The present invention provides a novel composition which is a hybrid heat labile enterotoxin comprising the A-subunit of the heat labile toxin of Escherichia coli (LT-A) and the B-subunit of the cholera enterotoxin of Vibrio cholerae (CT-B). The hybrid toxin is designated LT-A/CT-B. The LT-A subunit, the CT-B subunit, or both subunits of the hybrid toxin may be mutant subunits, e.g., differing from wild-type subunits by amino acid substitutions, deletions or additions. Also provided are methods of using the novel LT-A/CT-B comprising compositions of the invention as adjuvants for vaccines, methods of making the LT-A/CT-B hybrid holotoxin, and kits.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a composition and methods for its use to promote the production of serum and/or mucosal antibodies as well as cell-mediated immune responses against antigens that are simultaneously administered with a hybrid bacterial toxin. The hybrid toxin combines the A-subunit of the heat-labile enterotoxin of Escherichia coli (LT-A) with the B-subunit of the cholera enterotoxin of Vibrio cholerae (CT-B). This hybrid molecule has unexpectedly reduced enterotoxicity and enzymatic activity yet retains its ability to act as an immunological adjuvant. The invention is based, in part, on the discovery that LT-A/CT-B has utility as an adjuvant. LT-A/CT-B can be administered in any manner known to those of skill in the art, preferably by mucosal, for example but not limited to oral, administration. The mode of administration may be mucosal (i.e., intranasal, oral, rectal) or parenteral (i.e., subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal). Administration results in the production of antibodies as well as cell-mediated immune responses against the antigens with which LT-A/CT-B is delivered.

Hybrids consisting of the A-subunit of one toxin combined with the B-subunit of the other toxin were constructed. It was unexpectedly found that LT-A/CT-B has significantly reduced enterotoxicity in the patent mouse assay when compared to native LT, native CT, or the alternate hybrid CT-A/LT-B. In order to more fully evaluate this unexpected finding, LT-A/CT-B was evaluated for the ability to induce cAMP in cultured Caco2 cells. Activity in this assay has been shown to be directly correlated with the toxicity of these molecules. Despite having an unaltered, intact A-subunit, the hybrid molecule LT-A/CT-B induced significantly less cAMP in this assay when compared to native LT, native CT, or the alternate hybrid CT-A/LT-B.

As described above, an unexpected finding was that LT-A/CT-B has significantly reduced toxicity and enzymatic activity despite having an unmodified LT-A subunit. Also, surprisingly, even when the LT-A subunit is modified (for example, when the LT-A subunit has an R192G mutation), the hybrid LT-A/CT-B has even less toxicity than the non-hybrid LT with that same mutation, e.g., LT-A(R192G)/CT-B has reduced toxicity as compared to LT-A(R192G)/LT-B. Thus, the present invention also encompasses LT-A/CT-B hybrid toxins, wherein the LT-A subunit, the CT-B subunit, or both subunits of the hybrid toxin are mutant subunits, e.g., differing from wild-type subunits by one or more amino acid substitutions, deletions or additions. Suitable mutant LT-A subunits include LT-A(R192G), LT-A(R192G/L211A), LT-A(S63K), LT-A(E112K), LT-A (.DELTA.192-194), LT-A(A69G) and LT-A(A72R). The use of hybrids containing mutant, detoxified LT-A subunits provides the advantage of two separate means for reducing the toxicity of the LT-A/CT-B hybrid. The mutation of LT-A alone renders the molecule safe for administration; when combined with CT-B to form a hybrid, the resulting molecule can be even less toxic because both the mutation of LT-A and the association of LT-A with CT-B reduce toxicity. In addition, the use of mutant CT-B subunits is also-encompassed.

The present invention supersedes the prior art in that LT-A/CT-B retains adjuvanticity for induction of antigen-specific antibody and cell mediated immune responses against a co-administered antigen. In the illustrative example presented in section 6, LT-A/CT-B induces antigen-specific Th1 type responses equivalent to those induced by native LT and significantly greater than those induced by native CT or the alternate hybrid CT-A/LT-B. Furthermore, LT-A/CT-B induces antigen-specific Th2 type responses greater than those induced by native LT, native CT or the alternate hybrid CT-A/LT-B.

The present invention also provides a method of producing LT-A/CT-B comprising the steps of a) culturing a cell comprising a vector capable of expressing in said cell an LT-A subunit and culturing a cell comprising a vector capable of expressing in said cell a CT-B subunit, but not capable of expressing an LT-B subunit or a CT-A subunit under conditions that allow for the expression by said cell or cells of said LT-A and CT-B molecules, and b) isolating the LT-A/CT-B produced by the cell or cells.

It will be apparent to the skilled artisan that both subunits may be produced in one cell from one vector, or in one cell from two different vectors, or in different cells from two different vectors.

Preferably the cell is a bacterial cell, most preferably an E. coli cell, and the vector is a plasmid.

Variations of this method include separate expression and isolation of the LT-A and CT-B subunits from different cell cultures and subsequent association of said subunits to form LT-A/CT-B, and methods wherein the LT-A subunit, the CT-B subunit, or both subunits are mutant subunits.

5.1 Production of LT-A/CT-B

LT-A/CT-B can be produced by a number of means apparent to those of skill in the art. For example, plasmid pLT-A/CT-B, fully described in Example 6.1, can be utilized to produce substantially pure LT-A/CT-B in E. coli. LT-A/CT-B can be isolated by agarose affinity chromatography from bacteria expressing an LT-A/CT-B encoding plasmid. Alternate methods of purification will be apparent to those skilled in the art.

The LT-A/CT-B of the present invention is preferably isolated LT-A/CT-B. As used herein, an "isolated" LT-A/CT-B means LT-A/CT-B separated from that which it is normally associated, or separated from the material normally present during the production of LT-A/CT-B by a cell. More preferably, the LT-A/CT-B is substantially purified, i.e., LT-A/CT-B is 5%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the total protein present in an LT-A/CT-B comprising preparation.

5.2 Mode of Administration of LT-A/CT-B and Unrelated Antigens

In accordance with the present invention, LT-A/CT-B can be administered in conjunction with any biologically relevant antigen and/or vaccine, such that an increased immune response, as compared to the immune response achieved by administration of said antigen and/or vaccine without LT-A/CT-B, to said antigen and/or vaccine is achieved. In a preferred embodiment, LT-A/CT-B and antigen are administered simultaneously in a pharmaceutical composition comprising an effective amount of LT-A/CT-B and an effective amount of antigen. The mode of administration is mucosal (i.e., intranasal, oral, rectal) or parenteral (i.e., subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal). The respective amounts of LT-A/CT-B and antigen will vary depending upon the identity of the route of administration, antigen employed and the species of animal to be immunized. In one embodiment, the initial administration of LT-A/CT-B and antigen is followed by a boost of the relevant antigen. In another embodiment no boost is given. The timing of boosting may vary, depending on the route, antigen and the species being treated. The modifications in route, dosage range and timing of boosting for any given species and antigen are readily determinable by routine experimentation. The boost may be of antigen alone or in combination with LT-/CT-B.

The methods and compositions of the present invention are intended for use both in immature and mature vertebrates, in particular birds and mammals, including but not limited to humans. Useful antigens, as examples and not by way of limitation, include antigens from pathogenic strains of bacteria (Streptococcus pyogenes, Streptococcus pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Haemophilus influenzae, Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis, Staphylococcus aureus, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter jejuni, Aeromonas hydrophila, Bacillus cereus, Edwardsiella tarda, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Treponema pallidum, Treponema pertenue, Treponema carateneum, Borrelia vincentii, Borrelia burgdorferi, Leptospira icterohemorrhagiae, Mycobacterium tuberculosis, Toxoplasma gondii, Pneumocystis carinii, Francisella tularensis, Brucella abortus, Brucella suis, Brucella melitensis, Mycoplasma spp., Rickettsia prowazeki, Rickettsia tsutsugamushi, Chlamydia spp., Helicobacter pylori); pathogenic fungi (Coccidioides immitis, Aspergillus fumigatus, Candida albicans, Blastomyces dermatitidis, Cryptococcus neoformans, Histoplasma capsulatum); protozoa (Entamoeba histolytica, Trichomonas tenas, Trichomonas hominis, Trichomonas vaginalis, Trypanosoma gambiense, Trypanosoma rhodesiense, Trypanosoma cruzi, Leishmania donovani, Leishmania tropica, Leishmania braziliensis, Pneumocystis pneumonia, Plasmodium vivax, Plasmodium falciparum, Plasmodium malaria); or Helminths (Enterobius vermicularis, Trichuris trichiura, Ascaris lumbricoides, Trichinella spiralis, Strongyloides stercoralis, Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and hookworms) either presented to the immune system in whole cell form or in part isolated from media cultures designed to grow said organisms which are well know in the art, or protective antigens from said organisms obtained by genetic engineering techniques or by chemical synthesis.

Other relevant antigens would be from pathogenic viruses (as examples and not by limitation: Poxviridae, Herpesviridae, Herpes Simplex virus 1, Herpes Simplex virus 2, Adenoviridae, Papovaviridae, Enteroviridae, Picornaviridae, Parvoviridae, Reoviridae, Retroviridae, influenza viruses, parainfluenza viruses, mumps, measles, respiratory syncytial virus, rubella, Arboviridae, Rhabdoviridae, Arenaviridae, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Non-A/Non-B Hepatitis virus, Rhinoviridae, Coronaviridae, Rotoviridae, and Human Immunodeficiency Virus) either presented to the immune system in whole or in part isolated from media cultures designed to grow such viruses which are well known in the art or protective antigens therefrom obtained by genetic engineering techniques or by chemical synthesis.

Further examples of relevant antigens include, but are not limited to, vaccines. Examples of such vaccines include, but are not limited to, influenza vaccine, pertussis vaccine, diphtheria and tetanus toxoid combined with pertussis vaccine, hepatitis A vaccine, hepatitis B vaccine, hepatitis C vaccine, hepatitis E vaccine, Japanese encephalitis vaccine, herpes vaccine, measles vaccine, rubella vaccine, mumps vaccine, mixed vaccine of measles, mumps and rubella, papillomavirus vaccine, parvovirus vaccine, respiratory syncytial virus vaccine, Lyme disease vaccine, polio vaccine, malaria vaccine, varicella vaccine, gonorrhea vaccines schistosomiasis vaccine, rotavirus vaccine, mycoplasma vaccine pneumococcal vaccine, meningococcal vaccine, Campylobacter vaccine, Helicobacter vaccine, cholera vaccine, enterotoxigenic E. coli vaccine, enterohemorrhagic E. coli vaccine, Shigella vaccine, Salmonella vaccine and others. These can be produced by known common processes. In general, such vaccines comprise either the entire organism or virus grown and isolated by techniques well known to the skilled artisan or comprise relevant antigens of these organisms or viruses which are produced by genetic engineering techniques or chemical synthesis. Their production is illustrated by, but not limited to, as follows:

Influenza vaccine: a vaccine comprising the whole or part of hemagglutinin, neuraminidase, nucleoprotein and matrix protein which are obtainable by purifying a virus, which is grown in embryonated eggs, with ether and detergent, or by genetic engineering techniques or chemical synthesis.

Pertussis vaccine: a vaccine comprising the whole or a part of pertussis toxin, hemagglutinin and K-agglutinin which are obtained from avirulent toxin with formalin which is extracted by salting-out or ultracentrifugation from the culture broth or bacterial cells of Bordetella pertussis, or by genetic engineering techniques or chemical synthesis.

Diphtheria and tetanus toxoid combined with pertussis vaccine: a vaccine mixed with pertussis vaccine, diphtheria and tetanus toxoid.

Japanese encephalitis vaccine: a vaccine comprising the whole or part of an antigenic protein which is obtained by culturing a virus intracerebrally in mice and purifying the virus particles by centrifugation or ethyl alcohol and inactivating the same, or by genetic engineering techniques or chemical synthesis.

Hepatitis B vaccine: a vaccine comprising the whole or part of an antigen protein which is obtained by isolating and purifying the HBs antigen by salting-out or ultracentrifugation, obtained from hepatitis carrying blood, or by genetic engineering techniques or by chemical synthesis.

Measles vaccine: a vaccine comprising the whole or part of a virus grown in a cultured chick embryo cells or embryonated egg, or a protective antigen obtained by genetic engineering or chemical synthesis.

Rubella vaccine: a vaccine comprising the whole or part of a virus grown in cultured chick embryo cells or embryonated egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.

Mumps vaccine: a vaccine comprising the whole or part of a virus grown in cultured rabbit cells or embryonated egg, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.

Mixed vaccine of measles, rubella and mumps: a vaccine produced by mixing measles, rubella and mumps vaccines.

Rotavirus vaccine: a vaccine comprising the whole or part of a virus grown in cultured MA 104 cells or isolated from the patient's feces, or a protective antigen obtained by genetic engineering techniques or chemical synthesis.

Mycoplasma vaccine: a vaccine comprising the whole or part of mycoplasma cells grown in a liquid culture medium for mycoplasma or a protective antigen obtained by genetic engineering techniques or chemical synthesis.

Those conditions for which effective prevention may be achieved by the present method will be obvious to the skilled artisan.

The vaccine preparation compositions of the present invention can be prepared by mixing the above illustrated antigens and/or vaccines with LT-A/CT-B at a desired ratio. Pyrogens or allergens should naturally be removed as completely as possible. The antigen preparation of the present invention can be used by preparing the antigen per se and the LT-A/CT-B separately or together.

Further, the present invention encompasses a kit comprising an effective amount of antigen and an adjuvant effective amount of LT-A/CT-B. In use, the components of the kit can either first be mixed together and then administered or the components can be administered separately within a short time of each other.

The vaccine preparation compositions of the present invention can be combined with either a liquid or solid pharmaceutical carrier, and the compositions can be in the form of tablets, capsules, powders, granules, suspensions or solutions. The compositions can also contain suitable preservatives, coloring and flavoring agents, or agents that produce slow release. Potential carriers that can be used in the preparation of the pharmaceutical compositions of this invention include, but are not limited to, gelatin capsules, sugars, cellulose derivations such as sodium carboxymethyl cellulose, gelatin, talc, magnesium stearate, vegetable oil such as peanut oil, etc., glycerin, sorbitol, agar and water. Carriers may also serve as a binder to facilitate tabletting of the compositions for convenient administration.
 

Claim 1 of 2 Claims

1. A composition comprising a LT-A/CT-B hybrid enterotoxin holotoxin recombinantly produced using plasmid pLT-A/CT-B assigned ATCC accession number PTA-6116, which holotoxin has immunologic adjuvant activity and is less toxic than native E. coli heat-labile enterotoxin holotoxin as measured in the patent mouse assay.

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