to CA 02244800 l998-07-3l W O 97/29771 PCT~B97/00183 integrate, and preferably contain two homologous sequences flanking the expression construct. Integrations appear to result from recombinations between homologous DNA in the vector and the yeast chromosome [Orr-Weaver et al. (1983) 5 Methods in EnzYmol. 101:228--245]. An integrating vector may be directed to a specific locus in yeast by selecting the appropriate homologous sequence for inclusion in the vector.
See Orr-Weaver et al., supra. One or more expression construct may integrate, possibly affecting levels of recombinant protein produced [Rine et al. (1983) Proc. Natl.
Acad. Sci. USA 80:6750]. The chromosomal sequences included in the vector can occur either as a single segment in the vector, which results in the integration of the entire vector, or two segments homologous to adjacent segments in the chromosome and flanking the expression construct in the vector, which can result in the stable integration o~ only the expression construct.

Usually, extrachromosomal and integrating expression constructs may contain selectable markers to allow for the selection of yeast strains that have been transformed.
Selectable markers may include biosynthetic genes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2, TR~1, and A~G7, and the G418 resistance gene, which confer resistance in yeast cells to tunicamycin and G418, respectively. In addition, a suitable selectable marker may also provide yeast with the ability to grow in the presence of toxic compounds, such as metal. For example, the presence of CUP1 allows yeast to grow in the presence o~
copper ions [Butt et al. (1987) Microbiol, Rev. 51:351~.

Alternatively, some o~ the above described components can be put together into transformation vectors. Transformation vectors are usually comprised o~ a selectable marker that is either maintained in a replicon or developed into an integrating vector, as described above.

Expression and transformation vectors, either W O 97129771 PCT~B97/00183 extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts. For example, expression vectors have been developed for, inter alia, the following yeasts:Candida albicans ~Kurtz, et al. (1986) Mol.
Cell. Biol. 6:142], Candida maltose [Kunze, et al. (1985) J.
Basic Microbiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen- Microbiol. 132:34Sg; Roggenkamp et al.
(1986~ Mol. Gen. Genet- 202:302], Kluyveromyces fragilis CDas, et al. (1984) J. Bacteriol. 158:1165], Xluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol. 154:737;
Van den Berg et al. (1990) Bio/Technoloqv 8:135], Pichia guillerimondii ~Kunze et al. (1985) J. Basic Microbiol.
25:141], Pichia pastoris [Cregg, et al. (1985) Mol. Cell.
Biol. 5:3376; U.S. Patent Nos. 4,837,148 and 4,929,555~, Saccharomyces cerevisiae [Hinnen et al. (1978) Proc. Natl.
Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol.
153:163], Schizosaccharomyces pombe [Beach and Nurse (1981~
Nature 300:706], and Yarrowia lipolytica [Davidow, et al.
(1985) Curr. Genet. 10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known in the art, and usually include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformation procedures usually vary with the yeast species to be transformed. See e.g., [Kurtz et al. (1986) Mol. Cell~ Biol. 6:142; Kunze et al. (1985) J. Basic Microbiol. 25:141; Candida]; ~Gleeson et al. (1986) J. Gen. Microbiol. 132:3459; Roggenkamp et al.
(1986) Mol. Gen. Genet. 202:302; Hansenula]; [Das et al.
(1984) J. Bacteriol. 158:1165; De Louvencourt et al. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (19gO) Bio/Technoloqy 8:135; Kluyveromyces]; [Cregg et al. ~1985) Mol. Cell. Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Patent Nos. 4,837,148 and 4,929,555;
Pichia]; [Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA
75;1929; Ito et al. (1983) ~. Bacteriol. 153:163 Saccharomycesl; ~Beach and Nurse (19813 Nature 300:706;

W O 97/29771 rCT~B97/00183 Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet.
10:39i Gaillardin et al. (1985) Curr. Genet. 10:49;
Yarrowia].

1 Preparation of CT-S106 mutant 1.1 Source of CT DNA

A 1.1 kb XbaI-~indIII fragment of pJM17 plasmid (Pearson et lOal . (1982) PNAS USA 79: 2976--2980), containing the ctxAB
gene, was amplified by the polymerase chain reaction (PCR) using the following oligonucleotide primers:
5 GGCAGATTCTAGACCTCCTGATGAAATAAA3 (ctxA) and 155TGAAGTTTGGCGAAGCTTCTTAATTTGCCATACTAATTGCG3 (ctxB).

~The 5' XbaI site corresponds to the site in pJM17 and the 3' HindIII site was created by the PCR procedure). The amplified XbaI-HindIII fragment was subcloned in pEMBL 19 vector (Dente et al. (1983) NAR 11: 1645-1655), generating the pEMBL19-CT vector which was used for site-directed mutagenesis (Zoller et al . ( 1982) NAR 10:6487).

1.2 Methods of mutation Site-directed mutagenesis was performed according to the method of Zoller (see above) on single-stranded DNA of pEMBL19-CT plasmid. The oligonucleotide used:
5 GGCATACAGTAGCCATCCAGA3 (oligoCT-S106) m~tates the codon for ProlO6 to a Ser codon.

1.3 Expression and purification of the CT-S106 mutant.

The mutated XbaI-~indIII fragment containing the S106 mutation (ProlO6-Ser) was subcloned under the control of CT
promoter into pGEM-3 vector (Promega, Madison, USA), generating the pGEM/CT-S106 vector. V. chol erae 0395-NT
strains were transformed with pGEM/CT-S106 plasmid ~y W O 97/29771 PCT~B97/00183 electroporation (Sambrook et al. (1989~ Molecular cloning -A laboratory manual. Cold spring Harbor Laboratory Press, Cold Spring Harbor, NY). The mutant protein was purified from the culture supernatant of V. cholerae strain and S purified.

After culturing transformed V. cholerae in Syncase modified medium (Lebens et al . (1993) Biotechnology 11: 1574-1578), the culture was centrifuged and the secreted soluble CT-S106 10 protein was precipitated from the culture supernatant by adding 2.5 g/l sodium hexametaphosphate and then adjusting to pH 4.5 with concentrated HCl (Rappaport et al . (1974) Infect. Immun. 9: 294) . After centrifuging, the resulting precipitate was re-dissolved in 0.1 M sodium phosphate, pH
15 8, dialyzed against 10 mM sodium phosphate, pEI 7 (Mekalanos et al . (1988) Methods Enzymol . 165 : 169-175), and loaded on a CM-Sepharose column (Pharmacia LKB, Uppsala, Sweden). The column was eluted first with 20 mM sodium phosphate buffer, pH 7.5, and then with 40 mM sodium phosphate buffer, pH 7.5.

2 Properties of the CT-S106 mutant 2.1 Toxicity 25 Toxic activity on Yl cells.
The morphological change caused by CT on Yl adrenal cells (Donta et al. (1973) Science 183:334) was used to detect the toxic activity of the CT-Sl06 mutant. 25,u1 of medium (nutrient mixture Ham's F-lO supplemented with 2mM
30 glutamine, 50mg gentamycin, 1.596 horse serum) was added to each well of the microtitre plate. 25,u1 of protein solution, containing 80pg of wild-type CT or 18.75,ug of CT-S106, was added in the first well and then twelve serial 1:2 dilutions were made (till 1:4096). 50,000 cells were added to each 35 well (200,~1 volume) and then the plate was incubated at 37~C
in a humidified atmosphere of 95% air, 5% C02. The results were recorded after 48hr of incubation by visual inspection of the wells using an inverted microscope.

W O97/29771 PCT~B97/00183 Wild-type CT was toxic until the 1:4 dilution and was non-toxic from the 1:8 dilution; CT-S106 was toxic until 1:32 dilution, and was non-toxic from 1:64 dilution.

CT is therefore toxic at concentrations of lOpg/well and greater, and non-toxic at concentrations of 5pg/well and below; CT-S106 is toxic at concentrations of 0.6~g/well and greater, and non-toxic at 0.3~g/well and below. This represents a 30,000x reduction in toxicity.
Rabbit ileal loop assay.
New Zealand adult rabbits (ca. 2.5 kg) were used for the assay. The rabbits were starved for 24 hrs before the experiment. Before the operation, the rabbits were anaesthetised and fixed on the operation table. The abdomen of the rabbit was opened with a scalpel and the intestine was extracted. The caecum was located, and 20-30 cm away from this tract of intestine (towards the stomach) 12-14 loops were made (each 5-6 cm in length) up to the approximate end of the intestine proximal to the stomach.

Five lml samples of wild-type CT in PBS buffer were prepared with concentrations of l.00, 0.50, 0.25, 0.10, 0.05 ~g/ml.
Mutant CT-S106 was similarly diluted to give five lml samples with concentrations of 750, 100, 10, 1, 0.5 ~g/ml.
The samples was injected into separate ileal loops, with a control loop receiving lml PBS. The abdomen was then closed.

After 18-20 hrs, the volume of liquid accumulated in each loop was measured with a syringe. The length of each loop was measured again. The results, from 4 different rabbits, expressed as volume of liquid per unit length of the loop (ml/cm) are reported in the Tables 1 and 2.

These data show that as little as 50ng of wild-type CT was able to induce a fluid accumulation in the intestinal loop of rabbits, whereas lOO~g of CT-S106 did not produce CA 02244800 l998-07-3l W O 97/29771 PCT~B97/00183 significant fluid accumulation, this requiring 750 ~g of CT-S106.
I

~ABLE 1: TOXICITY CUR~E OF WILD-TYPE CT

~g of toxin 1.00 0.50 0.25 0.10 0.05 liquid in the 2.0 Z.0 2.0 1.8 1.3 loop (ml/cm) 10 TABLE 2: TOXICITY CUR~E OF MUTANT CT-S106 ~g of toxin 750 100 10 1 0.5 liquid in the 1.6 0.1 0.0 0.0 0.0 loop (ml/cm) 2.2 Immunogenicity and Adjuvanticity.

The mucosal immunogenicity of CT-S106 and its adjuvanticity was tested using the protocol of Douce et al. (PNAS 92, 1644-1648 (1995)). Five mice were i ln;sed intranasally with l~g CT or CT-S106 and 10~g Fragment C of tetanus toxin.
All the animals were immunized on day 1 and day 22.
Responses were followed by assaying sample bleeds collected on day 0 and day 21. On day 35 the mice were challenged with 100xLDs0tetanus toxin (The tetanus toxin was not completely active, so 100xLD50 was used instead of 10xLD50). The results are shown Tables 3 and 4.

Titres of anti-CT specific IgG in sera of immunised mice Mean antibody titre to cholera toxin after Immunogen 21 days 35 days Wild-type CT 1:5800 1:87000 CT-S106 1:1250 1:11000 W O 97/29771 PCT~B97/00183 Immunogen Animals with Mean Antibody Survival at measurable titre to lOOxLD50 antibody tetanus with titres to toxin tetanus (Intranasal tetanus toxin Immunisation) toxin Fragment C 5/5 1:129399 5/5 +CT
Fragment C 5/5 1:23413 5/5 ~CT-S106 Fragment C 2/5 1:393 2/5 These data show that intranasal immunisation with Fragment C of tetanus toxin alone does not afford protection against subsequent challenge. The additional presence of CT-S106, however, protects against lethal challenge with tetanus toxin, showing that CT-S106 acts as mucosal adjuvant.
CT-specific antibodies were measured using a GM1 capture ELISA (Douce et al.~. Plates were coated overnight at 4~C
with 100~1/well of 1.5~g/ml GMl ganglioside solution (Sigma Chemical Co., St. Louis, USA). Plates were washed three times with PBS/T (PBS + 0.05% Tween 20). 200~1/well of 1%
BSA were added and the plates were incubated for 1 hour at 37~C. 100~1/well of CT were added and incubated overnight at 4~C. Dilutions of serum from each mouse (1:50 dilution and eight subsequent 1:5 dilutions~ were added to the wells. The 3S plates were then incubated for 2 hours at 37~C, washed as described above, and incubated with anti-mouse immunoglobulin G conjugated to alkaline phosphatase (Sigma).
After three washes, the substrate of alkaline phosphatase (pNPP) was added and the absorbancies were read at 405nm.
ELISA titres were determined arbitrarily as the dilution corresponding to OD405= 0-3 Fragment C specific antibodies were measured using an ELISA.
Plates were coated overnight at 4~C with 50~1/well of lO~g/ml tetanus toxoid diluted in PBS. Plates were washed three times with PBS/T. 200~1/well of 1% BSA were added and the plates were incubated for 1 hour at 37~C. Dilutions of serum from each mouse (1:50 dilution and eight subsequent 1:5 dilutions) were added to the wells. The plates were incubated for 2 hours at 37~C, washed as described above, and incubated with anti-mouse immunoglobulin G conjugated to alkaline phosphatase (Sigma). After three washes, the substrate of alkaline phosphatase (pNPP) was added and the absorbancies were read at 405nm. ELISA titres were determined arbitrarily as the dilution corresponding to OD40~ 0-3-It will be understood that the invention is described aboveby way of example only and modifications may be made within the scope and spirit of the invention.

Claims (15)

1. An immunogenic detoxified protein comprising the amino acid sequence of subunit A of a cholera toxin (CT-A) or a fragment thereof, wherein at least one amino acid is substituted with another amino acid, characterised in that, in purified form, the immunogenic detoxified protein exhibits a residual toxicity greater than 10000 fold lower than its naturally occuring counterpart.

2. An immunogenic detoxified protein according to claim 1 wherein the amino acid at, or in a position corresponding to Pro-106 is replaced with another amino acid.

3. A vaccine composition comprising an immunogenic detoxified protein according to claim 1 or 2 and a pharmaceutically acceptable carrier.

4. A vaccine composition according to claim 3 further comprising an adjuvant.

5. A vaccine composition according to claim 3 further containing a second immunogenic antigen.

6. A DNA sequence encoding an immunogenic detoxified protein according to claim 1 or 2.

7. A vector carrying a DNA according to claim 6.

8. A host cell line transformed with a vector according to claim 7.

9. A process for the production of an immunogenic detoxified protein according to claim 1 or 2 comprising culturing a host cell according to claim 8.

10. A process for the production of a DNA according to claim 6 comprising the steps of subjecting a DNA encoding a CT-A or a fragment thereof to site-directed mutagenesis.

11. A method of vaccinating a mammal against Vibrio cholerae comprising administering an immunologically effective amount of an immunogenic detoxified protein according to claim 1 or 2.

12. A method for the prevention or treatment of disease in a subject comprising administering to the subject an immunologically effective dose of a composition according to any one of claims 3 to 5.

13. A process for the formulation of a vaccine composition according to claim 3 to 5 comprising bringing an immunogenic detoxified protein according to claim 1 into association with a pharmaceutically acceptable carrier.

14. A process for the formulation of a vaccine according to claim 4 comprising bringing an immunogenic detoxified protein according to claim 1 into association with an adjuvant.

15. A process for the formulation of a vaccine according to claim 5 comprising bringing an immunogenic detoxified protein according to claim 1 into association with a second antigen.