Compositions comprising the mutant ABs toxin B subunit proteins are also discussed. Further, use of these compositions as vaccines and bioactive molecule delivery agents is also discussed.

BACKGROUND OF THE INVENTION Mucosal Immunity Most licensed vaccines are delivered parenterally, a route of immunization that, in general, is extremely effective at inducing systemic protection against invasive disease. However, as many pathogens replicate or invade at mucosal surfaces, the efficacy of vaccines against these organisms may be improved if both systemic and mucosal immune responses were induced. Vaccines may have to be administered at a mucosal site, for example by oral or nasal delivery, in order to induce mucosal immune responses. Indeed, this may be a prerequisite for the production of vaccines against certain pathogens for which no vaccine is currently available (e. g., respiratory syncytial virus and even possibly HIV). An additional potential advantage of mucosal vaccines is that they would be easier to deliver, an extremely important consideration in mass immunization programs.

Unfortunately, delivery of many antigens via mucosal routes is often characterized by low immune responsiveness, due in part to inefficient uptake of antigen. As a result, various strategies for effective delivery of antigens by mucosal routes have been investigated. Antigens that are successfully delivered across the barrier of epithelial cells lining mucosal tracts stimulate underlying inductive sites of the mucosa-associated lymphoid tissue (MALT). Antigen-specific lymphocytes that are sensitized in the MALT migrate through the circulatory system to populate distant mucosal sites, thus mucosal immunization may provide both local and systemic protection.

Because many microorganisms invade their hosts via mucosal membrane surfaces, the development of a protective mucosal immune response can provide a preemptory line of defense against a variety of infectious diseases. Successful oral vaccination must induce the production of mucosal antibodies and/or cellular immunity. However, oral administration of many purified antigens fails to result in any substantial immune response.

Use of cholera toxin in mucosal immunity Cholera toxin (CT) has long been considered responsible for inducing an effective, long- lasting protective mucosal immune response to subsequent cholera infection and disease. It was soon discovered that an enhanced mucosal immune response to orally administered antigens can be

achieved when the antigens are mixed with CT or conjugated to cholera toxin B protein (CTB).

The ability of CT and CTB to act as mucosal adjuvants may stem from the adhesive and epithelial cell membrane-permeabilizing effects of these proteins; however, it appears that active toxin is more effective than the B subunit in exerting this immunostimulatory activity. It is therefore important to determine the events associated with CTB-membrane interaction and to identify mechanisms related to the potential translocational pathways of CTA 1.

Cholera Toxin The overt symptoms of cholera are well-known and the cellular events responsible for toxicity of the Vibrio cholerae enterotoxin (CT) have been thoroughly described. In contrast, molecular details of how the different subunits of the hexameric AB5 complex of CT act together to initiate cellular toxicity remain poorly understood. What is known is that CT (Mr = 85 620) binds to gangliosides on the apical surface of intestinal epithelial cells via its homopentameric B subunit (CTB). The unusually strong avidity of CTB for gangliosides, particularly for GM-1, is most likely due to the cooperative affinities of each of the monomeric subunits in their pentameric form. Upon binding, CTB forms a planar ring structure on the membrane surface which possesses a highly polar central pore that interacts intimately with the A subunit of CT. The A subunit is composed of a toxic CTA1 subunit linked to the CTA2 subunit by a disulfide bond. Liberation of CTAI by reduction of this disulfide linkage occurs during transcytosis through the cell to its basolateral membrane. This leads to permanent activation of adenylate cyclase and ribosylation of the GTP-binding regulatory protein Gga-These events result in the activation of sodium pumps via cAMP-dependent protein kinases and subsequent expulsion of sodium and water into the intestinal lumen. While CTB is clearly responsible for toxin binding to ganglioside GM-1 receptors, an essential step in cholera toxicity, the mechanism for transport of CTAI into the cytoplasmic compartment of target cells, remains unclear.

Studies of CT-membrane interactions have shown that CTB binding to GM-I incorporated into lipid bilayers induces the formation of membrane channels. These findings suggest that CTAI is translocated into the target cell cytoplasm by passing through a protein-lined pore that traverses the membrane. However, on the basis of investigations using membrane-imbedded photoactivatable probes, as well as low-resolution structural studies, it does not appear that CTB enters the membrane following its association with GM-1 receptors. These studies could indicate that CT binds with CTA1 oriented toward the cell surface, thereby forcing CTAI into and through the membrane upon CTB-GM-1 interaction; however, such a mechanism is not consistent with the crystal structures of CT, CTB, and the closely related heat-labile enterotoxin of Escherichia coli (LT). High-resolution structural analyses show that if the B pentamer of the toxin were bound with the Al polypeptide oriented toward the membrane, a significant portion of the B subunit would

also have to enter the membrane. Moreover, recent evidence has demonstrated that functional CT binds with CTA1 facing away from the cell surface. These observations and the fact that CT does not appear to undergo significant structural changes upon receptor association have prompted many questions concerning the mechanism by which CTAI moves from a site that is some distance from the cell surface to a position within the target cell cytoplasm.

Evidence is now accumulating that supports a model in which endocytosis plays a major role in mediating cellular toxicity by CT. The role of endocytosis in mediating CT intoxication is not entirely clear ; however, endosomal processing appears to involve CT migration to the Golgi and possibly other membrane-lined regions of the target cell cytoplasm. It now appears that endocytosis allows CT to encounter environmental stimuli that influence the course of cellular toxicity. The precise nature of these signals remains to be clearly established, but temperature sensitivity and pH have been suggested to be important in CT intoxication of rat hepatocytes and lung epithelial cell lines and may influence the cytoplasmic routing of CTB. Moreover, low pH has been suggested to have a role in cellular toxicity by other ABs toxins.

The mechanisms responsible for CTA1 transport are important because, in addition to its well-known toxic effect, CT possesses potent mucosal adjuvant properties that can be exploite in oral and other immunization strategies.

Production of mucosal vaccines using CT: previous strategies One strategy to develop mucosal vaccines has been to employ bacterial enterotoxins such as cholera toxin (CT) from Vibrio cholera and the related E. coli heat-labile enterotoxin (LT), which are highly immunogenic when delivered mucosally and which can act as carriers to potentiate responses to non-related antigens. Both biotoxins are composed of an ADP-ribosylating A subunit which mediates toxicity, linked to a cyclic pentamer of B subunits which interacts specifically with monosialoganglioside (GM-1) sugars expressed on eukaryotic cells. During a natural infection, the function of the B subunits is to facilitate the binding and entry of the attached A subunit into GM-1-expressing cells. Although the holotoxins are potent adjuvants for a range of antigens and can modulate different immune functions including antigen presentation, cytokine production, and B-cell switching, their practical use is limited by the toxicity of the A subunit.

Attempts to diminish toxicity through mutations to the A subunit have raised considerable debate over the direct relationship between the toxicity and adjuvanticity of this subunit. If successful, these adjuvants would be relative simple to formulate. Other attempts to solve the toxicity problem have included an attempt to develop non-toxic subunits as adjuvants. While vaccines incorporating B subunits appear to enhance the toxin's adjuvant activity by diminishing the toxin dosages required, B subunits have failed to gain popularity. Many investigations, however, only consider the B subunits as adjuvants, rather than as vehicles for antigen delivery.

Recent evidence indicates that B subunits might effectively serve to deliver antigen to the mucosal immune system when physicaily coupled to that antigen. In such case, the B subunit is known to significantly enhance the mucosal immune response to the carried antigen.

It is assumed that some of the adjuvant properties of LTB and CTB can be explained by the increased uptake of B subunit across GM-I-expressing epithelial cells lining mucosal tracts, thereby enhancing the amount of antigen passively delivered to the MALT inductive sites and the subsequent stimulation of antigen-specific B-and T-lymphocytes. LTB can also modulate immune activities such as cytokine production, lymphocyte apoptosis, and expression of B-cell activation molecules. These properties may also add to their effectiveness as adjuvants. What is clear is that the highly immunogenic nature of LTB and CTB and their ability to modulate lymphocyte activity is dependent upon their ability to bind GM-1.

Antigens can be physically attached to B5 subunits by either genetic or chemical means.

Genetic fusion of epitopes to LTB has been successful in some cases, but genetic coupling of heterologous epitopes can interfere with the structure, secretion, GM-1-binding and immunogenicity of the LTB fusion proteins. Also, there are limitations to the size and type of antigen that can be attached to LTB by genetic fusion. An alternative and more flexible approach is chemical coupling, which may be used to attach larger and a wider range of antigens, including polysaccharides. CTB has been chemically coupled to antigens such as whole Sendai virus and purified Agl/II from Streptococcus mutar7s and these conjugates have been shown to stimulate strong immune responses at mucosal sites (gut, respiratory tract, genital tract) and in the circulation.

O'Dowd et al., 1999, Vaccine 17, pp. 1442-1453 generated a panel of LTB fusion proteins in which individual amino acids within an epitope tag were replaced with cysteine. Horseradish peroxidase (HRP) was chemically coupled to these cysteine residues. Following intranasal administration, high titer mucosal and systemic antibody responses to HRP were generated when HRP was chemically coupled to LTB. There was no antibody response when HRP was co- administered with LTB.

SUMMARY OF THE INVENTION The invention provides novel sequence modifications to recombinant CTB or other AB5-type B5 subunit proteins through site directed mutations, deletions or additions. The invention also contemplates a method for producing said proteins which can be cross-linked chemically, hydrophobically, or genetically, or otherwise associated with an immunogen adjuvant, microcapsule, drug or immune response modifier in a manner in which the CTB product or other Abus-type B5 retains ganglioside specificity. In one embodiment, the recombinant B subunit proteins are specifically altered so that chemical conjugation to an immunogen, drug or immune

response modifier occurs in a residue specific manner and can be attached without eliminating the ganglioside affinity of the resultant conjugate. The recombinant B subunit proteins can be expressed and isolated in large amounts. This allows for a conjugate that is more homogeneous in composition and is known to bind to the ganglioside with an acceptable affinity. The vaccine (s) derived from this conjugate thereby target the mucosal tissues and promote both systemic and mucosal immune responses. The preferred embodiment of this invention is particularly advantageous for the recruitment of immunity to the predominant site of infection for infectious diseases, most of which typically colonize mucosal tissue surfaces.

One embodiment of the invention is a novel plasmid construct of a B5 expression system in which a B5 protein is expressed at high levels. In one embodiment, a recombinant cholera toxin B (rCTB) subunit protein including at least one mutation, addition, or deletion of residues between positions 1-103 of CTB is disclosed. Advantageously, the alteration allows chemical conjugation of said recombinant protein without interference to the ganglioside binding of CTB. In one embodiment, the mutation diminishes the ability of rCTB to combine at that site with any compound, drug, immunogen, immunomodulatory molecule, adjuvant or other bioactive molecule.

However, the compound can bind more specifically at another site and deliver these compounds to the mucosal epithelium. Preferably, the mutation, deletion or addition is to positions 1-103 of wild type CTB.

In a further embodiment, at least one mutation includes K23U, K34U, K43U, K62U, K63U, K69U, K8] U, K84U, C86U or K91U ; wherein"U"is any amino acid that fails to promote chemical modification at those sites or wherein"U"represents the deletion of the residue, and the deletion of the amino acid results in a rCTB protein with a reduced ability to be covalently modified at those sites.

In one embodiment, advantageously, the at least one mutation, deletion or addition facilitates rCTB to combine at the site of the mutation, deletion or addition with any compound, drug, immunogen, immunomodulatory molecule, adjuvant or other bioactive molecule and to deliver the complex to the mucosal epithelium. Advantageously, the mutation, deletion or addition is to positions 1-103 of wild type CTB.

A further embodiment of the invention is a recombinant cholera toxin B (rCTB) subunit protein including at least one mutation or deletion that reduces the number of potential chemical modification sites in the recombinant protein as compared to a wild type CTB protein. Preferably, the mutation or deletion is at positions 1-103 of CTB. Advantageously, at least one mutation or addition increases the number of potential chemical modification sites in the recombinant protein at the mutation sites as compared to a wild type CTB protein. In one aspect of the invention, the mutations or additions are at positions 1-103 of CTB.

A further embodiment of the invention is a method of making a recombinant cholera toxin B (rCTB) subunit gene encoding a rCTB protein with a reduced number of modification sites as compared to a wild type CTB protein. The method includes providing a cholera toxin B (CTB) subunit gene encoding a CTB protein ; selecting codons encoding amino acid residues favoring chemical modification of the CTB protein; and mutating or deleting the codons such that the resulting amino acid residues are unfavorable for chemical modification.

A further embodiment of the invention is a method of making a recombinant cholera toxin B (rCTB) subunit gene encoding a rCTB protein with an increase in chemical modification sites as compared to a wild type CTB protein, comprising providing a cholera toxin B (CTB) subunit gene encoding a CTB protein ; selecting codons encoding amino acid residues unfavorable to chemical modification of the CTB protein; and mutating or adding said codons such that the resulting rCTB protein possesses favorable chemical modification capabilities; selecting codons encoding amino acid residues favorable to chemical modification of the CTB protein; and mutating or deleting said codons such that the resulting amino acids are incapable of chemical modification.

A further embodiment of the invention is a method of producing a recombinant cholera toxin B (rCTB) subunit gene encoding a rCTB protein with decreased chemical modification sites as compared to a wild type CTB protein, comprising providing a cholera toxin B (CTB) subunit gene encoding a CTB protein ; selecting codons encoding amino acid residues unfavorable to chemical modification of the CTB protein; mutating or adding said codons such that the resulting rCTB protein possesses favorable chemical modification capabilities; selecting codons encoding amino acid residues favorable to chemical modification of the CTB protein; and mutating or deleting said codons such that the resulting amino acids are incapable of chemical modification.

A further embodiment of the invention is a method for producing a recombinant cholera toxin B (rCTB) subunit protein including the steps of obtaining a gene encoding an rCTB protein or mutated protein of the invention, adding a promoter, thereby producing an expression cassette; introducing the expression cassette into a suitable host cell, and cultivating the host cell under conditions whereby the expression cassette is translated into protein.

A further embodiment of the invention is a gene construct for producing a recombinant binding subunit protein of cholera toxin (CTB), having a promoter and a DNA sequence which encodes a rCTB protein operably linked in the proper reading frame.

A further embodiment of the invention is a method for producing a recombinant binding sub-unit protein of cholera toxin (CTB). The method includes expressing a gene construct in a suitable host cell and recovering CTB or mutations thereof.

A further embodiment of the invention is the creation of complexes of rCTB or variants thereof and compounds of whole or part, live, killed or reconstituted bacterium or virus, virus-like

particle, protein, peptide, glycoprotein, carbohydrate, polysaccharide, phospholipid, DNA, drug, biological response modifier, microparticles or adjuvant by genetic, covalent, hydrophobic or associative mechanisms.

A further embodiment of the invention is a gene fusion vector which includes a promoter, a DNA sequence encoding the cholera toxin binding subunit protein or mutant rCTB of the invention and an immunogenic peptide coding sequence, wherein the cholera toxin binding protein subunit and the immunogenic peptide coding sequences are operably linked in the proper reading frame, whereby a gene fusion protein results, and the gene fusion protein is expressed and secreted within inclusion bodies or medium.

A further embodiment of the invention is a method of generating an antibody or cellular immune response to an immunogen or immunogenic complex. Advantageously, the method includes providing the recombinant CTB protein of the invention, modifying the protein covalently with a heterobifunctional cross-linking reagent with a first and a second functional groups, wherein the first functional group is in chemical association with the recombinant CTB protein, covalently modifying the second functional group covalently with the immunogen, and administering the CTB protein modified in such a way that the animal generates an immune response.

A further embodiment of the invention is a method of generating an antibody or cellular immune response to an immunogen or immunogenic complex including providing the recombinant CTB protein of the invention, modifying the protein with a dimeric cross-linking reagent with a first and a second functional groups, wherein the first functional group is in chemical association with the recombinant CTB protein, covalently modifying the second functional group with an epitope, and administering the protein to the host until an immune response is generated.

A further embodiment of the invention is a method of generating an antibody or cellular immune response to an immunogen or immunogenic complex which includes providing the recombinant CTB protein of the invention, modifying the protein hydrophobically by prenylation or by covalent linkage in a CTB site directed manner to an amphipathic side group, mixing with an immunogenic complex containing any lipid or phospholipid micellar formulation, and administering the CTB-immunogen to the host until an immune response is generated.

A further embodiment of the invention is an rCTB expression vector including an MS-0 vector, and SEQ ID NO: 1. The vector may be pML-CTBtacl.

A further embodiment of the invention is a method for generating an immune response to an immunogen comprising providing a recombinant ABs toxin B protein, prenylating the toxin B protein, mixing the prenylated toxin B protein with an immunogen to produce a hydrophobically coupled protein, and administering the protein to an animal to generate an antibody or cellular response. In one embodiment, the ABs toxin B protein may be cholera toxin B protein (CTB), E.

coli heat labile toxin B protein (LTB), LT type IIa B protein, LT type IIb B protein, Shiga toxin B protein, Shiga-like toxin B protein, or pertussis toxin B protein. Preferably, the ABC toxin B protein is cholera toxin B protein (CTB).

A further embodiment of the invention is a method of making a recombinant ABC toxin B (rB) subunit gene encoding a rB protein with an enhanced number of modifications sites as compared to the wild type rB protein, comprising providing a B subunit gene encoding a B protein, selecting codons encoding amino acid residues not involved in covalent modification of the B protein, and mutating said codons such that the resulting rB protein possesses enhanced modification capabilities.

A further embodiment of the invention is a method of making a recombinant ABC toxin B (rB) subunit gene encoding a rB protein with altered chemical modification capabilities as compared to the wild type B protein, including providing a toxin B (B) subunit gene encoding a B protein, selecting codons encoding amino acid residues which favor covalent modification of the B protein, mutating the codons such that the resulting rB protein possesses enhanced chemical modification capabilities at that site, selecting codons encoding amino acid residues which favor chemical modification of the B protein; and mutating the codons such that the resulting amino acids do not favor chemical modification at that site. In one embodiment, the rB protein is cholera toxin B protein (CTB), E. coli heat labile toxin B protein (LTB), LT type lIa B protein, LT type IIb B protein, Shiga toxin B protein, Shiga-like toxin B protein, or pertussis toxin B protein. In a further embodiment, the rB protein is cholera toxin B protein (CTB). Advantageously, the mutations include changing lysine, histidine, arginine or cysteine residues to any other amino acid residues. In a further embodiment, the mutations include changing any amino acid to lysine, histidine, arginine or cysteine.

A further embodiment of the invention is a gene fusion vector comprising a promoter, a DNA sequence encoding an ABs toxin ganglioside binding subunit protein and an immunogenic peptide coding sequence, wherein said ABs toxin ganglioside binding protein subunit and said immunogenic peptide coding sequences are operably linked in the proper reading frame, whereby a gene fusion protein results; and said gene fusion protein is expressed and secreted within inclusion bodies or medium.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the translated coding sequence of the rCTB protein produced by the MS-0 (Maxim Secretory-0) genetic expression vector (SEQ ID NOs : I and 2). The encoded protein is identical to the wild-type sequence from V. cholera strain 569B.

Figure 2 is a plasmid map illustrating the MS-0 plasmid.

Figure 3 is the amino acid sequence of rCTB (SEQ ID NO : 2), further illustrating the predicted secondary structure, position of lysine residues, and regions involved in binding to GM-1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention contemplates introducing mutations, such as amino acid additions or deletions. throughout the nucleic acid sequence of the B subunits from the family of ABs toxins (hereinafter ABs B subunit proteins) to produce mutant proteins that possess enhanced conjugation characteristics. The term"enhanced conjugation characteristics''refers to an ABs B subunit protein that can be conjugated by chemical or other means to drugs, immunogens, cytokines or other bioactive compounds such that the conjugated compound possesses increased bioactivity.

Specifically, enhanced conjugation characteristics in an ABs B subunit protein results from the mutations taught here that are designed to decrease steric hinderance or the formation of inappropriate quaternary structures as a consequence of the process of conjugation. In this way, these mutations maximize the availability of the ligand binding sites on the mutant ABs B subunit proteins an conjugates composed thereof. One benefit of using such mutant AB5 B subunit proteins is to improve the yield of bioactive structures following conjugation.

The invention describes mutants of ABs B subunit proteins comprising single or multiple amino acid substitutions, deletions, or insertions, throughout its sequence that result in novel, predictable spatial orientations of the molecules to be conjugated relative to one another. The invention further contemplates mutant ABS B subunit proteins with enhanced or altered conjugation characteristics that enhance the bioactivity of the resulting conjugate. Such a mutant ABs B subunit protein coupled to an immunogen or other bioactive compound serves to enhance the compound's activity in a subject to whom the mutant protein conjugate is administered.

ABs toxins consist of hexameric assembles comprising a single catalytically active A- subunit and a pentamer of B-subunits. The B-pentamer of these toxins is capable of target cell recognition and binding even in the absence of the A-subunit. The toxin pentamers bind to saccharides, either to the olgosaccaride moieties of gangliosides in the cell membrane or to glycosylated proteins at the cell surface. The class of ABs toxins may be subdivided into families on the basis of sequence homology and catalytic activity. One family, the cholera toxin (CT) family includes, in addition to CT itself, the E. coll enterotoxins enterotoxins and and LT-11, and a less well-characterized toxin from Campylobacter jejuni. Another family, the shiga toxin family, comprises a number of toxins from Shigella dysenteriae and the shiga-like toxins (also known as verotoxins) from E. coli. Other ABs toxins include pertussis toxin from B. pertussis and the eukaryotic toxin ricin.

The ABs toxin LTB illustrates one relationship between this family of proteins. LTB shares extensive homology with CTB. The two toxins share 80% amino acid sequence identity, similar receptor specificity, catalytic activity, and immunological properties. Those ABC proteins with little sequence identity such as the shiga and pertussis toxins still share a high degree of structural similarity. This was determined based on the structure of the toxins in complex with the pentasaccharide from the toxin receptor and based on crystallographically determined details of toxin-receptor binding interactions (see Merritt et al. Current Opinion in Structural Biology, 1995, 5 : 165-171). Therefore, although other B5 subunit proteins can be distinct in amino acid sequence, they still possess comparable functional specificity. Some B5 holotoxins of this type are LT type lIa and lib. Additionally. Shiga toxin, shiag-like toxin and Pertussis toxin share a similar receptor specificity of B5 subunits. Further, mutations such as those set out herein for rCTB, to these subunits would create delivery molecules enhanced in their ability to bind other bioactive compounds but which still retain ganglioside specificity. The concepts taught in this application for rCTB may be applied to all examples of B5 subunits, some of which are shown below in Table 1.

TABLE 1 : ABs TOXIN SEQUENCES AB5 family Protein NCBI accession # SEQ ID NO. CTB CTB GI : 209555 32 GI: 758351 4 GI:1827850 5 GI : 808900 6 GI:229616 7 GI : 998409 8 GI : 2144685 9 GI: 1421511 10 CTB classic (596B) GI: 48890 I I CTB Ogawa 41 GI: 2781121 12 CTB Ogawa 41 R35D GI : 1421525 13 LTB Classic LTB GI : 3062900 15 GI:1169505 16 AB5 family Protein NCBI accession # SEQ ID NO. 17 GI : 1395122 G1 : 145833 18 LT87 Gl : 1648865 19 GI : 223254 20 GI : 408996 21 GI : 494265 22 GI : 69630 23 LT-IIa GI : 146671 24 LT-Ilb GI : 576584 25 Shiga toxin Shiga toxin beta subunit GI : 152784 26 Shiga-like toxin from E. Shiga-like 01 : 4877349 27 coli Pertussis Pertussis toxin chain S2 GI : 144070 28 S3 GI : 144070 29 S4 GI : 144070 30 S5 GI : 144070 31

In many cases several toxin variants that differ in amino acid sequence have been characterized. The first CTB sequence shown (SEQ ID NO: 33) is the original sequence published by Sanchez and Holmgren that is identical to the one used in this application, except for the amino terminus. (SEQ ID NO : 2). The first LTB sequence (SEQ ID NO: 15) is derived from the sequence of the cloned gene. The other sequences listed in the table are variants of either CTB or LTB found in the NCBI gene bank. The list is not intended to show all variants, but to demonstrate the natural variation among these toxins.

Polvnucleotides Encoding Mutant AB B Subunit Proteins The invention also relates to nucleic acids molecules comprising polynucleotide sequences encoding mutant ABC B subunit proteins, fragments or derivatives thereof. In one embodiment, the ABs B subunit proteins, fragments or derivatives contemplated for use with the invention possess mucosal tissue binding or association capabilities. These sequences generally contain at least one base insertion, deletion or substitution, or combinations thereof that result in single or multiple amino acid addition, deletion and substitution relative to the wild type ABs B subunit protein.

Due to the degeneracy of nucleotide coding sequences, any other DNA sequences that encode the same amino acid sequence for a mutant subunit may be used in the practice of the invention.

These include but are not limited to nucleotide sequences comprising all or portions of the coding region of a ABs B subunit which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.

AB5 B Subunit Gene Cloning Polynucleotides encoding ABs B subunits can be obtained by standard procedures from sources of cloned DNA, as would be represented by a"library"of biological clones, by chemical synthesis, by cDNA cloning. or by the cloning of genomic DNA purified from a desired cell type.

Methods useful for conducting these procedures have been detailed by Sambrook et al., in Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); by Glover, D. M. (ed.), in DNA Cloning. A Practical Approach, MRL Press, Ltd., Oxford, U. K. (1985); and by Current Protocols in Molecular Biology, Eds. Ausubel, et al., John Wiley & Sons, Inc. (1998). The polymerase chain reaction (PCR) can be used to amplify sequences encoding an ABs B subunit in a genomic or cDNA library. Synthetic oligonucleotides can be utilized as primers in a PCR protocol using RNA or DNA, or a cDNA library, as a source of target sequences.

The nucleic acid sequence being amplifie can include cDNA or genomic DNA from any ABs B toxin producing bacterium. After successful isolation or amplification of a polynucleotide encoding a segment of an ABs B subunit, that segment can be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit characterization of the nucleotide sequence of the ABs B subunit-encoding polynucleotide, and the production of the ABs B subunit protein product for functional analysis and/or therapeutic or diagnostic use.

Alternatives to isolating the coding regions for the subunits include chemically synthesizing the gene sequence itself from the published sequence. Other methods are possible and within the scope of the invention. The above-methods are not meant to limit performance of the methods by which mutants of the Bs subunits may be obtained.

The identified and isolated polynucleotide can be inserted into an appropriate cloning vector for amplification of the gene sequence. A large number of vector-host systems known in the art may be used for this purpose. Possible vectors include plasmids or modified viruses. Of course, the vector system must be compatible with the host cell used in these procedures. Such vectors include bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the pBLUESCRIPT vector (Stratagene, La Jolla, CA). The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment of interest into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically

modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and mutant subunit gene may be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells via transformation, transfection, infection or electroporation so that the copies of the vector containing the gene sequence of interest is increased.

In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a"shot gun"approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinant DNA molecules that comprise the mutant subunit gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the ABs B subunit-encoding polynucleotide may be obtained in large quantities by growing transformed host organisms, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. Copies of the gene are used in mutagenesis experiments to study the structure and function of mutant ABC B subunit proteins.

Mutagenesis The mutations present in mutant ABC B subunits, fragments and derivatives of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned coding region of the subunits can be modified by any of numerous strategies known in the art (see Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The polynucleotide sequence can be cleaved at appropriate sites using restriction endonucleases, followed by further enzymatic modification if desired, isolated, and ligated in vitro.

In the production of a mutant subunit, care should be taken to ensure that the modified gene remains within the same translational reading frame, uninterrupted by translational stop signals in the gene region where the subunit is encoded.

Additionally, the polynucleotide sequence encoding the subunits can be mutated in vitro or in vivo, to create variations in coding regions (e. g. amino acid substitutions), and/or to create and/or destroy translation, initiation, and/or termination sequences, and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C., et al., 1978, J. Biol. Chem 253: 6551), PCR-based overlap extension (Ho et al., 1989, Gene 77: 51-59), PCR-based megaprimer mutagenesis (Sarkar et al., 1990,

Biotechniques, 8: 404-407), or similar methods. The presence of mutations can be confirmed by double stranded dideoxy DNA sequencing.

Manipulations of the mutant subunit sequence may also be made at the protein level.

Included within the scope of the invention are mutant ABs B subunits that are differentially modified during or after translation, e. g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4 ; acetylation, formylation, oxidation, reduction; or metabolic synthesis in the presence of tunicamycin.

In addition. mutant ABs B peptide sequences or chemical analogs can be synthesized. For example, a peptide corresponding to a portion of a mutant subunit which comprises the desired mutated domain can be synthesized using an automate peptide synthesizer. Optionally, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the mutant subunit sequence. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, y-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, P-alanine, fluoro-amino acids, designer amino acids such as ß-methyl amino acids, Ca-nethyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Expression of Mutant ABs B Subunit-Encoding Polynucleotides The polynucleotide sequence encoding a mutant subunit of CTB or a functionally active fragment or other derivative thereof can be inserted into an appropriate expression vector. In the context of the invention, appropriate expression vectors will contain the necessary elements for the transcription and translation of the inserted protein-coding sequence. The necessary transcriptional and translational signals may also be supplied by the native ABs B subunit cDNA or gene, and/or genomic sequences flanking the subunit gene. A variety of host-vector systems may be utilized to express the protein-coding sequence. These include established or experimental bacterial expression systems; mammalian cell systems infected with a recombinant virus such as a vaccinia virus or adenovirus, insect cell systems infected with a virus such as a recombinant baculovirus, and microorganisms such as yeast containing vectors capable of replication in yeast.

The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements

may be used. In specific embodiments, a mutant subunit coding region or a sequence encoding a mutated and functionally active portion of the respective mutant subunit is expressed.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include ij7 vitro recombinant DNA synthetic techniques. Expression of polynucleotide sequences encoding mutant ABs B subunits or peptide fragments thereof may be regulated by a second polynucleotide sequence so that the mutant subunit (s) or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a mutant ABC B subunit or peptide fragments thereof may be controlled by any promoter/enhancer element known in the art. Promoters which may be used include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290: 304-310), the promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto. et al.. 1980, Cell 22 : 787-797). the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Ncrtl. Acad Sci. U. S. A. 78: 1441-1445), and the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296: 39-42). in a specific embodiment, a vector is used that comprises a promoter operably linked to the coding region of a mutant ABs B subunit, and one or more selectable markers (e. g., an antibiotic resistance gene).

A host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers. In this matter, expression of the genetically engineered mutant subunits may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e. g., glycosylation, phosphorylation of proteins). Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

Expression in mammalian cells can be used to ensure"native"glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to different extents.

Once a recombinant host cell which expresses the mutant subunit gene sequence (s) is identified, the gene product (s) can be purified and analyzed. The analysis is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay or other techniques useful for detecting the biological activity of the mutant subunit.

Production of Antibodies to Mutant Subunits and Analogs Thereof According to the invention, mutant ABC B subunits, fragments or other derivatives thereof, alone or conjugated to an immunogenic molecule or some other protein of interest may be used as an immunogen to generate antibodies which immunospecifically bind such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a specific embodiment, antibodies to a mutant ABs B subunit, such as CTB, are produced. In another embodiment antibodies to a domain of a mutant subunit are produced. In another embodiment, antibodies to the immunogen are produced.

Various procedures known in the art may be used for the production of polyclonal antibodies directed against mutant ABs B subunits, fragments. or other derivatives thereof. For the production of antibodies, various host animals can be immunized by injection with the mutant subunits produced with the methods described herein. Appropriate host animals include rabbits, mice, rats, other mammals, as well as birds such as chickens. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, caprylic acid glycerides, peptides, polyoxyethylene sorbitan monoesters, oil and water emulsions, liposomes, poly-L-lactide co- glycolide (PLG) microspheres, protein carriers such as keyhole limpet hemocyanin or diphtheria toxin mutants, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette- Guerin) and corynebacterium parvum.

For preparation of monoclonal antibodies directed against mutant ABs B subunits, its fragments, or other derivatives thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture can be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256: 495-497). as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology (PCT/US90/02545). Human antibodies can be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U. S. A. 80: 2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). In fact, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U. S. A. 81: 6851-6855 ; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454) by splicing the genes from a mouse antibody molecule specific for the epitope together with genes from a human antibody

molecule of appropriate biological activity can be used. The antibody products of these techniques fall within the scope of this invention.

According to the invention, techniques described for the production of single chain antibodies (U. S. Patent 4,946,778) can be adapted to produce specific single chain antibodies against ABs B subunits, fragments, or derivatives thereof. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246: 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F (ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule, the Fab'fragments which can be generated by reducing the disulfide bridges of the F (ab') 2 fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent, and Fv fragments.

In the production of antibodies, screening for the desired antibody can be accomplished using standard techniques known in the art. For example, the ELISA (enzyme-linked immunosorbent assay) would be an appropriate screening technique. For example, to select antibodies which recognize a specific domain of a mutant subunit, one may assay hybridomas for a product which binds to a fragment of a mutant subunit containing such domain. For selection of an antibody that specifically binds a mutant ABs B subunit, but which does not specifically bind the wild type protein, one can select on the basis of positive binding to the mutant and a lack of binding to the wild type protein. Antibodies specific for a domain of a mutant ABs B subunit, or fragment thereof are also provided by the invention.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the mutant ABs B subunits of the invention. These methods can involve labeling of the proteins, measuring levels thereof in appropriate physiological samples in diagnostic methods.

Structure and Function Analvsis of Mutant ABS B Subunits Described herein are methods for analyzing the structure and function of mutant ABs B subunits and for analyzing the in vitro activities and in vivo biological functions of the foregoing.

Once a mutant ABs B subunit is identified, it can be isolated and purified by standard methods including chromatography (e. g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique useful for purifying proteins.

Functional properties of the protein can be evaluated using any suitable assay, including immunological, immunochemcial, biochemical and biological assays.

Alternatively, once a mutant ABs B subunit produced by a recombinant host cell is identified, the amino acid sequence of the subunit (s) can be determined using standard techniques for protein sequencing, including the use of an automated amino acid sequencer.

The functional activity of a mutant ABs B subunit, derivatives and fragments thereof can be assayed by various methods known in the art. For example, where a mutant ABs B subunit or fragment thereof is assayed for its ability to bind or compete with the corresponding wild type ABs B subunit. Alternatively, when ABC B subunits are assayed for antibody binding, various immunoassays known in the art can be used. These immunoassays include competitive and non- competitive assay systems using techniques such as radio-immunoassays, ELISA,"sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, if7 situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e. g., gel agglutination assays, hemagglutination assays) complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays. Antibody binding can be detected by detecting a label on the primary antibody. Alternatively, the primary antibody can be detected by detecting binding of a secondary antibody or reagent to the primary antibody, particularly where the secondary antibody is labeled.

One of ordinary skill in the art may use the various assays discussed above to measure the binding affinity of a mutant ABC B subunit for its target ligand. Binding affinities for the mutant ABs B subunits may be reduced by one, two, three, four, or five, orders of magnitudes. A mutant AB5 B subunit protein remains acceptable so long as the binding affinity of the mutant protein does not eliminate delivery of the mutant protein complex in vivo. The binding affinity of wild type CTB for Gm-1 has been calculated at approximately 4.6 x 10-12 as determined by BIAcor analysis (Duziemdo, et al. 1996, Biochem. 35: 6375-6384). Mutants of CTB with altered modification characteristics may demonstrate reduced binding affinities for GM-I, however, wild type affinity is so strong that significant reductions of affinity following mutation may not reduce the in vivo delivery of the mutant protein complexes. Accordingly, mutant CTB proteins with binding affinities of approximately 10-12, 1 O-11, 10-10, 10~9, 1 O-8, 10~7 are considered acceptable.

Mutant ABs B subunit proteins as vaccines Mutant ABC B subunit proteins can be used as vaccines. When used in this manner, a mutant ABs B subunit may be coupled to an immunogen of choice. The mutant ABs B subunit proteins described herein are combined in a desired weight ratio so as to produce a vaccine that can elicit an optimal protective mucosal and systemic immune response from an immunized subject. In one embodiment, the mutant ABC B subunit and the immunogen component are mixed in a ratio in which the proportion of carrier component exceeds that of the immunogen, for example, at a

molecular weight ratio of about 500,000: 1. In another embodiment of the invention, the mutant ABs B subunit and the immunogenic component are mixed in a molecular weight ratio of about 100,000: 1. In still another embodiment the mutant ABs B subunit and the immunogen are mixed in a molecular weight ratio of about 1000: 1, or 100: 1, or 10: 1. And in yet another embodiment, the molecular weight ratio can be that of 1: 1. Alternatively, the components of the vaccine can also be combined in a ratio where the proportion of carrier component is less than that of the immunogen.

For example, small compounds or peptides conjugated to a mutant rCtB described herein can be conjugated at a ratio of I rCtB homopentamer to 5, 10 or more immunogenic molecules.

Cross-Hnkins reagents for use with mutant ABs B subunits There are a variety of cross-linking procedures and reagents available to conjugate bioactive compounds, such as immunogens and vaccines to mutant ABC B subunits. Cross-linking procedures generally employ bifunctional reagents that modify amino acid side-chains. Typical amino acids modified include cysteine, lysine, and histamine, although any modifiable amino acid is contemplated for use with the invention. Bifunctional reagents are often classifie on the basis of (i) chemical specificity; (ii) length of cross-bridge formed; (iii) whether the compound is a homo-bifunctional or hetero-bifunctional compound; (iv) whether the groups are chemically or photochemically reactive; and (v) whether the reagents contain a cleavable bond.

Frequently, cross-linking reagents are reactive with amino-groups or sulphydryl-containing side chains of amino acids, for example, as with lysine or cysteine side chains. The choice of a cross- linking reagent will depend on the target amino acid in both the ligand to be cross-linked and the target ABs B subunit. Cross-linking reagents for use with the invention modify target amino acid residues, produce a cross-link between the immunogen of choice and a mutant ABC B subunit that produces an immunogenic product, and does not substantially reduce the ligand binding affinity of the mutant ABs B subunit. An example of a cross-linking technique for lysine and cysteine residues is shown in Example 5.

A number of reagents are available to cross-link proteins. Examples of suitable cross- linking reagents include cleavable and non-cleavable cross-linking reagents. Some suitable cleavable cross-linking reagents that primarily react with primary amines such as lysine side chains include: dimethyl-3, 3'-dithiobispropionimidate (DTBP), 2-iminothiolane (2-IT), N-succinimidyl- (4-azidophenyl)-1, 3-dithiopropionate (SADP), ethyl-4-azidophenyl-1, 4-dithiobutyrimidiate (EADB), I- [n-2-hydroxy-5-azidobenzoyl)-2-am (N-hydroxysuccinimidyl)-succinate (HAHS), and N- [-4-p-azidophenylazo) benzoyl]-3-aminopropyl-l\-oxysulphosuccinim ide ester. A suitable non-cleavable cross-linking agent that is specific for primary amines is succinimidyl trans- 4-(maleimidylmethyl) cyclohexane-l-carbodylate (SMCC). Examples of suitable cleavable cross-

linking reagents that are specific for cysteine side chains include : N- (4-azidophenyl) phthalimide (APTP), N-[4-(p-azidosalicylamido) butyl]-3'-(2'-pyridyldithio) propionamide (APDP), N, N'-bis (4- azidobenzoyl) cystine (ABC) 2, and N- (4-azidobenzoylglycl)-S- (2-thiopyridyl) cysteine (AGTC).

Cross-linking with mutant ABs B subunit proteins can be induced with an aldehyde. For example, suitable aldehydes include glutaraldehyde, formaldehyde, glyceraldehyde, acetaldehyde, phenylaldehyde, valeraldehyde, or 3,4-dihydroxyphenylacetaldehyde. In another aspect of the invention, ketones can be used to modify and cross-link the vaccine components. Suitable ketones would include acetone, methyl ethyl ketone, 3-pentanone, or any other ketone known to one of ordinary skill in the art. Use of other cross-linking or conjugation agents is also contemplated, including N-succinimidyl 3-[2-pyridyldithio] propionate (SPDP), ultraviolet cross-linking and other protein cross-linking methods known in the art.

Mutants of ABs B subunit proteins Three-dimensional structural analysis A number of mutations analysis studies have been conducted to understand the component residues involved in either the ganglioside receptor or the toxic activity of ABs toxins. Mutations to the A subunit of LT have attempted to eliminate toxicity while maintaining adjuvant activity. The toxicity of CT and LT requires the binding of the toxin to a cell through the B subunit and activation of the toxic Al subunit by cleavage from the A2 subunit. Mutations to the A subunit either prevent cleavage of the toxic Al from the A2 subunit or alter the active residues of the Al enzymatic site.

Numerous and varied mutations of LTA at residues A7, S61, Q 112, G 118, A 146 or A 192 have been claimed to retain adjuvant activity concurrent with the loss of toxic activity (Komase et al. 1998 Vaccine 16: 248-254). The adjuvant activity of many of these mutants is enhanced however, by the addition of rLTB (Verweij et al. 1998. Vaccine 16: 2069-2076.) Mutations to the ganglioside binding sites of the B subunits of the ABs toxins might adversely affect the ganglioside affinity characteristics of the protein. In fact, several mutations to CTB subunit residues influence receptor binding. For example, the mutation G33D ablates ganglioside affinity yet appears to retain polyglycosylceramide affinity (Backstrom et al. 1997 Mol Microbiol 24 : 489-497). Surprisingly, G33R mutations retain ganglioside affinity (Merritt et al. 1995 Structure 3: 561-570).

Alternative substitutions may have surprising effects on the quaternary structure of CTB. For example, G35D substitutions diminish homopentamer formation (Merritt et al. 1995. Structure 3: 561- 570). Further, an affinity of the CTB residues between 10-14, particularly H13, for the ganglioside receptor of CTB has been suggested to lead to pentamer-pentamer formations (Merritt et al. 1995.

Structure 3: 561-570). His 13 has also been crucially implicated in the affinity of rCTB for Zn+2 (Dertzbaugh et al. 1998 Prot Eng 11 : 577-581). In contrast, substitution of several novel peptide

sequences for positions 55-64 failed to influence ganglioside affinity (Backstrom et al. 1995. Gene 165: 163-171).

In another example, site specific mutagenesis of the B subunit of LT-IIa enterotoxin at positions T13 and T14 significantly diminished GDla ganglioside affinity. Similarly, mutagenesis of the LT-IIb positions T13, T14 and additionally T34 diminished ligand affinity. Interestingly, only serine substitutions at these sites allowed the retention of ligand affinity (Connell and Holmes. 1995.

Mol Microbiol 16: 21-31). Thus, mutation of residues known to be involved with ligand affinity may still be accomplished without loss of activity. Converse) y, mutations (insertions, deletions, substitutions) of residues not known to be involved with ligand affinity may influence receptor activity. Site directed mutational analysis must therefore be conducted on a site by site basis.

A number of mutational analysis studies have been conducted to understand the component residues involved in either the ganglioside receptor or the toxic activity of AB5 toxins. Mutations to the A subunit of LT attempt to eliminate toxicity while maintaining adjuvant activity. The toxicity of CT and LT requires the binding of the toxin to a cell through the B subunit and activation of the toxic Al subunit by cleavage from the A2 subunit. Mutations to the A subunit either prevent cleavage of the toxic A I from the A2 subunit or alter the active residues of the A I enzymatic site. Numerous and varied mutations of LTA at residues A7, S61, Q112, G118. A146 or A192 have been claimed to retain adjuvant activity concurrent with the loss of toxic activity (Komase et al. 1998 Vaccine 16: 248- 254). The adjuvant activity of many of these mutants is enhanced by the addition of rLTB (Verweij et al. 1998. Vaccine 16: 2069-2076.) Mutations to the ganglioside binding sites of the B subunits of the ABs toxins can adversely affect the ganglioside affinity characteristics CTB. Several mutations to CTB subunit residues influence receptor binding. For example, the mutation G33D ablates ganglioside affinity yet appears to retain polyglycosylceramide affinity (Backstrom et al. 1997. Mol Microbiol 24 : 489-497).

Surprisingly, G33R mutations retain ganglioside affinity (Merritt et al. 1995. Structure 3: 561-570).

Alternative substitutions may have surprising effects on the quaternary structure of CTB. For example, G35D substitutions diminish homopentamer formation (Merritt et al. 1995. Structure 3: 561-570). Further, an affinity of the CTB residues between 10-14, particularly H13, for the ganglioside binding site of CTB has been suggested to lead to pentamer-pentamer formations (Merritt et al. 1995, Structure 3 : 561-570). His 13 has also been crucially implicated in the affinity of rCTB for Zn+2 (Dertzbaugh et al. 1998, Prot Eng 11 : 577-581). In contrast, substitution of several novel peptide sequences for positions 55-64 failed to influence ganglioside affinity (Backstrom et al. 1995, Gene 165: 163-171).

Structural Features of CTB Amino acids 2-7 form a loop on the outer surface of rCTB. Residues Gln 3, Asn 4, Thr 6, and Asn7, all possess polar side chains which face out from the molecule. These side chains can be replace with another amino acid to introduce a chemically modifiable amino acid side chains into this region. Residues 10-14 are reported to form an amino acid sequence with ganglioside receptor affinity. Mutations to this site, particularly H13, might create a more hydrophobic rCTB (Merritt et al. 1995. Structure 3: 561-570). Amino acids 14-20 are thought to form a ?-sheet on the outer surface of rCTB with many of the side chains facing outward. Among these amino acids are many polar side chain groups, particularly His 18, which may be amenable to lysine additions.

Residues 21-25 of CTB form a surface loop on the surface of rCTB. The loop is comprised of Asn 21, an uncharged polar amine, Asp 22, an acidic side chain, and Lys 23, a basic amine. Each side group is thought to face outward from the surface of the protein. The orientation of this residue suggests that it may play some role in structural stability of the monomer. Amino acid residue Phe 25 also faces outward from the molecular surface with its uncharged, nonpolar benzyl ring and might be mutated to introduce a modifiable amino acid into the sequence of the protein. Residues 25-30 form a ßpleated sheet within the CTB monomer that is not accessible to the molecular surface. Mutations to this site might be expected to disrupt the quaternary structure of rCTB. Residues 31-35 are referenced within the specification with regard to the Gm-1 receptor.

Residues 36-40 of CTB are thought to form an internal (3-pleated sheet as in 25-30.

Residues 41-44 form a surface exposed loop containing Lys 43 next to Asn 44. Asn 44 may be a good candidate for lysine due to its location, charge, and side chain similarity to Lys 43.

Residues 45-55 are thought to form a (3-pleated sheet within the monomer. Residues 55-64 are thought to form an insert permissive site. Lysines at positions 62 and 63 within this region may be deleted or mutated without apparent adverse effect on the ganglioside binding affinity of rCTB mutants (Backstrom et al. 1995, Gene 165 : 163-171). Residues 60-78 form an internal hydrophobic a-helix in the homopentamer. Significant changes to this sequence, particularly to residues 65-78, might be expected to interfere with homopentamer formation.

Residues 79-85 of CTB form a loop on the surface of the CTB monomer. Within the loop, Lys 81 and Lys 84 face outward from the monomeric structure. Lys 81 and 84 would appear to be an optimal target residues for conjugation due to their position contralateral to the receptor and surface location within the monomer.

CTB residues 86-95 form internalized side chains and inferior loop contact points with Gm-1, encompass the Lys 91 successfully produced and claimed herein. CTB residues 96-103 form an internal sheet which ends with an optimally positioned Asn 103. This Asn 103 is a key target for Lys insertion due to its similar charge, structure and on the molecule.

In view of the structural analysis discussed above, it is clear that a large number of mutations can result in a mutant CTB protein with altered conjugation characteristics. Some mutants will preserve, enhance, or at least not detrimentally reduce the Gm-1 binding affinity of the mutant CTB proteins described herein. On the other hand, some loss of Gm-1 affinity is tolerable given the extraordinarily high initial affinity (4.6 x 10-12) of the cholera toxin B for the Gm-1 receptor. Binding affinity of the mutant proteins can be determined using a number of assays well known in the art.

Mutants of Recombinant cholera Toxin Subunit B (rCTB) The invention contemplates mutations, substitutions, deletions, or insertions of one, two, three, four or more amino acid residues in the amino acid sequence of cholera toxin subunit B. The full length amino acid sequence of wild type rCTB is shown in FIGURE 1 (SEQ ID NO : 2).

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule.

Such mutations include: TIX, P2X, Q3X, N4X, ISX, T6X, D7X, L8X, C9X, AIOX, El IX, Y12X, H13X, N14X, TI5X, Q16X, I17X, H18X, T19X, L20X, N21X, D22X, K23X, 124X, F25X, S26X, Y27X, T28X, E29X, S30X, L31X, A32X, G33X, K34X, R35X, E36X, M37X, A38X, 139X, 140X, T41X, F42X, K43X, N44X, G45X, A46X, T47X, F48X, Q49X, V50X, E51X, V52X, P53X, G54X, S55X, Q56X, H57X, I58X, D59X, S60X, Q61X, K62X, K63X, A64X, 165X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, 174X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, 196X, A97X, A98X, ! 99X, SIOOX, MIOIX. A102X. N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

Lvsine Substitutions in rCTB Conjugation of rCTB to immunogens derived from infectious diseases can create vaccines which induce protection following mucosal delivery. Classic conjugation chemistry targets are reactive amino acid side chain groups such as primary amines, carboxylic acids, aldehydes, or sulfhydryls. The primary reactive amino acids in rCTB are lysines (see Figure 3). The B subunit of cholera toxin contains nine (9) lysine amino acid residues. A number of these residues are contemplated to play a role in facilitating conjugation of bioactive compounds to the rCTB protein.

These lysine residues are distributed at various places throughout the CTB sequence. Those lysine

residues that are near the Gm-1 binding site (e. g. K91) can couple to a bioactive compound of interest in a conformation which interferes with ganglioside binding. Further, any lysine residues to which the bioactive compound is coupled may induce a conformational change upon conjugation, which prevents or disrupts the formation of the quaternary structure required for ganglioside binding.

Accordingly, a number of mutant rCTB proteins with increased or decreased numbers of conjugation sites as compared to the wild type form of the protein are disclosed herein.

The present approach involves directing the covalent linkage of an ABs B subunit carrier and a bioactive molecule such that the bioactive molecule does not interfere with the ganglioside site of CTB. Site directed mutagenesis was employed to create a panel of mutations at Lys91 of the rCTB coding sequence. These mutations resulted in several mutants which produce levels of protein, in vitro, comparable to those of the wild type and which can effectively deliver bioactive CTB-immunogen complexes to the mucosal immune system.

Accordingly, one embodiment discussed herein articulates a number of mutant rCTB proteins with amino acid substitutions that replace the lysine residues found in the wild type rCtB protein. Of the 9 lysine residues present in wild type CTB, the invention contemplates recombinant CTB proteins with one or more of the following amino acid substitutions: K23X, K34X, K43X, K62X, K63X, K69X, K81X, K84X, and K91X ; wherein"X"is any amino acid that results in a rCTB protein with a reduced ability to conjugate at the mutant sites. These mutants are also contemplated to maintain a substantial degree of Gm-1 binding affinity as compared to the wild type or unmutated form of the protein. Moreover, as mentioned before, some loss of affinity is allowable due to the extraordinarily high initial affinity (4.6 x 10-12) of the wild type CTB for the Gm-1 receptor.

There are a number of lysine residues that are thought to result in particularly deleterious conjugation events given the proximity of these residues to the Gm-1 binding site. These residues include K34, K62, K63, and K91. Mutation of these residues favors conjugation events that will not interfere with Gm-I binding affinity.

The invention further contemplates mutating the lysine residue at K69 to eliminate conjugation at this position. Lysine 69 is thought to be distal from the Gm-1 binding site; however, conjugation to this residue might reduce rCtB homopentamer self-assembly. Incomplete quaternary structure formation is known to diminish Gm-1 affinity and likely also diminishes the bioactivity of a rCTB conjugated molecule.

The remaining lysine residues in CTB are K23, K43, K81, and K84. Each of these residues are contemplated as acceptable sites for conjugation. One or more of these residues can be mutated to reduce the total number of sites for conjugation.

McCann et al. (Biochem. 1997,36: 9169) demonstrated that Lys91 was the most reactive of all lysine residues in CTB when reacted with"small" (fluorescent) molecular probes. When CTB

is bound to Gm-l, Lys91 is positioned proximal to the terminal galactose residue of Gm-1.

Therefore, conjugation of large molecular weight bioactive molecules is likely to result in steric hinderance of the Gm-1 binding site of rCTB. Similarly, reaction of larger molecular weight compounds with Lys69, which is located within the hydrophobic core of the pentamer, would disfavor reformation of bioactive CTB homopentamers.

Lys91 possesses a basic amino side chain with which the hydroxylated galactose of Gm-1 may interact and facilitate the affinity of rCTB for Gm-1. A knockout of Lys91 might therefore be expected to significantly decrease the affinity of the mutant rCTB for Gm-l and effect a decrease in recombinant production levels.

Lvsine additions in rCTB The invention further contemplates the addition of amino acid residues throughout the amino acid sequence of rCTB to direct conjugation of immunogens, immunomodulators, drugs, or other bioactive molecules, to specified sites within rCTB. Any residue of rCTB can be mutated to introduce an amino acid residue that promotes acceptable conjugation events. Examples of these mutations include Q3X, N4X, T6X, N7X, H I SX, N21 X, F25X, N44X, M101X, and N103X wherein "X"is any amino acid that results in the addition of an acceptable conjugation site. Specific examples of suitable amino acids for use at these sites include lysine and cysteine. Accordingly, one or more of the following mutations: Q3K, N4K, T6K, N7K, H18K, N21K, F25K, N44K, MIOIK, N103K, Q3C, N4C, T6C, N7C, Hl 8C, N2 I C, F25C, N44C, MIOIC, and N 103C are contemplated.

Alternative Chemical Cross-linking Moieties A further approach to specific chemical coupling or site-directed chemical coupling is to create a site for chemical coupling using a cysteine residue rather than a lysine residue. Accordingly, the following cysteine insertions are created: H18C and MIOIC. The site-directed mutagenesis is performed as in Examples 1-2. Example 5 provides techniques for chemically coupling these residues to an immunogen of interest.

Mutants in the N-terminus of rCTB The wild type amino acid sequence of rCTB is shown in Figure 1. The invention contemplates various mutations throughout the rCTB amino acid sequence. In one embodiment, one or more mutations are introduced into the N-terminus of the protein sequence. For example, in one embodiment, an alanine residue is introduced at the first position of the rCTB amino acid sequence.

Introduction of this particular amino acid creates a defined signal sequence cleavage site, as opposed to the threonine residue at the amino terminus of the wild type form of rCTB. This cleavage site can be important in post-translational modifications.

Mutants of other CTB family members identified by NCBI accession number The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI:758351 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutants include : MIX, I2X, K3X, L4X, KSX, F6X, G7X, V8X, F9X, F10X, T11X, V1WX, L13X, L14X, S15X, S16X, A17X, Y18X, A19X, H20X, G21X, T22X, P23X, Q24X, N25X, I26X, T27X, D28X, L29X, C30X, A31X, E32X, S33X, H34X, N35X, T36X, Q37X, I38X, Y39X, T40X, L41X, N42X, D43X, K44X, 145X, F46X, S47X, Y48X, T49X, E50X, S51X, L52X, A53X, G54X, K55X. R56X, E57X, M58X, A59X, 160X, I61X, T62X, F63X, K64X, N65X, G66X, A67X, I68X. F69X, Q70X, V71X, E72X, V73X. P74X. S75X, S76X, Q77X, H78X, 179X, D80X, S81X, Q82X, K83X, K84X, A85X, 186X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, 195X, A96X, Y97X, L98X, T99X, E100X, AIOIX, K102X, V103X, E104X, K105X, L106X, C107X, V108X, W109X, N110X, N111X, K112XS, T113X, P114X, H115X, A166X, I117X, A118X, A119X, I 120X, S 121 X, M122X, A123X, N I 24X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 758351 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI: 1827850 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutants include : TIX, P2X, Q3X, N4X, ISX, T6X, D7X, L8X, C9X, AIOX, EI IX, Y12X, H13X, N14X, TISX, Q16X, 117X Y18X, T19Xb L20X N21Xt D22X, K23X, I24X, F25X, S26X, Y27X, T28X, E29X, S30X, L3 ! X, A32X, G33X, K34X, R35X, E36X, M37X. A38X, I39X, 140X, T41X, F42X, K43X, N44X, G45X, A46X, 147X, F48X, Q49X, V50X, E51X, V52X, P53X, S54X, S55X, Q56X, H57X, 158X, D59X, S60X, Q61X, K62X, K63X, A64X, I65X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, I74X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, T87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, I96X, A97X, A98X, I99X, S100X, M101X, A102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI: 1827850 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-I binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB G1 : 808900 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include: MIX, I2X, K3X, L4X, K5X, F6X, G7X, V8X, F9X, F10X, TI IX, V12X, L13X, L14X, S15X, S16X, A17X, Y18X, A19X, H20X, G21X, T22X, P23X, Q24X, N25X, I26X, T27X, D28X, L29X, C30X, A31X, E32X, Y33X, H34X, N35X, T36X, Q37X, 138X, H39X, T40X, L41X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, ESOX, S51X, L52X, A53X, G54X, N55X, R56X, E57X, M58X, A59X, I60X, I61X, T62X, F63X, K64X, N65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, 186X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, A96X, Y97X, L98X, T99X, EIOOX, A101X, K102X, V103X, E104X, K105X, <BR> <BR> <BR> <BR> L106X, C107X, V108X, W109X, NI IOX, Nl l IX, K112X T113X, P114X, H115X, A116X, 1117X, A118X, A119X, I120X, S121X, M122X, A123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI:808900 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-I binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 229616 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include: TIX, P2X, E3X, N4X, 15X, T6X, D7X, L8X, C9X, AIOX, E11 X, Y12X, H13X, N14X, T15X, Q16X, 117X, H18X, T19X, L20X, N21X, N22X, K23X, 124X, F25X, S26X, Y27X, T28X, E29X, S30X, L31X, A32X, G33X, K34X, R35X, E36X, M37X, A38X, 139X, I40X, T41X, F42X, K43X, D44X, G45X, A46X, T47X, F48X, E49X, V50X, E51X, V52X, P53X, G54X, S55X, E56X, H57X, 158X, D59X, S60X, E61X, K62X, K63X, A64X, 165X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, I74X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, I96X, A97X, A98X, I99X, SIOOX, MIOIX, A102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 229616 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 998409 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include : TIX, P2X, Q3X, N4X, I5X, T6X, D7X, L8X, C9X, AIOX, E11X, Y12X, H13X, N14X, T15X, Q16X, I17X, Y18X, T19X, L20X, N21X, D22X, K23X, 124X, F25X, S26X, Y27X, T28X, E29X, S30X, L31X, A32X, G33X, K34X, R35X, E36X, M37X, A38X, 139X, I40X, T41X, F42X, K43X, N44X, G45X, A46X, I47X, F48X, Q49X, VSOX, E51X, V52X. P53X, G54X, SSSX, Q56X, H57X, 158X. D59X S60X, Q61X, K62X, K63X, A64X, 165X, E66X, R67X. M68X, K69X, D70X, T71X, L72X, R73X, I74X, A75X, Y76X, L77X, T78X, E79X, A80X, K8] X. V82X, E83X. K84X, L85X, C86X, V87X, W88X. N89X. N90X, K91X, T92X, P93X, H94X, A95X, I96X, A97X, A98X, I99X, SIOOX MIOIX, A102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 998409 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-I binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 2144685 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include: MIX, I2X, K3X, L4X, KSX, F6X, G7X, V8X, F9X, F10X, T1 IX, V12X, L13X, L14X, SISX, S16X, A17X, Y18X, A19X, H20X, G21X, T22X, P23X, Q24X, N25X, I26X, T27X, D28X. L29X, C30X, A3 IX, E32X. S33X, H34X, N35X, T36X, Q37X, I38X, Y39X, T40X, L41X, N42X, D43X, K44X, 145X, F46X, S47X. Y48X, T49X, ESOX, S5 ! X, L52X, A53X, G54X, KSSX, R56X, E57X, M58X, A59X, 160X, 161X, T62X F63X K64X, N65X G66X, A67X, I68X, F69X, Q70X, V71X, E72X, V73X, P74X, S75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, A96X, Y97X, L98X, T99X, EIOOX, AIOIX, K102X, V103X, E104X, KIOSX, L106X, C107X, V108X, W109X, N110X, N11X, K112X, T113X, P114X, H115X, A116X, I117X, A18X, A119X, I120X, S121X, M122X, A123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 2144685 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 1421511 without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include: TIX, P2X, Q3X, N4X, I5X, T6X, D7X, L8X, C9X, AIOX, El IX, Y12X, H13X, N14X, T15X, Q16X, I17X, H18X, T19X, L20X, N21X, D22X, K23X, I24X, F25X, S26X, Y27X, T28X, E29X, S30X, L3 IX, A32X, D33X, K34X, R35X, E36X, M37X, A38X, I39X, 140X, T41X, F42X, K43X, N44X, G45X, A46X, T47X, F48X, Q49X, VSOX, E51X, V52X, P53X, G54X, S55X, Q56X, H57X, I58X, D59X, S60X, Q61X, K62X, K63X, A64X, I65X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, I74X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, 196X, A97X, A98X, I99X, S100X, MIOIX, A102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 1421511 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 48890 (classic 569B) without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include : MIX, I2X, K3X, L4X, K5X, F6X, G7X. V8X, F9X, FIOX, Tl IX, V12X, L13X, L14X, S15X, S16X, A17X, Y18X, A19X, H20Xb G21X, T22X, P23X, Q24X, N25X, I26X, T27X, D28X, L29X, C30X, A3 I X, E32X, Y33X, H34X, N35X, T36X, Q37X, I38X, H39X, T40X, L41X, N42X, D43X, K44X, I45X, F46X, S47X, Y48X, T49X, E50X, S51X, L52X, A53X, G54X, K55X, R56X, E57X, M58X, A59X, I60X, I61X, T62X, F63X, K64X, N65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, A96X, Y97X, L98X, T99X, E100X, AIOIX, K102X, V103X, E104X, K105X, <BR> <BR> <BR> <BR> LI06X, CI07X, V108X, W109X, N110X, N11 IX, Kl I2X, Tl 13X, PI 14X, HI I5X, AI I6X, 1117X, A118X, A119X, I120X, S121X, M122X, A123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI: 48890 (classic 569B) with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 2781121 (Ogawa 41) without deleterious effect to the ganglioside (Gm-1) binding affinity of the recombinantly produced molecule. Such mutations include : MIX, T2X, P3X, Q4X, N5X, I6X, T7X, D8X, L9X, CIOX, AIIX, E12X, Y13X, H14X, N15X, T16X, Q17X, 118X, H19X, T20X, L21X, N22X, D23X, K24X, 125X, F26X, S27X, Y28X, T29X, E30X, S3] X, L32X, A33X, G34X, K35X, R36X, E37X, M38X, A39X, 140X, I4 ! X, T42X, F43X, K44X, N45X, G46X, A47X, T48X, F49X, Q50X, V51X, E52X, V53X, P54X, G55X, S55X, Q56X, H57X, I58X, D59X, S60X, Q61X, K62X, K63X, A64X, 165X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, 174X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, I96X, A97X, A98X, 199X, SIOOX, MIOIX, A102X, N103X, wllerein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid,. The"X" represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI: 2781121 (Ogawa 41) with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rCTB GI : 1421525 (Ogawa 41 R35D) without deleterious effect to the ganglioside (Gm-l) binding affinity of the recombinantly produced molecule. Such mutations include : TI X, P2X, Q3X, N4X, 15X, T6X, D7X, L8X, C9X, AIOX, EIIX, Y12X, H13X, N14X, T15X, Q16X, 117X, H18X, T19X, L20X, N21X, D22X, K23X, I24X, F25X, S26X, Y27X, T28X, E29X, S30X, L31X, A32X, G33X, K34X, D35X, E36X, M37X, A38X, 139X, 140X, T41X, F42X, K43X, N44X, G45X, A46X, T47X, F48X, Q49X, V50X, E51X, V52X, P53X, G54X, S55X, Q56X, H57X, 158X, D59X, S60X, Q61X, K62X, K63X, A64X, 165X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, 174X, A75X, Y76X, L77X, T78X, E79X, A80X, K81X, V82X, E83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, H94X, A95X, 196X, A97X, A98X, 199X, SIOOX, MIOIX, A102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rCTB GI : 1421525 (Ogawa 41 R35D) with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its Gm-1 binding activity.

Mutants of LTB and LTB family members identified by NCBI accession number The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI : 3062900 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include : MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, FIOX, TIIX. A12X, L13X, L14X, S15X, S16X, L17X, C18X, A19X, Y20X, G21X, A22X, P23X, Q24X, S25X, 126X, T27X, E28X. L29X, C30X, S31X, E32X, Y33X, R34X, N35X, T36X, Q37X, 138X, Y39X, T40X, 141X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, E50X, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, I60X, I61X, T62X, F63X, K64X, S65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S8 ! X, Q82X, K83X, K84X, A85X, I6X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, 195X, T96X, Y97X, L98X, T99X, EIOOX, TlOlX, K102X, 1103X, D104X, K105X, L106X, C107X, V108X, W109X, N110X, N111X, K112X, T113X, P114X, N115X, SII6X, II17X, AI18X, A1I9X, II20X, S121X. MI22X, EI23X. N124X. wherein the first letter is the native amino acid appearing at the position of the indicated number. and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI : 3062900 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion. or insertion that selectively alters the conjugation characteristics of rLTB G1 : 1169505 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include : MIX, N2X, K3X, V4X, Ksi, C6X, Y7X, V8X, L9X, F10X, T11X, A12X, L13X, L14X, S15X, S16X, L17X, Y18X, A19X, H20X, G21X, A22X, P23X, Q24X, T25X, I26X, T27X, E28X, L29X, C30X, S31X, E32X, Y33X, R34X, N35X, T36X, Q37X, I38X, Y39X, T40X, I41X, N42X, D43X, K44X, I45X, L46X, S47X, Y48X, T49X, E50X, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, I60X, I61X, T62X, F63X. K64X, S65X, G66X, E67X, T68X, F69X, Q70X. V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, T96X, Y97X, L98X, T99X, E100X, TlOlX, K102X, I103X, D104X, K105X, L106X, C107X, V108X, W109X, N110X, N111X, K112X, T113X, P114X, N115X, S116X, I117X, A118X, A119X, I120X, S121X, M122X, K123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of

any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI : 1169505 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI : 1395122 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include: MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, FIOX, TllX, A12X, 1. 13X, L14X, S15X, S16X, L17X, C18X, A19X Y20X, G21X, A22X, P23X, Q24X, S25X, 126X, T27X, E28X, L29X, C30X, S31X, E32X, Y33X, R34X, N35X, T36X, Q37X, I38X. Y39X, T40X, 141X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, E50X, SS 1 X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, 160X, I61X, T62X, F63X, K64X, S65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, 179X, D80X, S81X, Q82X, K83X, K84X, A85X, 186X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, 195X, T96X, Y97X, L98X, T99X, E100X, T101X, K102X, I103X, D104X, K105X, L106X, C107X, V108X, W109X, N110X, N111X, K112X, T113X, P114X, N115X, S116X, I117X, A118X, A119X, I120X, M121X, E122X, N123X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The "X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI : 1395122 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB G1 : 145833 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include: MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, FIOX, TIIX, A12X, L13X, L14X, S15X, S16X, L17X, Y18X, A19X, H20X, G21X, A22X, P23X, Q24X, T25X, I26X, T27X, E28X, L29X, C30X, S31X, E32X, Y33X, R34X, N35X, T36X, Q37X, I38X, Y39X, T40X, 141X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, E50X, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X. V59X, I60X, 161X, T62X, F63X, K64X, S65X, G66X, E67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, 195X, T96X, Y97X, L98X, T99X, EIOOX, TIOIX, K102X, 1103X, D104X, K105X, L106X, C107X, V108X, <BR> <BR> <BR> <BR> W109X, N1IOX, N111X, K1I2X, T113X, P114X, NI15X, SI16X, I1I7X, A118X, AI19X, I120X, S121X, M122X, K123X, N124X, wherein the first letter is the native amino acid appearing at the

position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI: 145833 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI: 1648865 (LT87) without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include : MIX, N2X, K3X, V4X, K5X, F6X, Y7X, V8X, L9X, F10X, TI IX, A12X, L13X, L14X, SI SX, S 16X, L17X, C18X, A19X, H20X, G21X, A22X, P23X, Q24X, S25X, 126X, T27X, E28X, L29X, C30X. S31X, E32X, Y33X, H34X, N35X, T36X, Q37X, 138X, Y39X, T40X, I14X, N42X, D43X, K44X, I45X, L46X, S47X, Y48X, T49X, ESOX, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, I60X, 161X, T62X, F63X, K64X, S65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, 179X, D80X, S81X, Q82X, K83X, K84X, A85X, 186X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, T96X, Y97X, L98X, T99X, E100X, T101X, K102X, I110X, D104X, K105X, L106X, C107X, V108X, W109X, NI IOX, Nl llX, K112X, T113X, P114X, N115X, S116X, 1117X, A118X, A119X, 1120X, S121X, M122X, E123X, N] 24X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI : 1648865 (LT87) with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI:223254 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include: MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, F10X, T11X, A12X, L13X, L14X, S15X, S16X, L17X, Y18X, A19X, H20X, G21X, A22X, P23X, Q24X, T25X, I26X, T27X, E28X, L29X, C30X, S31X, E32X, Y33X, R34X, N35X. T36X, Q37X. I38X. Y39X, T40X, 141X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, E50X, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, 160X, 161X, T62X, F63X, M64X, S65X, G66X, E67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, T96X, Y97X, L98X, T99X, E100X, T101X, K102X, I103X,

D104X, K105X, L106X, C107X, V108X, W109X, NI IOX, Nl l IX, K112X, T113X, P114X, NI ISX, S116X, 1117X, A118X, A119X, 1120X, S121X, M122X, K123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI:223254 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI:408996 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include: MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, F10X, T11X, A12X, L13X, L14X, S15X, S16X, L17Xt C18X, A19X, Y20X, G21X, A22X, P23X, Q24X, S25X, 126X, T27X, E28X, L29X, C30X, S31X, E32X, Y33X R34X, N35X, T36X, Q37X, 138X, Y39X T40X 141X, N42X, D43X, K44X, 145X, L46X, S47X. Y48X, T49X, E50X, SS 1 X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, 160X, I61X, T62X, F63X, K64X, S65X, G66X, A67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, I86X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, I95X, T96X, Y97X, L98X, T99X, E100X, T101X, K102X, I103X, D104X, K105X, L106X, C107X, V108X, W109X, N110X, N111X, K112X, T113X, P114X, N115X, S116X, I117X, A118X, A119X, I120X, S121X, M122X, E123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI:408996 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI : 494265 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include : AIX, P2X, Q3X, T4X, I5X, T6X, E7X, L8X, C9X, S1OX, E11X, Y12X, R13X, N14X, T15X, Q16X, 117X, Y18X, T19X, I20X, N21X, D22X, K23X, I24X, L25X, S26X, Y27X, T28X, E29X, S30X, M31X, A32X, G33X, K34X, R35X, E36X, M37X, V38X, I39X, I40X, T41X, F42X, K43X, S44X, G45X, E46X, T47X, F48X, Q49X, VSOX, E51X, V52X, P53X, G54X, SSSX, Q56X, H57X, I58X, D59X, S60X, Q61X, K62X, K63X, A64X, I65X, E66X, R67X, M68X, K69X, D70X, T71X, L72X, R73X, I74X, T75X, Y76X, L77X,

T78X, E79X, T80X, K81X, I82X, D83X, K84X, L85X, C86X, V87X, W88X, N89X, N90X, K91X, T92X, P93X, N94X, S95X, I96X, A97X, A98X, I99X, SIOOX, MIOIX, K102X, N103X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB G1 : 494265 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLTB GI : 69630 without deleterious effect to the ganglioside and glycosphingolipid binding affinity of the recombinantly produced molecule. Such mutations include : MIX, N2X, K3X, V4X, K5X, C6X, Y7X, V8X, L9X, FIOX, T11X, A12X, L13X, L14X, S15X, S16X, L17X, Y18X, A19X, H20X, G21X, A22X, P23X, Q24X, T25X, I26X, T27X, Q28X, L29X, C30X, S31X, E32X, Y33X, R34X, N35X, T36X, Q37X, I38X, Y39X, T40X, 141X, N42X, D43X, K44X, 145X, L46X, S47X, Y48X, T49X, ESOX, S51X, M52X, A53X, G54X, K55X, R56X, E57X, M58X, V59X, I60X, I61X, T62X, F63X, M64X, S65X, G66X, E67X, T68X, F69X, Q70X, V71X, E72X, V73X, P74X, G75X, S76X, Q77X, H78X, I79X, D80X, S81X, Q82X, K83X, K84X, A85X, 186X, E87X, R88X, M89X, K90X, D91X, T92X, L93X, R94X, 195X, T96X, Y97X, L98X, T99X, EIOOX, TIOIX, K102X, 1103X, D104X, K105X, L106X, C107X, V108X, <BR> <BR> <BR> <BR> W109X, Nl lOX, Nl l IX, K112X, T113X, P114X, N115X, S116X, 1117X, A118X, A119X, I120X, S121X, M122X, K123X, N124X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid,. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLTB GI : 69630 with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside and glycosphingolipid binding activity.

Mutants of LT-IIa The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLT-Ila NCBI Accession No. GI : 146671 without deleterious effect to the ganglioside (GDI b, among others) binding affinity of the recombinantly produced molecule. Such mutations include : MIX, S2X, S3X, K4X, KSX, I6X, I7X, G8X, A9X, F10X, V11X, L12X, M13X, T14X, G15X, 116X, L17X, S18X, G19X, Q20X, V21X, Y22X, A23X, G24X, V25X, S26X, E27X, H28X, F29X, R30X, N31X, I32X, C33X, N34X, Q35X, T36X, T37X, A38X, D39X, I40X, V41X, A42X, G43X, V44X, Q45X, L46X, K47X, K48X, Y49X, ISOX, A51X, D52X, V53X, N54X, T55X. N56X, T57X, R58X, G59X, I60X, Y61X,

V62X, V63X, S64X, N65X, T66X, G67X, G68X, V69X, W70X, Y71X, I72X, P73X, G74X, G75X, R76X, D77X, Y78X, P79X, D80X, N81X, F82X, L83X, S84X, G85X, E86X, I87X, R88X, K89X, T90X, A91X, M92X, A93X, A94X, I95X, L96X, S97X, D98X, T99X, KIOOX, V101X, N102X, L103X, C104X, A105X, K106X, T107X, S108X, S109X, S110X, PIIIX, N112X, H113X, I1114X, W115X, A116X, M117X, E118X, L119X, D120X, R121X, E122X, S123X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLT-Ila with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its GDI b binding activity.

Mutants of LT-IIb The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of rLT-llb NCBI accession no. GI : 576584 without deleterious effect to the ganglioside (GDla among others) binding affinity of the recombinantly produced molecule. Such mutations include: MIX, S2X, F3X, K4X, K5X, I6X, 17X, K8X, A9X, FIOX, V11X, 112X, M13X, A14X, A15X, L16X, V17X, S18X, V19X, Q20X, A21X, H22X, A23X, G24X, A25X, S26X, Q27X, F28X, F29X, K30X, D31X, N32X, C33X, N34X, R35X, T36X, T37X, A38X, S39X, L40X, V41X, E42X, G43X, V44X, E45X, L46X, T47X, K48X, Y49X, I50X, S51X, D52X, I53X, N54X, NSSX, N56X, T57X, D58X, G59X, M60X, Y61X, V62X, V63X, S64X, S65X, T66X, G67X, G68X, V69X, W70X, R71X, 172X, S73X, R74X, A75X, K76X. D77X, Y78X, P79X, D80X. N81X, V82X, M83X, T84X, A85X, E86X, M87X. R88X, K89X, 190X, A91X, M92X, A93X. A94X, V95X, L96X, S97X, G98X, M99X, R1OOX, VlOlX, <BR> <BR> <BR> <BR> N102X, M103X, C104X, A] 05X, S106X, P107X, A] 08X, S109X, SHOX. PH IX, N112X, VI 13X, 1114X, W1ISX, A116X, 1117X, E118X, L119X, E120X, A121X, E122X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant rLT-IIb with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside GDla binding activity.

Mutants of Shiga toxin Beta subunit The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of Shiga toxin Beta subunit NCBI Accession No. GI : 152784 without deleterious effect to the carbohydrate binding affinity of the recombinantly produced molecule for Gal alpha 1----4Gal beta 1 ----Glc and globotrioside trisaccharides. Such mutations include: MIX, K2X, K3X, T4X, L5X, L6X, I7X, A8X, A9X, S1OX,

LllX, S12X, F13X, F14X S15X, A16X, S17X, A18X, L19X, A20X, T21X, P22X, D23X, C24X, V25X, T26X, G27X, K28X, V29X, E30X, Y31X, T32X, K33X, Y34X, N35X, D36X. D37X, D38X, T39X, F40X, T41X, V42X, K43X, V44X, G45X, D46X, K47X, E48X, L49X, F50X, T51X, N52X, R53X, W54X, N55X, L56X, Q57X, S58X, L59X, L60X, L61X, S62X, A63X, Q64X, I65X, T66X, G67X, M68X, T69X, V70X, T71X, I72X, K73X, T74X, N75X, A76X, C77X, H78X, N79X, G80X, G81X, G82X, F83X, S84X, E85X, V86X, I87X, F88X, R89X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X''represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant Shiga toxin Beta subunit with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its carbohydrate binding activity.

Mutants of verotoxin (Shiga-like toxin) beta subunit from E. coli The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of Shiga-like toxin Beta subunit from E. coli NCBI Accession No. GI : 4877349 without deleterious effect to the carbohydrate binding affinity for globotriaosyl ceramide, Gb3 or blood group PI antigenic trisaccharide of the recombinantly produced molecule. Such mutations include: MIX, K2X, K3X, 14X, FSX, V6X, A7X, A8X, L9X, FIOX, AIIX, F12X, V13X, S14X, V15X, N16X, A17X, M18X, A19X, A20X, D21X, C22X, A23X, K24X, G25X, K26X, 127X, E28X, F29X, S30X, K31X, Y32X, N33X, E34X, N35X, D36X, T37X, F38X, T39X, V40X, K41X, V42X, A43X, G44X, K45X, E46X, Y47X, W48X, T49X, N50X, R51Xs W52X, N53X, L54X, Q55X, P56X, L57X, L58X, Q59X, S60X, A61 X, Q62X, L63X, T64X, G65X, M66X, T67X, V68X, T69X, 170X, K71 X, S72X, N73X, T74X, C75X, A76X, S77X, G78X, S79X, G80X, F81X, A82X, E83X, V84X, Q85X, F86X, N87X, N88X, D89X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X" represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant Shiga-like toxin Beta subunit from E. coli with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its carbohydrate binding activity.

Mutants of pertussis toxin B subunits The amino acid substitutions contemplated in the invention encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of pertussis toxin S2 subunit NCBI Accession No. GI : 144070 without deleterious effect to the ganglioside (sialoglycoproteins) binding affinity of the recombinantly produced molecule. Such mutations

include: MIX, P2X, I3X, D4X, R5X, K6X, T7X, L8X, C9X, H10X, L11X, L12X, S13X, V14X, L15X, P16X, L17X, A18X, L19X, L20X, G21X, S22X, H23X, V24X, A25X, R26X, A27X, S28X, T29X, P30X, G31X, I32X, V33X, I34X, P35X, P36X, Q37X, E38X, Q39X, I40X, T41X, Q42X, H43X, G44X, S45X, P46X, Y47X, G48X, R49X, C50X, A51X, N52X, K53X, T54X, R55X, A56X, L57X, T58X, V59X, A60X, E61X, L62X, R63X, G64X, S65X, G66X, D67X, L68X, Q69X, E70X, Y71X. L72X, R73X, H74X, V75X, T76X, R77X, G78X, W79X, S80X, I81X, F82X, A83X, L84X, Y85X, D86X, G87X, T88X, Y89X, L90X, G91X, G92X, E93X, Y94X, G95X, G96X, V97X, I98X, K99X, D100X, G101X, T102X, P103X, G104X, G105X, A106X, F107X, D108X, L109X, K110X, T111X, T112X, F113X, C114X, I115X, M116X, T117X, T118X, R119X, N120X, T121X, G122X, Q123X. P124X. A125X, T126X. D127X, H128X, Y129X, Y130X, S131X, N132X, V133X, T134X, A135X, T136X, R137X, L138X, L139Xw S140X, S14] X, T142X, N143X S144X, R145X, L] 46X, C147X, A148X, V149X, F150X, V151X, R152X, S153X, G154X, Q155X, P156X, V157X, 1158X, G159X, A160X, C161X, T162X, S163X, P164X, Y165X, D166X, G167X, K168X, Y169X, W170X, S171X, M172X, Y173X, S174X, R175X, L176X, R177X, K178X, <BR> <BR> <BR> <BR> M179X, L180X, Y181X, L182X, 1183X, Y184X, V185X, A186X, G187X, 1188X, S189X, V19OX, R191X, V192X, H193X, V194X, S195X, K196X, E197X, E198X, Q199X, Y200X, Y201X, D202X, Y203X, E204X, D205X, A206X, T207X, F208X, E209X, T210X, Y211X, A212X, L213X, T214X, G215X, I216X, S217X, I218X, C219X, N220X, P221X, G222X. S223X, S224X, L225X, C226X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant pertussis toxin S2 subunit with altered characteristics for convalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside (sialoglycoproteins) binding activity.

The amino acid substitutions encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of pertussis toxin S3 subunit NCBI Accession No.

GI : 144070 without deleterious effect to the ganglioside (sialoglycoproteins) binding affinity of the recombinantly produced molecule. Such mutations include: MIX, L2X, I3X, N4X, NSX, K6X, K7X, L8X, L9X, HIOX, HIIX, 112X, L13X, P14X, 115X, L16X, V17X, L18X, A19X, L20X, L21X, G22X, M23X, R24X, T25X, A26X, Q27X, A28X, V29X, A30X, P31X, G32X, I33X, V34X, I35X, P36X, P37X, K38X, A39X, L40X, F41X, T42X, Q43X, Q44X, G45X, G46X, A47X, Y48X, G49X, RSOX, C51X, P52X, N53X, G54X, T55X, R56X, A57X, L58X, T59X, V60X, A61X, E62X, L63X, R64X, G65X, N66X, A67X, E68X, L69X, Q70X, T71X, Y72X, L73X, R74X, Q75X, I76X, T77X, P78X, G79X, W80X, S8 X, I82X, Y83X, G84X, L85X, Y86X, D87X, G88X, T89X, Y90X, L91X, G92X, Q93X, A94X, Y95X, G96X, G97X, I98X, I99X, K100X,

DIOIX, A102X, P103X, P104X, G105X, A106X, G107X, F108X, I109X, Y110X, R111X, E112X, T113X, F114X, C115X, I116X, TII7X, T1I8X, II19X, Y120X, K121X, T122X, G123X, Q124X, P125X, A126X, A127X, D128X, H129X, Y130X, Y131X, S132X, K133X, V134X, T135X, A136X, T137X, R138X, L139X, L140X, A141X, S142X, T143X, N144X, S145X, R146X, L147X, C148X, A149X, V150X, F151X, V152X, R153X, D154X, G155X, Q156X, S157X, V158X, I159X, G160X, <BR> <BR> <BR> <BR> A161X, C162X, A163X, S164X, P165X, Y166X, E167X, G168X, R169X, Y170X, R171X, D172X, M173, Y174X, D175X, A176X, L177X, R178X, R179X, L180X, L181X, Y182X, M183X, I184X, <BR> <BR> <BR> <BR> YI85X, M] 86X, S] 87X, G188X. LI89X, AI90X, VI9 ! X, R192X, V193X. H194X, V] 95X, S196X, K196X, E197X, E198X, Q199X, Y200X, Y201X, D202X, Y203X, E204X, D205X, A206X, T207X, F208X, Q209X, T210X, Y211X, A212X, L213X, T214X, G215X, 1216X, S217X, L218X, C219X, N220X, P221X, A222X, A223X, S224X, I225X, C226X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant pertussis toxin S3 subunit with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside (sialoglycoproteins) binding activity.

The amino acid substitutions further encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of pertussis toxin S4 subunit NCBI Accession No. G1 : 144070 without deleterious effect to the ganglioside (sialoglycoproteins) binding affinity of the recombinantly produced molecule. Such mutations include: MIX, L2X, R3X, R4X, F5X, P6X, T7X, R8X, T9X, T10X, A11X, P12X, G3X, Q14X, G15X, G16X, A17X, R18X, R19X, S20X, R21X, V22X, R23X, A24X, L25X, A26X, W27X, L28X, L29X, A30X, S31X, G32X, A33X, M34X, T35X, H36X, L37X, S38X, P39X, A40X, L41X, A42X, D43X, V44X, P45X, Y46X, V47X, L48X, V49X, K50X, T51X, N52X, M53X, V54X, V55X, T56X, S57X, V58X, A59X, M60X, K61X, P62X, Y63X, E64X, V65X, T66X, P67X, T68X, R69X, M70X, L71X, V72X, C73X, G74X, I75X, A76X, A77X, K78X, L79X, G80X, A81X, A82X, A83X, S84X, S85X, P86X, D87X, A88X, H89X, V90X, P91 X, F92X, C93X, F94X, G95X, K96X, D97X, L98X, K99X, RIOOX, PIOIX, G102X, S103X, S104X, P1OSX, M106X, E107X, V108X, M109X, LI10X, R111X, A112X, V113X, F114X, Ml lSX, Q116X, Q117X, R118X, P119X, L120X, R121X, M122X, F123X, L124X, G125X, P126X, K127X, Q128X, L129X, T130X, F131X, E132X, G133X, K134X, P135X, A136X, L137X, E138X, L139X, 1140X, R141X, M142X, V143X, E144X, C145X, S146X, G147X, K148X, Q149X, D1SOX, C151X, P152X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The"X"represents the deletion or replacement of any amino acid residue that, when

introduced in place of the wild type amino acid residue, results in a mutant or variant pertussis toxin S4 subunit with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside (sialoglycoproteins) binding activity.

The amino acid substitutions contemplated in the invention likewise encompass any such substitution, deletion, or insertion that selectively alters the conjugation characteristics of pertussis toxin S5 subunit NCBI Accession No. GI :] 44070 without deleterious effect to the ganglioside (sia) ogtycoproteins) binding affinity of the recombinantly produced molecule. Such mutations include : MIX, Q2X, R3X, Q4X, A5X, G6X, L7X, P8X, L9X, K) OX. AHX, N12X, P13X, M14X, H15X, T16X, 117X, A18X, S19X, 120X, L21X, L22X, S23X, V24X, L25X, G26X, 127X, Y28X, S29X, P30X, A31X, D32X, V33X, A34X, G35X, L36X, P37X, T38X, H39X, L40X, Y41X, K42X, N43X, F44X, T45X, V46X, Q47X, E48X, L49X, ASOX, L51X, K52X, L53X, K54X, G55X, K56X, N57X, Q58X, E59X, F60X, C61X, L62X, T63X, A64X, F65X, M66X, S67X, G68X, R69X, S70X, L71 X, V72X, R73X, A74X, C75X. L76X, S77X, D78X, A79X. G80X, H81X, E82X, H83X, D84X, T85X. W86X, F87X, D88X, T89X, M90X, L91X, G92X, F93X, A94X, I95X, S96X, A97X, Y98X, A99X, L1OOX, KIOIX, S102X, R103X, 1104X, A105X, L106X, T107X, V108X, E109X, D110X, S111X, P112X, Y113X, P114X, G115X, T116X, P117X, G118X, D119X, L120X, L121X, E122X, L123X, Q124X, 1125X, C126X, P127X, L128X, N129X, G130X, Y131X, C132X, E133X, wherein the first letter is the native amino acid appearing at the position of the indicated number, and the last letter is the amino acid substituted for the native amino acid. The "X"represents the deletion or replacement of any amino acid residue that, when introduced in place of the wild type amino acid residue, results in a mutant or variant pertussis toxin S5 subunit with altered characteristics for covalent modification or conjugation and, when coupled to bioactive compounds at that site, retains its ganglioside (sialoglycoproteins) binding activity.

Lysine Substitutions in the rCTB, rLTB, Shiga. Shiga-like, and pertussis family Conjugation of members of the rCTB, rLTB, Shiga, Shiga-like, and pertussis family to immunogens derived from infectious diseases can create vaccines which induce protection following mucosal delivery. Classic conjugation chemistry targets are reactive amino acid side chain groups such as primary amines, carboxylic acids, aldehydes, or sulfl1ydryls. The lysine residues are distributed at various places throughout the sequence. Those lysine residues that are near the Gm-1 binding site can couple to the ligand in a conformation which interferes with ganglioside binding. Accordingly, the invention articulates a number of mutant conjugation characteristics, primarily at lysine residues found in the rCTB protein family.

Therefore, the present approach involves the idea of directing the covalent linkage of carrier and immunogen away from the ganglioside receptor of CTB family. Site directed mutagenesis is employed to create a panel of mutations at various lysines of the rCTB family

coding sequences. These mutations will result in mutants which produce variable levels of protein, in vitro, comparable to those of the wild type and which can effectively deliver bioactive CTB- immunogen complexes to the mucosal immune system.

Accordingly, the invention articulates a number of mutant rCTB family proteins that with amino acid substitutions that replace the lysine residues found in the wild type rCTB family protein.

Preferably, the following amino acid substitutions are contemplated for rCTB family proteins GI : 758351, 808900,2144685, and 48890 (wherein"X"is any amino acid that results in a rCTB protein with a reduced ability to conjugate at the mutant sites): K44X, KSSX, K64X, K83X, K84X, K90X, K102X, KIOSX, and K112X. For rCTB family proteins GI : 1827850,998409, and 1421511, preferably K34X, K43X, K62X, K63X, K69X, K81X, K84X, and K91X. For rCTB family proteins GI : 229616, preferably K43X, K63X, K69X, K81, K84X. and K91X. For rCTB family protein GI : 2781121, preferably K35X, K44X, K62X, K69X, K81 X, K84X, and K91X. For rCTB family protein GI : 1421525. preferably K43X, K62X, K63X, K69X. K81X, K91X. For all rCTB family B subunit proteins if they have a lysine at position 91, most preferably K91X.

For LTB and the LTB family proteins GI : 3062900,1169505,1395122,145833,1648865, 223254,408996, and 69630 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites, K44X, KSSX, K64X, K83X, K84X, K90X, K102X, and KlOSX. For LTB family protein 494265 preferably K34X, K43X, K62X, K63X, K69X, K81X, K84X, and K91X. For all LTB family proteins if they have a lysine at position 91, most preferably K91X For TL-IIa protein GI : 146671 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites, K47X, K48X, K89X, K100X, and K107X.

For LT-IIb protein GI : 576584 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites, K48X, K76X, and K89X.

For Shiga toxin beta subunit protein GI : 152784 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites, K43X, K47X, and K73X.

For Shiga-like toxin from E. coli subunit B protein G1 : 4877349 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites, K41X, K45X, and K71X.

For pertussis toxin protein GI : 144070 wherein"X"is any amino acid that results in a protein with a reduced ability to conjugate at the mutant sites subunit S2: K98X, K167X, K177X, K195X.

S3: K38X, K100X, K121X, K133X, and K196X. S4: KSOX, K61X, K78X, K96X, K99X, K127X, K134X, and K148X. S5: K42X, K52X, K54X, K56X, and K101X.

Lysine additions in rCTB familv subunits The invention further contemplates the addition of amino acid residues throughout the amino acid sequence of the rCTB family, the LTB family, Shiga toxin, Shiga-like toxin, and pertussis toxin to direct conjugation of various bioactive compounds such as immunogens, immunomodulators, or drugs to specified sites. Any residue can be mutated to introduce amino acid residues that promotes acceptable conjugation events.

Hydrophobic coupling of rCTB Although chemical coupling is an excellent way to produce Gm-1 targeted immunogens, it would be highly advantageous to alter rCTB in such a way that it no longer requires chemical coupling, but instead strongly binds to other proteins or immunogens non-covalently. As an example, if rCTB could be prenylated it would then display up to 5 hydrophobic isoprene tails per pentamer. It is expected that in the properly folded and assemble protein, these tails would be present on the surface of the protein in opposite orientation to the Gm-1 binding surfaces. The commonly occurring isoprene groups include farnesyl, geranyl, geranyl-geranyl : all other prenylation or hydrophobic modification are considered to achieve the same outcome. Cholera toxin thus modified is expected to have an affinity for hydrophobic structures and surfaces.

Prenylated rCTB would bind to bacterial cell walls, cell membranes, virus membrane coats, and other hydrophobic structures such as hydrophobic proteins and hydrophobic phospholipids or adjuvants. The isoprene groups on rCTB would spontaneously insert into any lipid membrane, or hydrophobic miscelle, to non-covalently associate rCTB with the hydrophobic structure in such a manner as to present the ligand binding site of rCTB on the outer surface of the micelle. This concept could be applied to making conjugates between rCTB and vaccine antigens or to promote ganglioside specificity in microsome or liposome delivery systems.

Prenylatioll naturally occurs on selected proteins in eukaryotic cells, such as GTP binding proteins. The post-translational modification of proteins occurs at the C-Terminus, with a consensus sequence of CAAX (SEQ ID NO: 34) where C=cysteine, A=aliphatic amino acid, and X=any amino acid. The isoprenyl group is attached to the cysteine via a thioether link and after modification the cysteine becomes the last amino acid in the sequence. Other post-translational modifications such as modification of proteins with lipids, fatty acids, and glycosyl phospholipids also occur and could potentially serve the same purpose as prenylation.

In one embodiment, the gene for cholera toxin B is modified to include a prenylation signal that would permit in vitro prenylation. This may include a C-terminal cysteine, C-terminal CAAX box sequence (SEQ ID NO: 34), or any other sequence necessary or sufficient to achieve in vitro or in vivo prenylation. A plasmid containing the mutant cholera toxin B gene is transfected into a suitable host for expression. For example, a high expression rCTB vector is used to express

the mutated rCTB in bacteria. Once the mutated rCTB has been expressed and purified, it is used as a substrate for in vitro prenylation. In vitro prenylation is performed with the appropriate prenylation enzymes and substrates. The prenylated rCTB product is purified as necessary and used to produce the desired conjugated product. Alternatively, if the prenylation reactions permit, prenylation of rCTB is performed in the presence of the conjugation partner thus allowing immediate binding of the modified rCTB to the immunogen or vaccine. This may serve to stabilize prenylated rCTB or facilitate production and purification.

An alternative embodiment involves co-transfecting bacterial or eukaryotic cells to produce rCTB and the necessary proteins for prenylation. In this embodiment it may be necessary to construct the proteins for the special requirements of each system. For example, in any expression system both the rCTB and prenylation proteins may need to be targeted to the same compartment for efficient prenylation. This could be the cytoplasm, periplasm, or extracellular milieu of bacterial, or the cytoplasm, any of the secretory compartments or other organelles in eukaryotic expression systems.

Confirmation of hydrophobic coupling, purification, and Gm-1 binding assays would be performed essentially the same as chemically coupled rCTB, with variations in buffer and pH.

Vaccination would also be performed in essentially the same way.

Vaccines These concepts have been contemplated as methods for producing cholera toxin B based vaccines. In particular, these prenyl-rCTB constructs lend themselves to whole cell, whole virus, or hydrophobic particle vaccine conjugates. Vaccines contemplated include influenza virus vaccines, both whole and split, because influenza virus acquires a membrane coat and is thus capable of binding prenyl-rCTB to the membrane coat. The split virus vaccines commonly contain the membrane coat protein hemagglutinin. This protein binds to prenyl-rCTB through hydrophobic patches on its surface. Another suitable vaccine is whole cell pertussis, where prenyl-CTB could insert the hydrophobic groups into the outer membrane of B. pertussis. Also, hydrophobic particles such as water in oil mulsion based vaccines and liposome based vaccines would be attractive candidates for this technology.

Genetic fusion of rCTB with an immunogen Another option for production of an rCTB coupled immunogen is genetic fusion. Genetic fusion would occur using a vector that contains a promoter for expressing the fusion protein, the DNA sequence of the cholera toxin binding subunit rCTB, and an immunogenic peptide coding sequence. The rCTB and the immunogenic peptide coding sequence would be linked such that they were in the proper reading frame producing a fusion protein. The fusion protein would be expressed, secreted, and purified for use as a vaccine. rCTB fusion proteins are as shown in Table 2.

TABLE 2: CTB FUSION PROTEINS

Expression levez Plasmid Bacterial host Product Form Protein (ig/m 1) NH2-GST-linker #17 GST1.10* XL-1 Inclusion bodies 96 #1-A8VD4-C NH2-GST-linker #17 GST2.9** XL-1 Inclusion bodies 58 #2-A8VD4-CT CTB with internal P55-64gp12*** V. cholera Secreted 110 HIV epitope * Other antigenic sequences may be substituted for A8VD4. Linker is GAPAS (SEQ ID NO 34).

** Other antigenic sequences may be substituted for A8VD4. Linker is PGSTRAADRDGAPAS (SEQ ID NO : 35).

*** HIV epitope is flanked by Kpni and Mscl restriction enzyme sites, allowing easy insertion of other coding sequences into CTB. rCTB vaccines Microorganisms which invade via mucosal membrane surfaces are the best targets for rCTB vaccines. However, it could be used for almost any type of vaccine. Examples of diseases and antigens that could be coupled to rCTB for production of a vaccine are listed below in Table 3.

As mentioned above, larger, whole and killed cell vaccines or whole virus vaccines could be produced particularly using the hydrophobic coupling. Vaccines which couple the rCTB protein in some way to an immunogen of choice would be particularly useful for any type of disease which is targeted to mucosal surfaces.

Production of a specific vaccine involves the steps of 1) identifying an antigen or immunogen ; 2) coupling the antigen or immunogen to rCTB in some way (chemically, hydrophobically, or genetically) ; 3) isolating the coupled rCTB/immunogen and confirming that it still binds to Gm-1 ; 4) identifying that it works in vivo by injecting it into a mouse or other model animal ; and 5) testing it for efficacy in humans or other primates. Examples of vaccine targets are shown in Table 3 with potential antigens.

TABLE 3: HUMAN INFECTIOUS DISEASES AND VACCINE ANTIGENS Oral/Intestinal Exemplary Infectious agent Diseases Antigen Urease, 50 kDa citrate synthatase homologue protein, bacterial lysates, whole organisms E. coli LPS, enterotoxin type I, II, whole organisms V. cholera Killed bacteria, virulent live RP2, VP2, VP4, VP6, VP7, whole viral particles, Rotavirus VLPs Salmonella typhi Whole or fragments of organism Shigella dysenteria Whole or fragments of organism Respiratory Diseases H. influenza Current vaccines are conjugates RSV F/G protein, whole viral particles, VLPs Corynebacteria Toxoid, whole or fragments of organism diphtheria Toxoid, filamentous hemagglutinin, pilus, Boredetella pertussis pertactin, S1 subunits serotyopes, whole or (Whooping couth) fragments of organism Polysaccharide ags (23) 23 most common serotypes of 90 capsular serotypes currently Streptococcus targeted. New vaccines target 7-11 serotypes peneumonia bound to a protein carrier, whole or fragments of organism Killed, lysates, HA (hemagglutinin), NA Influenza virus (neuraminidase), trivalent or more based on most common serotypes that year; whole virus Anthrax Whole or fragments of organism Urogenital diseases (STD's) Gp41, Gpl20 env, Gpl60env, Tat protein, HIV multiepitope polypeptide TAB9, Gag, Pol, and Env proteins Chlamydia Whole or fragments of organism Treponema 47 kDa antigen, whole or fragments of pallidum (Syphilis) organism Neisseria gonorrhea whole or fragments of organism Herpes whole viral particles, VLPs, Tk

Use of rCTB in treatment of autoimmunitv and the induction of tolerance Autoantigen specific peripheral T cell tolerance is induced by CTB-autoantigen conjugates. Peripheral tolerance suppresses autoinflammatory disease by down regulating Thl.

CTB-autoantigens can even reverse existing inflammatory cell proliferation in animals.

Autoimmune diseases considered for treatment using CTB include without limitation : rheumatoid arthritis, encephalomyelitis (or other neuron demyelinating diseases), diabetes, and female anti- paternal immuno-contraception.

Reversa of inflammatory cell proliferation (thl tolerization) was shown in autoimmune models for diabetes in NOD mice (insulin-CTB conjugates; Bergerot et al. PNAS 1997 94: 4610- 14.). Additionally it was shown for rheumatoid arthritis (HSP 60, Haque et al. 1996 EurJImmunol 26: 2650-2656; collagen type 11 conjugates) and experimental autoimmune encephalomyelitis (EAE) in Lewis rats (Sun et al., Myelin basic protein MBP-peptide CTB conjugate, 1996 PNAS 93: 7196-7201). These conjugates were fed as single doses to animals before and after induction of disease.

It is contemplated that the rCTB of the invention can be chemically, hydrophobically, or genetically coupled to the above-mentioned antigens and fed to animal models to test the efficacy.

It is believed that because such a treatment was effective in the above examples it will be as effective or more so using the advantageous rCTB of the invention. Additionally, the rCTB conjugates of the invention are easier to make, more likely to work because the site of conjugation can be controlled, and yield a higher percentage of conjugated products that in turn will induce both a systemic and mucosal response.

The rCTB or other B subunits of the invention can also be used to induce tolerance to infection. For example, tolerization may be induced by administering the Leshmania major produced immunodominant antigen LACK, which generates a CD4+ T cell dominant response and drives a th2 response to associated antigens. Tl12 facilitates lesion development and leads to unresolved infection. LACK+ transgenic mice are tolerant of LACK and resolve infection. LACK fed animals are tolerized and resolve infection. (McSorley et al, Eur. J. Immunol. 1998,28: 424- 32.) showed that CTB-LACK, fed or given nasally, down regulated the T cell proliferative response to LACK and allowed animals to resolve (fight off) disease more effectively.

Additionally, responses to diseases associated with the development of acute autoimmunity such as Reiter's syndrome or Lyme disease can be treated with tolerizing conjugates.

EXAMPLES EXAMPLE 1 Expression and Purification of Mutant rCTB Production of Recombinant CTB in Bacteria The expression plasmid MS-0 (see Figure 2) was used to express rCTB and variants. MS-0 containing the rCTB gene is named pML-CTBtacl. The plasmid pML-CTBtacl surprisingly generates up to five times the product which was generated by a comparable plasmid (SBL's vector pJS162). PML-CTBtacl was constructed by cloning a portion of the CTB genomic region and the complete CTB coding region into plasmid MS-0 creating a 3.66 Kb expression plasmid. The PvuII

site in the polylinker was destroyed during cloning. The plasmid contains a tac promoter from pKK223, an EcoRI-BamHI polylinker fragment, and can be found at genbank accession No M77749.

The encoded protein is identical to the sequence from V. cholera strain 569B (SEQ ID NO: 2). The signal sequence (SEQ ID NO: 3) is a ! so from the CTB V. cholera classic strain 569B CTB gene. The complete nucleotide sequence of V. cholera strain 569B CTB gene is shown in Figure 1 (SEQ ID NO: 1). The signal sequence for LTB (SEQ ID NO : 15) is MNKVKCYVLFTALLSSLCAYG and can be used in the production of mutants or variants of LTB.

Measurement of Expression Level of rCTB mutant or variant It is advantageous to purify large quantities of the immunogen of interest quickly, easily and inexpensively. Accordingly, the ability to isolate milligram quantities of rCTB and rCTB variants for use in production of vaccines is discussed below.

A protocol for the isolation of rCTB and rCTB variants from shaker flasks is presented.

Surprisingly, is was possible to obtain up to 800 Lg of rCTB per ml of culture supernatant. Many recombinant expression systems are considered very productive with yields exceeding 10 pg/ml. The expression level of rCTB mutants thus far produced are moderately lower than wild type.

The concentration of all proteins tested is measured by comparison to a standard curve of rCTB at defined concentrations in the Gm-1 ELISA. The values recorded for a serial dilution of the unknown sample are then used to determine the sample concentration which is then multiplie by the dilution factor to obtain the correct concentration of the unknown sample.

One limitation of the Gm-1 ELISA is that it only identifies the relative binding concentrations of wild type vs. mutant rCTB. Thus, actual concentrations must be confirmed by A280 or by Lowry, Bradford, BCA or similar colorimetric protein assays known by those practiced in the art.

Any mutation to the rCTB sequence might result in an increase or decrease in receptor binding affinity for Go- ! and other ligands (Backstrom et al. 1997 Mol Microbiol 24 : 489-497; Merritt et al. 1995 Structure 3: 561-570). Any significant change in receptor affinity of a newly created mutant rCTB for Gm-1 may cause the loss of binding in a Gm-1 ELISA and result an "apparent"decrease in concentration. In such case, each test protein is initially purified to homogeneity using methods discussed below. From these, the purity is demonstrated by comassie or silver stained polyacrylamide gel electrophoresis, a technique well known in the art. The relative concentration of each protein is determined by A280 and confirmed by protein concentration assays. Equivalent concentrations of each protein. sufficient to saturate the ganglioside moiety (e. g.-50 fJ. g/ml), are incubated in a Gm-l ELISA plate. A two-fold serial dilution of each mutant or unlabeled wild type protein is then incubated in solution with a constant concentration of horse radish peroxidase labeled wild type CTB (at concentrations which alone

generate a maximum signal, usually >-10 ug/ml). From this assay, a standard curve for each mutant can be generated and compared to a similar curve for unlabeled wild type (positive standard curve control). Discrepancies from the positive standard curve control reflect significant changes in the affinity of the mutant rCTB for gm-1. The difference between curves can be quantified to determine the quantitative differences in affinity assuming the wild type affinity of 4. 6xl0-12M.

Purification of rCTB and rCTB variants A number of CTB purification protocols are available and known in the art. Three purification methods are discussed below.

Ammonium Sulphate Precipitation of rCTB This procedure worked well for cultures grown in baffled shaker flasks containing 100-500 g/ml rCTB in the supernatant. Culture volumes between 50-250 ml have been successfully processed using this protocol. The supernatant was processed immediately after growth and clarification of the culture.

Briefly, the bacteria expressing rCTB or mutants of rCTB were grown in a shaker flask.

The supernatant was clarifie by centrifuging the cells at 7000 x g (avg.) for 15 min at 4°C into a pellet. A step wise precipitation of protein with increasing concentrations of ammonium sultate results in highly purified rCTB product. Accordingly, ammonium sulfate was gradually added to the culture supernatant to bring the ammonium sulfate concentration up to 30% (vol/vol) while stirring at room temperature. After 1 hour of precipitation without stirring, the sample was centrifuged at 14,000 x g (avg.) for 15 minutes at 4°C. The supernatant was removed and a concentrated ammonium sulfate solution was added to bring the concentration to 50% (vol/vol) while stirring. This mixture was allowed to stand at room temperature for 1 hour. The sample was again centrifuged at 14, 000 x g (avg.) for 15 min at 4°C. A third precipitation step was performed, bringing the concentration up to 65% (101 g/L total volume). This mixture was again stirred and allowed to stand for 1 hour before centrifugation as before. The pellet of highly purified rCTB was then resuspended in a suitable buffer, for example, if additional purification by ion exchange chromatography is desired, the sample was brought up in 10 mM NaP04 buffer. The sample was then placed in dialysis tubing (7000 MWCO). If the sample is not immediately used, it can be stored undialyzed for up to one month at 4°C until ready to use. When dialysis was performed, one-liter volumes of buffer were used for at least one hour each, changing the buffer three times.

Purification of rCTB by DEAE Ion-exchange Chromatography This part of the protocol applies specifically to ammonium sulfate precipitated rCTB. A DEAE Sepharose Fast Flow gel of choice was packed on an Amersham Pharmacia Biotech column following the manufacturer's directions. 15 ml of a well-mixed gel slurry produced about 10 ml bed volume. The flow rate for packing was at least 4 ml/min. A column size of 1.5 cm (inner

diameter) and 20 cm (length) with a bed volume of 10 ml bound approximately 30 mg of rCTB from culture processed by ammonium sulfate precipitate. A flow rate adapter was used with the column. The column was washed with 1 M NaCI to activate the column bed, or 2 M NaCI to regenerate a spent column bed. The flow rate for all washes did not exceed 75% of the packing rate, or 3 ml/min. The column was extensively equilibrated with Buffer A (10 mM Sodium Phosphate Buffer, pH 7.5) to remove all salt. The sample containing rCTB was applied at a rate of 1-1. 5 ml/min. Each fractions was tested for the presence of rCTB. Following sample application, the column was washed with Buffer A at a flow rate of 3 ml/min until the optical density at A280 dropped below 0.02, confirming the removal of all non-specific and cationic species. The column was washed with Buffer A containing 50 mM NaCI, until the optical density at A280 dropped below 0.02. RCTB was eluted from the column with Buffer A containing 100 mM NaCI. All fractions were collected and pooled. Approximately 80% of the total column bound rCTB was recovered in these fractions. The column was washed with Buffer A containing 1 M NaCI until the optical density at A280 dropped below 0.02. Before regeneration of the column, the DEAE bed was washed with 10 mM sodium phosphate, pH 7.5 to remove all of the salt. Alternatively, CTB bound to the DEAE column can be eluted with a salt gradient from 0.02M NaCI to I M NaCI in buffer A. Fractions containing rCTB are pooled, dialyzed or concentrated in the buffer of choice.

Detailed protocol for purification of rCTB using affinitv chromatography on a ganglioside- Affigel column In this technique, clarified bacterial culture supernatant was directly poured onto the column. rCTB or rCTB mutants bound specifically to the immobilized gangliosides and the column was washed. rCTB was then eluted with 5M guanidine thiocyanite and dialyzed against a phosphate buffer. This technique was very easy and a high purity was achieved. However, there was a limited capacity of approximately 7 mg rCTB per run and it required denatured protein to be able to fold properly, which may be a problem with some mutants.

The Ganglioside-Affigel columns were produced using Affigel (Pierce, Rockford, IL).

The smaller Ganglioside-Affigel column (0.5 ml bed volume) had the capacity to bind 1-2 mg of rCTB, while the larger (5 ml bed volume) will bind at least 7 mg. The following buffers were used: 0.1 M Na-phosphate buffer, pH 7.5; 0. 1 M Na-phosphate buffer, pH 7.5,0.5 M Na Cl ; 5 M guanidine thiocyanite in DI water, freshly made in a 50 ml conical tube.

The culture supernatant was centrifuged at 10,000 rpm for 10 min (SS-34). Particulate matter was avoided as it clogs the column. The column was equilibrated with 0. 1 M Na-phosphate buffer, pH (2-5 column volumes). The sample containing rCTB was added and the flow through was collected and applied once more to allow quantitative binding to the column. The column was washed with 0.1 M Na-phosphate buffer, pH 7.5 until the A280 was close to 0, the column was

washed again with 0.1 M Na-phosphate buffer, pH 7.5,0.5 M NaCI until the A280 was close to 0.

The column was equilibrated with 0.1 M Na-phosphate buffer, pH 7.5 (2 column volumes). Fresh guanidine thiocyanite solution was prepared in 1 ml steps, 1 ml fractions were collected. The column was allowed to sit for 10 min after addition of the first two I ml additions of the elution buffer to the column. This ensured complete elution of rCTB from the ganglioside resin. The A280 containing fractions were pooled and dialyzed against 500 ml of 0.1 M Na-phosphate buffer, pH 7.4, with no stirring. A total of 4 buffer changes were performed with the last two in the desired buffer in which to store the protein.

Optionally, rCtB can be purified by cation exchange on a CM Sepharose Fast Flow ion exchange column. Briefly, NH4S04 salt precipitated or DEAE eluted rCTB can be dialyzed against 20 mM Tris-HCI, 10 mM NaCI, pH 8.4 and the dialysate applied to the column.

After washing, a highly purified rCtB product can be eluted with a continuous salt gradient of 10 mM to 300 mM NaCI in 20 mM Tris-HCI, pH 8.4.

This method resolves the remaining few contaminant products we see following DEAE anion exchange chromatography.

EXAMPLE 2 Mutavenesis of rCTB Mutagenesis was initially performed by oligonucleotide-directed in vitro mutagenesis according to Kunkel, TA, Roberts, J., and Zakour, R."Rapid and efficient site-specific mutagenesis without phenotypic selection", Methods Enzymol., 154, 367-382,1987. Subsequently mutagenesis was performed essentially according to Nelson, M. and McClelland, M., Methods Enzymol. 216, 279-303,1992. The results of the mutagenesis were confirmed by DNA sequencing.

EXAMPLE 3 Lysine Mutation of rCTB The following mutant rCTB proteins are made using the methods described in Example 2.

A lysine residue is inserted into the rCTB sequence of SEQ ID NO 2 to replace amino acid Asn 103. Asparagine's polarity comes from an amino side chain which is uncharged at neutral pH.

Replacement of Asn 103 with a lysine may prove to be an effective and novel conjugation site. A lysine residue is inserted into the rCTB sequence of SEQ ID NO 2 to replace the amino-terminal amino acid Ala 102. A lysine residue is inserted at position Alal. A lysine residue is inserted at position Pro2. Although this mutant changes the structure of the amino terminus, Gm-l binding affinity and antigenicity of the resulting composition is not compromised by the mutation.

Amino acids 14-20 form a P-sheet on the outer surface of rCTB with many of the side chains facing outward. Of the amino acid residues that form this structure, His 18, is particularly suitable for mutagenesis. Accordingly, a mutant rCTB protein is generated having the mutation

H18K. Substitution of His 13 for any other amino acid may effect Zn+2 binding characterisitics of rCTB as well as pentamer-pentamer self association. Elimination of either of these characteristics might be desired for purposes of biochemical isolation of the mutant proteins.

EXAMPLE 4 Determining Immunosenieity of rCTB Mutants Coupled to Immunogens Immunogenicity is determined by injection of the rCTB: Immunogen into balb/c mice. 1: 1, 1: 6, and 1: 3 ratios of rCTB : immunogen conjugate are injecte. Additionally, a control using immunogen alone is injected injecte. After ten days, blood is drawn from the immunized mice and antibodies to the rCTB: immunogen conjugate are identified and measured via an antibody assaying protocol such as ELISA or any other suitable assay widely known by one of skill in the art.

EXAMPLE 5 Chemical Couplinq of Mutant rCTB Proteins to an Immunogen Lysine residues Mutant CTB proteins CTB K9 I G and CTB-K62G, 63G, 9] G were modified, expressed, and purified using the method described in Examples 1-2. Mutation of K91 was chosen as a site for substitution because of its proximity to the Gm-1 binding site of CTB and it known interaction with the terminal glactose of Gm-1. Further, as discussed above, K91 is highly reactive with small molecular conjugates, such as FITC or SPDP. Accordingly, conjugations with heterobifunctional reagents are likely to create molecular complexes with diminished Gm-l binding affinities. some important modifications to protocols which create a simpler, more efficient coupling reaction with SPDP. In brief, the pH of the conjugation must be > 8.0, and the SPDP (N-Succinimidyl 3- [2- pyridyldithio] propionate) modified rCTB and its associated molecule must be highly concentrated.

Employing these modifications, rCTB wild type was successfully coupled to bovine serum albumin (BSA) and to Diphtheria toxoid (Dt).

In contrast to established protocols, neither BSA nor Dt was SPDP modified prior to conjugation. These proteins were determined to have free sulfllydryl groups which will readily react with the SPDP modified rCTB.

Modification of rCTB using SPDP chemistry rCTB or variant rCTB was dialyzed into Borate-Phosphate buffer. Borate-Phosphate buffers: a IOX stock solution (2L) was made by adding 12.4 g. Boric acid, 175.3 g NaCI, and 136.09 g Potassium phosphate (monobasic) into 1.5 liters of water. The solution was adjusted to a pH of 6.6 with 5M KOH and then brought up to a final volume of 2 liters with water. The solution was filtered through 0.2 micron filter. The pH of a I X solution was 7.0. Borate-Phosphate Buffer, pH 8.5 was IX buffer adjusted with 5M KOH. The rCTB was modified using ten-fold molar excess of SPDP over rCTB. The SPDP was weighed and diluted to 5 mg/ml using anhydrous

N, N-dimethylformamide (DMF), containing the molecular sieves (Grade 562, Type 3a, 4-8 mesh beads, Fisher Scientific, Tustin, CA). Thus, 25 ti of 5 mg/ml SPDP would be added to a 2 mg rCTB/ml solution of rCTB. The SPDP and rCTB were incubated and allowed to react for at least 1 hour at room temperature.

Excess SPDP was removed by running a G-25 gel filtration column (1.5 cm x 9 cm bed volume is the minimum required to separate excess SPDP). 10 mM sodium phosphate buffer, pH 7.5 containing 50 mM NaCI was used for column running buffer. The SPDP modified rCTB was concentrated by using Amicon centricon c-30 (30 kD cut-off) to a volume between 1-2 ml.

Conjugation of protein to SPDP modified rCTB Unmodified protein (the immunogen-i. e. BSA or Dt) was dialyzed against 3 x 1000 volumes Borate-Phosphate buffer, pH 7.0. SPDP modified rCTB and unmodified protein (immunogen) were combined at selected molar ratios and incubated overnight. The next the sample was concentrated using a Centricon 30 (30,000 MWCO Millipore, Bedford, MA).

Confirmation of Chemical Coupling The concentrated sample was separated into its component parts by gel electrophoresis on a 4-20% Tris-Glycine gel to confirm the formation of conjugates. A number of techniques can be used to confirm chemical coupling of the two proteins including: Gm-1 ELISA using immunogen- specific antibodies which recognize only immunogen when bound to rCTB, and molecular seive gel filtration.

Cysteine residues Mutant CTB proteins CTB-H18C and CTB-MIOIC were modified, expressed, and purified using the methods described in Examples 1-2. The rCTB proteins were treated with 100 mM DTT for 2 hours at room temperature. DTT was removed by centrifugation for 2 min at 2000g through a 1 ml spin column packed with Biogel P6 polyacrylamide gel (BioRad, Cambridge, MA), equilibrated with 25 mM sodium phosphate buffer pH 7.0/10 mM ethylenediaminetetraacetic acid (EDTA). The collected fraction was incubated for 2 hours at room temperature with maleimide-activated immunogen (i. e. immunogen functionalized with the cross-linker SMCC, Sigma). The reaction was stopped by the addition of P-mercaptoethanol ((3-me) to a final concentration of 1.5 mM and N- ethylmaleimide (NEM) suspended in N, N-dimethylformamide to a final concentration of 3 mM.

Confirmation of Chemical Coupling Conjugation of immunogen to rCTB was assessed by ELISA and Western blotting. (I) ELISA: conjugates of Immunogen and rCTB were incubated with GLM I-coated microtitre plates and immobilized Immunogen was detected with the chromogenic substrate 3,3', 5,5' tetramethylbenzidine (TMB, KPL). Detection of plate bound immunogen which was dependent on a coating of GM-1 was interpreted as evidence that rCTB was physically linked to the Immunogen. (II) Western Blots:

conjugates were separated by electrophoresis on 7.5% SDS polyacrylamide gels and transferred to nitrocellulose. rCTB was detected with polyclonal anti-rCTB sera, followed by goat anti-mouse Ig- alkaline phosphatase (A) (Amersham, Buckinghamshire, UK) and AP-specific substrate (BCIP/NBT, BioRad, Cambridge, MA). Immunogen was detected with antibodies specific to the immunogen, goat anti-mouse Ig-AP and BCIP/NBT.

Purification of immunogen-rCTB Conjugates Conjugates were purified by size exclusion chromatography based on the expected size of the conjugate. Additional purification was performed within a GM-1 or ganglioside affinity column to eliminate nonconjugated immunogen and within an antigen or immunogen specific column to eliminate residual free rCTB and rCTB-rCTB conjugates, respectively. This left only the bioactive conjugates which were assessed by SDS-PAGE and ELISA as described in Example 6.

EXAMPLE 6 Assaying Mutant rCTB Conjugated Complexes for Gm-1 Bindine Relative to Wild Type rCTB Maintenance of the ganglioside affinity by the GM-1 binding sites in mutant CTB, wild type CTB, or conjugates derived therefrom can be assayed by ELISA analysis. In the first example, the monosialoganglioside, GM-I, was used at a concentration of 0.3 nmol/ml PBS. One hundred microliters of ganglioside solution was added to each well of a 96 well microtiter plate and incubated overnight at 22°C. The following day, the plate was washed twice with PBS utilizing a Bioplate Autowasher EL-404 (Bio-Tec Instruments, Winooski, VT). All wells of the plate were then coated with 200 LU of a 0.1% BSA in PBS solution for 30 minutes at 37°C. The plate was washed three times in PBS containing 0.05% tween-20 and evacuated of remaining solution. All wells, other than the initial test wells, received 100 il of a solution of 0.] % BSA, 0.05% tween-20 in PBS. Test wells then received 150 p1 of 15 g CTB/ml test mutant, wild type, or conjugate. A three-fold serial dilution was then conducted across the plate, sequentially transferring and diluting 50 Stl from the second test well into 100 pI buffer in the third well and so on for up to twelve dilutions. The plate was incubated for 90 minutes at 22°C. The plate was then washed three times as described above. All wells then received either 100 ) of a 1/20, 000 dilution of mouse anti-rCTB antiserum or a pre-determined dilution of mouse anti-serum to the coupled compound. Wells were incubated one hour at 22°C and subsequently washed three times as above.

Test wells then received 100 Stl of a 1/3000 dilution of horseradish peroxidase conjugated goat anti-mouse IgG and were allowed to incubate one hour at 22°C. Plates were then washed again and incubated for 20 minutes with 100 p1 of a 1 mg/ml solution of othophenylenediamine dihydrochloride (OPD) and 0.01% H202 in 0.1 M sodium citrate buffer, pH 4.5.50 u. l of a 3N HC1 solution was then added to stop the enzymatic reaction. Plates were then read at 492 nm and 650 nm in a SpectroMax 250 microplate reader (Molecular Devices, Inc., Sunnyvale, CA). Results for mutants or conjugates thereof were reported as a function of endpoint dilution (apparent concentration) relative to wild type or conjugates thereof, respectively. The results indicated that mutated rCTB conjugated complexes retained their ability to bind to GM-I binding sites.