WO1998045324A1

WO1998045324A1 - Use of cholera toxin mutants as mucosal adjuvants

Info

Publication number: WO1998045324A1
Application number: PCT/US1998/006725
Authority: WO
Inventors: Jerry R. Mcghee; Shingo Yamamoto; Yoshifumi Takeda; Hiroshi Kiyono
Original assignee: The Uab Research Foundation
Priority date: 1997-04-04
Filing date: 1998-04-03
Publication date: 1998-10-15

Abstract

The invention provides a nontoxic mutant of cholera toxin that is efficacious as an adjuvant. Also provided are immunogenic compositions comprising the nontoxic mutant of cholera toxin and methods of using the immunogenic compositions.

Description

USE OF CHOLERA TOXIN MUTANTS AS MUCOSAL ADJUVANTS

Background of the Invention

The mucosal immune system can be divided into inductive sites, where vaccines/adjuvants are taken up for initial sensitization of B and T cells, and into effector sites where actual B cell and antibody responses actually occur. Oral or intranasal immunization leads to vaccine uptake into gut-associated or nasal- associated lymphoid tissues (GALT or NALT) which is followed by migration of vaccine-specific B and T cells into multiple mucosal effector sites such as the lamina propria regions of the gastrointestinal (GI), upper respiratory and genitourinary tracts as well as glandular tissues. In these effector regions polymeric IgA is produced and transported into the external secretion with specificity for the antigen initially encountered in GALT or NALT. This circular pathway, termed the Common Mucosal Immune System is where vaccine plus adjuvant are used to induce responses in a particular mucosal inductive site. These responses in turn result in antibody responses in multiple external secretions. The development and commercialization of mucosal vaccines has been hampered by the lack of an effective, nontoxic, mucosal adjuvant.

Both cholera toxin (CT) produced by Vibrio cholerae and Escherichia coli heat-labile enterotoxin (LT) induce significant antibody (Ab) responses and also function as potent mucosal adjuvants for co-administered, unrelated antigens (Ags), especially when given orally (C. O. Elson et al., J. Immunol.. 132. 2736-2741 (1984); C. O. Elson et al, J. Immunol. 133. 2982-2897 (1984); N. Lycke et al., Immunology. 59, 301-308 (1986); J. D. Clements et al, Vaccine. 6, 269-277 (1988)). It is known that serum IgG and IgA and mucosal IgA Ab responses are induced in mice given protein antigens orally with CT as adjuvant. It is also known that CT elicits adjuvant responses by inducing Ag-specific CD4⁺ T cells secreting the cytokines IL-4, IL-5, IL-6 and IL-10, which characterize the so-called Th2-type (J. Xu-Amano et al, J. Exp. Med.. 178. 1309-1320 (1993); M. Marinaro et al., J. Immunol.. 155. 4621-4629 (1995); J. L. VanCott et al., L Immunol.. 156. 1504-1514 (1996)). The induction of Ag-specific CD4⁺ Th2 cells correlated directly with serum IgGl and IgG2b subclass, IgE and mucosal IgA responses in mice orally immunized with Ag and CT as adjuvant (M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995)). A recent report (H. F. Staats et al, J. Immunol. 157. 462-472 (1996)) indicates that CT enhances serum IgG and mucosal IgA responses to a peptide antigen of HIV- 1 which was poorly immunogenic when intranasally administered alone.

Both CT and LT, however, have been shown to cause severe diarrhea in humans; it has been estimated, for example, that as little as 5 micrograms of native CT given orally to an adult human would be toxic, and that a 25 microgram dose would elicit a 20 liter diarrhea. Thus, the native toxins are not suitable for use as an adjuvant in humans.

CT and LT are multi-subunit macromolecules composed of two structurally, functionally and immunologically separate A and B subunits (Figure 1). The B subunit of each toxin consists of five identical 11.6 kD peptides, but differ from each other in that the B subunit of CT (CT-B) only binds to GM1 ganglioside, while the B subunit of LT (LT-B) binds GM1 as well as asialo GM1 and GM2. Following binding of the B subunit to epithelial cell GM1 or GM2, the A subunit reaches the cytosol and following activation, binds to NAD and catalyzes ADP-ribosylation of Gsα. This GTP -binding protein activates adenylate cyclase, resulting in elevation of intracellular cyclic AMP (cAMP) levels, which in epithelial cells causes secretion of water and chloride ions into the small intestine yielding a characteristic watery diarrhea.

Since the A subunit is responsible for toxicity, it has been suggested that CT-B be used as an adjuvant. Although CT-B alone initially appeared to have adjuvant properties (S. Tamura et al, Vaccine. 6, 409-413 (1988); A. Lee et al, Infect. Immun.. 62. 3594-3597 (1994)), CT-B prepared from holotoxin is now known to contain a small but sufficiently immunogenic amount of the toxic subunit CT-A (S. Spiegel, J. Cell Biochem.. 42, 143-152 (1990)).

In vitro studies in a variety of cells, e.g., B cells (N. Lycke et al, L Immunol. 142. 3781-3787 (1989)), T cell lines (H. J. Lee et al, J. Immunol. 151. 6135-6142 (1993); E. Munoz et al, J. Exp. Med.. 172. 95-103 (1990)), macrophages (A. Bromander et al, J. Immunol. 146. 2908-2914 (1991)), and epithelial cells (D. W. McGee et al, Infect. Immun.. 61, 4637-4644 (1993)), were carried out to assess the potential mechanism whereby CT and LT enhance the immune response. In most of these studies, it was concluded that adjuvanticity of CT resulted from the ADP-ribosyltransferase activity, i.e., induction of increased intracellular cAMP formation. In Bromander et al, L Immunol. 146. 2908-2914 (1991), CT as well as forskolin was shown to inhibit T cell receptor-mediated IL-2 production and proliferation in cloned Thl cells, but not to inhibit IL-4 production and proliferation in a clone of Th2 cells. This indicates that Thl and Th2 cells differ in their sensitivity to increases in intracellular cAMP .

Attempts to dissociate diarrhoeagenicity of these molecules from adjuvanticity have to date been unsuccessful. For example, a mutant LT toxin El 12K, which involved a single amino acid substitution in the ADP- ribosyltransferase active center (T. Tsuji et al, J. Biol Chem.. 265. 22520-22525 (1990); T. K. Sixma et al, Nature. 355. 561-564 (1992)) was nontoxic but also lacked adjuvanticity (N. Lycke et al, Eur. J. Immunol. 22, 2277-2281 (1992)). This led to the conclusion that ADP-ribosyltransferase activity was essential for adjuvanticity of both LT and CT (N. Lycke et al, Eur. J. Immunol. 22, 2277- 2281 (1992)). Two single amino acid substitution mutants of LT, 7K, (G. Douce et al,

Proc. Nat'l. Acad. Sci. U.S.A.. 92, 1644-1648 (1995) and R192G (B. L. Dickinson et al, Infect Immun.. 63. 1617-1623 (1995)) were recently shown to be nontoxic and still retain adjuvant properties when co-administered with protein by intranasal or oral routes, respectively. However, these amino acid substitutions are located outside the ADP-ribosyltransferase cleft, and the mutants retained low ADP-ribosyltransferase activity. This potentially could cause diarrhea in humans, were the mutants to be administered orally. Another LT mutant designated S63K (M. Pizza et al, Mol. Microbiol. 14, 51-60 (1994)) was shown to be without toxicity; however, this mutant exhibited poor mucosal adjuvant properties when administered intranasally (A. Di Tommaso et al, Infect. Immun.. 64, 974-979 (1996)). Mucosal vaccines are often safer and more effective than vaccines administered subcutaneously. However, at present there are no commercially available mucosal adjuvants for use in humans. What is needed therefore is an adjuvant for use with single or multiple vaccines given by mucosal routes.

Summary of the Invention The present invention provides a nontoxic mutant of cholera toxin that is effective as an adjuvant, preferably a mucosal adjuvant, for immunogenic compositions such as vaccines. Specifically, the present invention provides a mutant cholera toxin selected from the group consisting of S6 IF, El 12K, a nontoxic subunit of S61F, a nontoxic subunit of El 12K, a nontoxic derivative of S61F and a nontoxic derivative of El 12K, wherein the nontoxic subunit or derivative is effective as an adjuvant when co-administered with an immunogenic amount of an antigen. A "subunit" of a selected CT mutant means a continuous sequence of amino acids that is present within the full polypeptide sequence of the selected CT mutant. A "derivative" of a selected CT mutant is defined as a chemically or enzymatically altered variant of the full CT sequence or of subunit of the full sequence, such as, for example, an oxidized, reduced, amidated, esterified, or conjugated variant of S61F or El 12K. The CT mutants of the present invention include variants of S61F and El 12K that contain various amino acid additions, deletions, or substitutions as long as the resultant molecule is nontoxic (i.e., lacks ADP-ribosyltransferase activity) and retains substantial adjuvanticity, as described herein. Preferably, the nontoxic subunit or derivative of S61F or El 12K of the invention is an effective mucosal or parenteral adjuvant, more preferably an effective mucosal adjuvant.

The cholera toxin mutants of the invention are nontoxic in that they lack ADP-ribosyltransferase activity and as a result are not diarrhoeagenic in mammals. S61F and El 12K are each single-site mutants of cholera toxin wherein the single amino acid substitutions are present in the ADP- ribosyltransferase active center of the cholera toxin A subunit. Like the native cholera toxin, however, the mutant cholera toxin of the invention serves as an effective adjuvant when co-administered with another antigen. An adjuvant is a compound that enhances the immune response mounted by the recipient against a co-administered antigen. The present cholera toxin mutants are particularly suited for use as mucosal adjuvants, and the present invention thus provides a method for use of the mutant cholera toxin as an adjuvant, preferably a mucosal adjuvant.

The invention also provides a vaccine comprising (i) the mutant cholera toxin of the invention (ii) an immunogenic amount of an antigen, and (iii) a pharmaceutically acceptable carrier. Administration of the vaccine can take any convenient form, such as parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) or mucosal administration. Preferably, the vaccine is formulated for mucosal administration, more preferably for either oral or intranasal administration. The vaccine of the invention can be administered to any bird or mammal, and is preferably administered to a domesticated animal or a human.

Brief Description of the Figures

Figure 1 shows the amino acid sequence nCT strain 569B (A) A subunit and (B) B subunit. Amino acid differences for porcine LT (LTp) and human LT (LTh) are shown below the homologous CT sequences (B. D. Spangler, Microbio Rev.. 56, 622-647 (1992)).

Figure 2 shows serum OVA- and CT-B-specific Ab responses following subcutaneous immunization with OVA combined with mCTs or nCT as adjuvants. Groups of C57BL/6 mice were immunized subcutaneously with 100 micrograms of OVA alone (□) or together with 10 micrograms of rCT-B (E3), 1 microgram of nCT (□), or 10 micrograms of mCTs, S61F (1!) or El 12K (■), on days 0 and 14.

Figure 3 shows OVA- and CT-B-specific CD4⁺ T cell proliferative responses following subcutaneous immunization with OVA combined with mCTs or nCT as adjuvants. Groups of C57BL/6 mice were immunized subcutaneously with 100 micrograms of OVA alone (□) or together with 10 micrograms of rCT-B (E3), 1 microgram of nCT (□), or 10 micrograms of mCTs, S61F (M) or El 12K (■). on days 0 and 14.

Figure 4 shows cytokine production from OVA-specific splenic CD4⁺ T cells. Figure 5 shows serum OVA-and CT-B-specific IgA, IgM and IgG (A) and IgG subclass (B) responses on day 21 following intranasal immunization with OVA combined with mCT S61F or nCT as adjuvants. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (E3), 0.5 microgram of nCT (H), or 5 micrograms of mCT, S61 F (■), on days 0, 7 and 14.

Figure 6 shows numbers of OVA- and CT-B-specific IgG (A) and IgA (B) AFC in CLN, lung tissues and spleen following intranasal immunization with OVA combined with mCT S61F or nCT as mucosal adjuvants. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (E2), 0.5 microgram of nCT (lϋ), or 5 micrograms of mCT, S61F (■), on days 0, 7 and 14.

Figure 7 shows OVA- and CT-B-specific IgA Ab responses in mucosal secretions following intranasal immunization with OVA combined with mCT S61F or nCT as adjuvants. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B

(123), 0.5 microgram of nCT (H), or 5 micrograms of mCT, S61F (■), on days 0, 7 and 14.

Figure 8 shows OVA- and CT-B-specific CD4⁺ T cell proliferative responses isolated from lung (A) and spleen (B) following intranasal immunization with OVA combined with mCT S61F or nCT as adjuvants.

Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (E3), 0.5 microgram of nCT (II), or 5 micrograms of mCT, S61F (■), on days 0, 7 and 14.

Figure 9 shows cytokine production from OVA-specific CD4⁺ T cells isolated from lung tissue. Detailed Description

The mutant cholera toxins of the present invention retain the high adjuvanticity of native cholera toxin but lack ADP-ribosyltransferase activity and are therefore expected to lack the diarrhoeagenicity associated with the native peptide. The mutant cholera toxins of the invention are thus suitable for use in any vaccine formulation, preferably those designed to elicit immunity via mucosal delivery, and are particularly useful as adjuvants in vaccines given either intranasally or by the oral route for induction of immunity via the Common Mucosal Immune System. Examples of human vaccines containing immunogenic antigens are found in the Mayo Clinic Family Health Book, D. Larson, M.D. ed., William Morrow & Co. Inc., pp. 338-340, 867 (1990) and R. Atlas, Microbiology Fundamentals and Applications, Macmillan Publishing Company, p. 527 (1984). Methods of preparing vaccines are well-known in the art and disclosed, for example, in U.S. Pat. No. 5,419,907 (May 30, 1995). Examples of pharmaceutically acceptable carriers and suitable dosages are listed therein at col. 5. Immunization as a means of disease prevention is described, for example, in R. Atlas, Microbiology Fundamentals and Applications, Macmillan Publishing Company, pp. 525-530 (1984).

Mutant CT S61F (mCT S61F) was made by replacing serine with phenylalanine at position 61 of the cholera toxin A subunit. Mutant CT El 12K (mCT El 12K) was made by replacing glutamic acid with lysine at position 112 of the A subunit. Both amino acid substitutions caused the A subunit to lose ADP-ribosyltransferase activity and diarrhoeagenicity. Both mutants supported Ag-specific responses which were comparable to native CT (nCT) when given parenterally. mCT S61F is an effective mucosal adjuvant when administered intranasally and induces mucosal and systemic Ab responses which are mediated by CD4⁺ Tlι2-type cells. mCT El 12K is also an effective adjuvant when administrated by either the intranasal or oral routes. Nontoxicity and adjuvanticity of other S61F and El 12K CT variants of the invention can be conveniently established using the assays described herein below. Cholera toxin mutants S61F and El 12K are devoid of ADP- ribosyltransferase activity, are unable to induce increases in intracellular cAMP, and fail to elicit fluid accumulation in mouse ligated ileal loops. Advantageously, these mCTs retain the ability of native CT to boost Ab responses to the co-administered antigenic protein ovalbumin (OVA), as well as enhance Ab responses to the CT molecule itself, e.g., to the CT-B subunit. These adjuvant effects of mCTs are presumably due to the A subunit portion of the molecule, since co-administration of rCT-B at much higher concentrations with OVA does not enhance anti-OVA Ab responses.

Native CT (nCT) is known to elicit adjuvanticity by induction of Ag- specific CD4⁺ Th cells secreting Th2-type cytokines, which in turn provide B cell help for serum IgGl, IgG2b, IgA and IgE and mucosal S-IgA Ab responses. S61F and El 12K also both induce significant OVA-specific CD4⁺ T cell proliferative responses, with subsequent production of cytokines IL-4, IL-5, IL-6 and IL-10 (Th2-type) at levels comparable to the Th2-type response observed when nCT is used as adjuvant. The mutant CTs (mCTs) of the present invention thus retain adjuvanticity despite a lack of ADP-ribosyltransferase activity. These observations suggest that both the mCTs of the present invention and nCT enhance the immunogenicity of a co-administered, unrelated protein via the same mechanism, and that, contrary to the previous teachings in the field (N. Lycke et al, Eur. J. Immunol. 22. 2277-2281 (1992)), this mechanism is independent of the ADP-ribosyltransferase activity exhibited by nCT.

Furthermore, the IgG subclasses of the Abs induced by mCTs comprise largely IgGl and IgG2b, and OVA-specific CD4⁺ T cells from mice given mCT were of the Th2 type, a pattern essentially identical to that obtained with nCT. Thus, in marked contrast to the previous observations, the present findings indicate that adjuvanticity of CT can be dissociated from ADP-ribosyltransferase activity and enterotoxicity, and the CD4⁺ Th2-type T cell responses induced by CT are due to a pathway separate from the adenyl cyclase system.

It is noteworthy that the present mCT El 12K exhibits significant adjuvant activity, while a related LT mutant El 12K does not (N. Lycke et al, Eur. J. Immunol. 22, 2277-2281 (1992)). CT and LT share a significant degree of homology (approximately 80% amino acid sequence identity) and some antibodies induced to CT-B cross react with LT-B and vice versa. However, although CT and LT are both potent adjuvants, the molecules differ in terms of the nature of CD4⁺ Th cell subsets induced and the profile, isotype and subclass of Abs induced. For example, CT elicits adjuvanticity by promoting Ag-specific CD4⁺ Th2-type responses associated with high levels of IL-4 and IL-5 production with provision of help for IgGl subclass, IgE and S-IgA responses (J. Xu-Amano et al, J. Exp. Med.. 178, 1309-1320 (1993); M. Marinaro et al, 1 Immunol. 155. 4621-4629 (1995)), whereas LT promotes both Thl- and Th2- type responses with high levels of IFN-γ and IL-5 production and subsequent IgGl, IgG2a, IgG2b subclass and S-IgA Ab responses (I. Takahashi et al, 1 Infect. Pis.. 173. 627-635 (1996)). Furthermore, oral administration of CT as adjuvant failed to enhance Ag-specific S-IgA responses in IL-4 gene disrupted (IL-4T) mice (M. Vajdy et al, J. Exp. Med.. 181. 41-53 (1996)), whereas LT was able to induce Ag-specific mucosal S-IgA as well as serum IgG responses in both IL-47^" and IL-4 mice. These differences cannot be ascribed to ADP- ribosyltransferase activity which both molecules share.

Advantageously, mCT elicits significant protein-specific IgG, IgA and IgM Ab responses in serum and elicits IgA Abs in mucosal secretions after intranasal administration, and these responses are comparable to those induced when nCT is used as mucosal adjuvant. Significant enhancement is seen with different protein components, including poorly immunogenic OVA as well as TT and influenza virus. In contrast, rCT-B fails to enhance anti-protein Ab responses in serum or in the external secretions, indicating that the A subunit of CT is necessary for adjuvant activity.

Intranasal immunization, like other mucosal routes, offers several advantages when compared to parenteral immunization. For example, lower doses of immunizing proteins are required to induce effective Ab responses when compared with other routes, and this can decrease the cost for vaccination. In order to elicit intestinal mucosal IgA responses comparable to those induced by oral immunization with CT, typically only 5-10% of the quantity of vaccine is required when given intranasally. This intranasal does also effectively induces serum IgG Ab responses when compared with parenteral immunization which required more doses of OVA and mCT or nCT as adjuvants. In addition, the doses used in intranasal immunization induce lower total and Ag-specific IgE levels in serum than typical protocols used for oral administration (M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995)). This implies that intranasal immunization may have less risk for anaphylactic reactions. In this regard, mCT has an additional advantage over nCT, since serum CT-B-specific IgE levels elicited by mCT are significantly lower. Also, both total and OVA-specific IgE responses are less than those induced by nCT, although both molecules induce elevated Ag-specific Ab responses in serum. Further, intranasal immunization effectively induces not only systemic IgG but also mucosal IgA responses in mucosal effector tissues. Thus, intranasal vaccination using mCT could be useful in humans to prevent systemic, gastrointestinal or respiratory diseases as well as sexually transmitted diseases including HIV infection.

The mechanisms by which CT acts as a mucosal adjuvant are only partially understood. As noted above, native CT elicits adjuvanticity by induction of antigen-specific CD4⁺ T cells secreting IL-4, IL-5, IL-6 and IL-10, i.e., Th2-type cells, which in turn effectuate for serum IgGl, IgA and IgE and mucosal IgA Ab responses. The present mCT molecules of the invention induce significant OVA-specific CD4⁺ T cell proliferative responses, resulting in high levels of Th2-type cytokine production, which levels are comparable to those with nCT as adjuvant. On the other hand, OVA alone or OVA plus rCT-B do not induce this characteristic profile. These findings support the concept that both mCT and nCT boost Ab responses to the co-administered protein OVA through help provided by CD4⁺ Th2-type cells. Furthermore, although rCT-B does induce detectable CT-B-specific IgA Ab responses in mucosal secretions including nasal washes, saliva and fecal extracts, it fails to elicit anti-CT-B Abs in vaginal washes. The Th2-type CD4⁺ T cell responses elicited by mCT and nCT thus differ from those elicited by rCT-B, in that the former induce detectable responses in distant mucosal sites, such as sites within the reproductive tract system, as well as proximal mucosal sites such as the salivary gland and respiratory system sites.

The in vitro effects of CT on T cells has remained controversial. One study showed that cAMP activates the IL-5 promoter in EL-4, a thymoma T cell line (H. J. Lee et al, J. Immunol. 151. 6135-6142 (1993)). On the other hand, CT has been shown to inhibit mitogen- and anti-CD3 -stimulated T cell proliferative responses (D. L. Anderson et al, J. Immunol. 143. 3647-3652 (1989); (J. B. Imboden et al, Proc. Nat'1 Acad. Sci. USA. 83, 5673-5677 (1986)). In another study, CT as well as forskolin were shown to inhibit T cell receptor-mediated IL-2 production and proliferation in cloned Thl cells but not IL-4 production and proliferation in a clone of Th2 cells, indicating that Thl and Th2 cells differ in their sensitivity to an increase in cAMP (E. Munoz et al, 1 Exp. Med.. 172. 95-103 (1990)). Surprisingly, however, mCT elicited serum IgGl and IgG2b subclass and mucosal IgA Ab responses despite the lack of ADP-ribosyltransferase activity and resultant lack of cAMP induction. Further, OVA-specific CD4⁺ T cells from mice given mCT as adjuvant yielded a clear pattern of Th2-type responses which were identical to those induced when nCT was used as adjuvant. These findings indicate that the CD4⁺ Th2-type T cell responses induced by CT are elicited via a pathway separate from the adenyl cyclase system. CT may up-regulate of a yet to be characterized pathway which induces CD4⁺ T cells into a Th2-type subset, and this mechanism has not been elucidated by previous in vitro studies. Such adjuvant properties of CT appear to be associated with the A subunit, since rCT-B do not enhance anti-OVA Ab responses in the studies disclosed herein.

The present invention is described herein with reference to various specific embodiments, examples and techniques, however it should be understood that many variations and modifications may be made while remaining within the scope of the invention.

EXAMPLES Example 1: Construction and Purification of CT Mutants

A 3.1 kb Eco llPstl DNA fragment including the CT gene from V. cholerae OI strain GP14 was cloned into phage M13mpl9. Single strand DNA (ssDNA) was prepared from a culture supernatant of E coli CJ236 transfected with M13mpl9 including the CT gene and was subjected to a site-directed mutagenesis system using Mutan K (Takara Biomedicals, Kyoto, Japan) as described in T. A. Kunkel et al, Methods ΕnzvmoL. 154. 367-382 (1987). The sequences of oligonucleotides used for the serine to phenylalanine substitution at position 61 (S61F) and for the glutamate to lysine mutation at position 112 (Ε112K) were 5'-GGATATGTTTTTACCTCAATT-3' (SEQ ID NO:7) and 5'- GATGAACAAAAAGTTTCTGCT-3' (SEQ ID NO:8) , respectively. The amino acid mutation sites (i.e., 61 and 112) are both in the CT-A subunit, located in the proposed ADP-ribosyltransferase active center of CT, and substitution of these amino acids in LT have been shown to completely inactivate ADP- ribosyltransferase activity and enterotoxicity (S. Harford et al, Eur. J. Biochem.. 183. 311-316 (1989); T. Tsuji et al, J. Biol Chem.. 265-22520-22525 (1990)). After the DNA sequences were confirmed, pUCl 19 harboring the mutated CT genes at the EcoRI/.P-s'tl site were transformed into E. coli DH5α. E. coli strains containing the plasmids for the mutant CT genes were grown in LB medium (10 g NaCl, 10 g tryptone and 5 g yeast extract/L) with 100 micrograms/ml of ampicillin, and CT mutants were purified using a D-galactose immobilized column (Pierce, Rockford, IL) from a cell suspension prepared by sonication. A plasmid containing the rCT-B gene (M. T. Dertzbaugh et al, Gene. 82, 335-342 (1989)) was kindly provided by Dr. Charles O. Εlson at the University of Alabama at Birmingham and CT-B was also purified by use of a D-galactose immobilized column.

Example 2: Biologic. Enzymatic and Toxicity Assays of mCTs Chinese hamster ovary cell morphology. The ability of CT mutants

(mCTs) and native CT (nCT) to induce toxic effects in cultured Chinese hamster ovary (CHO) cells was assessed using the method of Guerrant et al. (R. L. Guerrant, et al, Infect. Immun.. 10, 320-327 (1974)). Briefly log₁₀ dilutions of each toxin were added to CHO cell cultures (2 x 10⁵ cells/0.5 ml of F10 medium containing 1% FCS) and cultured at 37° C in 5% CO₂ for 24 hours. Toxicity was defined as spindle formation in > 20% of cell cultures. For cAMP assessment, 1 x IO⁶ CHO cells in F10 medium containing 1% FCS were cultured with 1 nanogram/ml of mCTs or nCT for 24 hours as described above. The cellular protein precipitated with 5% trichloroacetic acid was dissolved in 0.2 N NaOH and the protein amount was determined (Bio-Rad Laboratories, Hercules, CA). The supernatants were assessed for cAMP with an enzyme immunoassay (EIA) system (Amersham International, Buckinghamshire, UK), and the levels of cAMP were expressed as picomoles of cAMP/milligram of protein.

ADP-ribosyltransferase activity of mCTs. The CT-A-catalyzed transfer of ADP-ribose from NAD to agmatine was done precisely in accordance with the method of Noda et al. (M. Noda et al, Biochemistry. 28, 7936-7940 (1989)). Briefly, each assay tube contained 10 micrograms of mCTs or nCT in a total volume of 300 microliters and a 50 microliter aliquot of the assay mixture was assessed for radioactive ADP-ribosylated agmatine by liquid scintillation counting.

Assessment of toxicity using mouse ileal loops. The enterotoxicity of mCTs and nCT was examined using a mouse ileal loop test (K. Fujita et al, 1 Infect. Pis.. 125. 647-655 (1972)). Groups of mice were anesthetized, and 100 microliters of PBS containing different doses of each toxin were injected into a 2 cm ileal loop which was isolated by suture. The mice were sacrificed 18 hours after the injection and the ratio of fluid to length was determined and defined as positive when the ratio was more than 40 microliters/cm.

Results. As expected, as little as 1.0 picogram/ml of nCT induced spindle cell formation in CHO cell cultures, a response previously shown to be dependent upon adenyl cyclase-mediated increases in cAMP. However, neither of the mCTs affected the appearance of CHO cells even at levels of 1.0 microgram/ml These results were confirmed by direct measurement of intracellular cAMP levels in CHO cells, which were sharply increased in nCT- treated, but not in mCT-treated cultures (Table 1). Quantitative analysis of ADP-ribosyltransferase activity was assessed and again increased enzymatic activity was associated with nCT but not with mCTs (Table 1). The toxicity of mCTs and nCT was also assessed in a mouse ileal loop assay, where as little as 100 nanogram of nCT induced significant fluid accumulation in ligated loops, while 1, 000-fold higher levels (100 micrograms) of mCTs were nontoxic (Table 1).

Table 1. Comparison of biologic, enzymatic and toxic activity ofmCTs and nCT

ADP- Ileal

Adjuvant CHO assay cAMP induction ribosyltransferase loop test assessed (pg/ml)* (pmol/mg)* activity (cmp)^§ (ng)ⁿ nCT 1 739 ± 127 4669 ± 256 100

S61F >10⁶ 8.3 ± 1.8 93 ± 12 >10⁵

E112K >10⁶ 6.2 ± 2.2 98 ± 15 >10⁵

PBS - 9.7 ± 2.2 98 ± 6 -

CHO cells were cultured in tissue culture chamber with log_]0 dilutions of each toxin for 24 hours and the toxic effects were defined as spindle formation in > 20% of cultured cells.

% CHO cells were cultured with 1 nanogram/ml of each toxin for 24 hours and cAMP assessed by an EIA system. The protein in 5% trichloroacetic acid precipitates was determined and concentrations of cAMP were expressed as the mean picomoles of cAMP/mg of protein ± SEM of 3 samples. The results are representative of three separate experiments.

§ The radioactivity of ADP-ribosylated agmatine induced by mCTs or nCT in a 50 microliter aliquot of the assay mixture was expressed as the mean cpm ± SEM of 6 samples. The results are representative of three separate experiments.

¹ ' The enterotoxicity ofmCTs and nCT was examined using an ileal loop test, where mice were anesthetized, and 100 microliters of PBS containing different levels of each toxin were injected into a 2 cm ileal loop. Loops were examined 18 hours later and the ratio of fluid to length was defined as positive when the ratio was more than 40 microliters/cm. Example 3; Immunological Response to Subcutaneous Administration of mCTs

Mice and their immunization. In order to assess the immunologic properties ofmCTs, groups of mice were immunized with OVA combined with each mCT or with nCT as a control. Specifically, C57BL/6 mice were obtained from the Frederick Cancer Research Facility (National Cancer Institute, Frederick, MD) at 5-6 weeks of age and were used at 8 to 12 weeks of age. Mice were immunized subcutaneously with 100 micrograms of OVA (Sigma Chemical Co., St. Louis, MO) alone or together with 10 micrograms of mCTs (S61F or El 12K), with 1 microgram of nCT (List Biological Laboratories, Campbell, CA), or with 10 micrograms of rCT-B on days 0 and day 14.

Detection of Ag-specific Abs by ELISA and antibody-forming cells (AFCs by enzyme-linked immunospot (ELISPOT) assay. Ab titers in serum were determined by ELISA and splenic AFCs by ELISPOT assay as described previously (J. Xu-Amano et al, J. Exp. Med.. 178. 1309-1320 (1993); M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995). Endpoint titers determined by ELISA were expressed as the reciprocal log₂ of the last dilution giving an optical density OD₄₅₀ of > 0.1 units above unimmunized controls. In the ELISPOT assay, the AFCs were determined by direct counting of spots.

Adjuvant properties of mCTs. Figure 2 shows OVA- and CT-B-specific Ab responses following subcutaneous immunization with OVA combined with mCTs or nCT as adjuvants. Serum IgM, IgG and IgA responses (A) and IgG subclass (B) responses were assessed by endpoint ELISA. Splenic Ag-specific AFCs (C) were determined by ELISPOT assay. Groups of C57BL/6 mice were immunized subcutaneously with 100 micrograms of OVA alone (□) or together with 10 micrograms of rCT-B (£3), 1 microgram of nCT (□), or 10 micrograms ofmCTs, S61F (BB) or El 12K (BB), on days 0 and 14. All assays were performed on samples from mice taken 1 week after the last immunization. Bars represent the mean Ab titer and mean number of AFCs ± SEM in each group of 10 mice and the data are representative of three separate experiments.

Immunization with OVA alone did not result in significant anti-OVA Ab responses, and admixture of OVA with rCT-B also failed to support anti-OVA Ab responses; however, both mCTs and nCT enhanced serum anti-OVA Abs and these responses were mainly of the IgG isotype (Fig. 2A). Further, anti-OVA Abs were largely restricted to IgGl with less IgG2b subclass response (Fig. 2B). Significant numbers of splenic OVA-specific IgG AFCs were noted in mice given OVA combined with mCTs or with nCT, whereas low numbers of AFCs were observed in mice given OVA alone or OVA with rCT-B (Fig. 2C). Thus, both mCTs induced an Ab pattern remarkably similar to the serum Ab responses which resulted from use of nCT as adjuvant. Mice immunized with OVA and mCTs or nCT as adjuvant also showed significant anti-CT-B-specific IgG responses. Although IgG anti-CT-B responses were also seen in mice given rCT-B, the titers were approximately 100-fold lower than seen when either mCTs or nCT were given (Fig. 2A). In addition, mCTs and nCT induced high levels of anti-CT-B specific IgGl and IgG2b Abs (Fig. 2B). Large numbers of CT-B-specific IgG AFCs were present in splenic cells from mice immunized with mCTs or with nCT, while much lower numbers of AFCs were seen in mice immunized with rCT-B (Fig. 2C).

IgE analysis. Past studies have shown that CT induces marked increases in both total and Ag-specific IgE Abs following mucosal immunization (M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995)). Two sensitive assays were used to detect increased total serum IgE as well as Ag-specific IgE Abs in mice given OVA combined with mCTs or with nCT as adjuvant. Total IgE levels were determined by ELISA as described in the preceding paragraph. Ag- specific IgE was detected by a modified IgE capture method (M. Sakaguchi et al, J. Immunol. Methods. 190. 189-197 (1989)). Briefly, 96 well microplates (Dynatech Microlite, Chantilly, VA) were coated with 1 microgram/ml of rat anti-mouse IgE mAb (PharMingen, San Diego, CA) in 50 mM carbonate- bicarbonate buffer (pH 9.5). After blocking with 3% BSA-PBS, serial dilutions of serum were added. Following incubation and washing, 2.5 microgram/ml of biotinylated-OVA or 1.5 microgram/ml of CT-B were added in 3% BSA-PBS- Tween 20. The plates were then washed with 2 mM EGTA-PBS-Tween 20 and incubated with 10 nanogram/ml streptaequorin (SeaLite Sciences, Inc., Bogard, GA) in 2 mM EGTA-PBS-Tween 20. Light development was carried out in a Dynatech ML-3000 luminometer by injection of Ca²⁺ buffer (50 mM Tris, 20 mM calcium acetate, pH 7.5) (R. J. Jackson et al, J. Immunol Methods. 190. 189-197 (1996)). Endpoint titers were determined as the dilution of each sample showing a 2-fold higher level of luminometric units above background. Maximum IgE responses peaked by 3 weeks and although differences in total IgE levels were not significant in mice given mCTs or nCT (Table 2), anti- OVA IgE titers were lower in mice given mCT S61F (p < 0.05), whereas CT-B- specific IgE Abs were depressed in mice given mCTs S61F or El 12K when compared with nCT (p < 0.01) (Table 2). In mice given OVA alone or OVA plus rCT-B, neither total nor Ag-specific IgE responses were noted.

* Mice were immunized subcutaneously with 100 micrograms of OVA alone or together with 10 micrograms of rCT-B, 1 microgram of nCT, or 10 micrograms ofmCTs, S61F or El 12K, on days 0 and 14. Each group contained 10 mice. The results are representative of three separate experiments

X IgE responses on day 21 were determined by ELISA (total) and luminometric assay (Ag-specific).

§ Significantly lower (p < 0.05 when compared with nCT).

¹¹ Significantly lower (p < 0.01 when compared with nCT).

OVA- and CT-B-specific splenic CD4+ T cell responses. Single spleen cell suspensions in complete RPMI 1640 medium were fractionated on a nylon wool column for 1 hour at 37°C to remove adherent cells. The CD4⁺ T cell subset (>98% purity) was then obtained by positive sorting using a magnetic bead activated cell separation system (MACS, Miltenyi Biotec Inc., Sunnyvale, CA) using biotinylated anti-CD4 mAb (GK1.5) and streptavidin coated microbeads (Miltenyi Biotec Inc.). Purified splenic CD4⁺ T cells were cultured at a density of 2 x IO⁶ cells/ml with 1 mg/ml of OVA or with 1 x IO⁷ CT-B- coated beads/ml, T cell-depleted, irradiated (3000 rads) splenic feeder cells (2.5 x IO⁶ cells/ml), and IL-2 (10 Units/ml) (PharMingen) in complete medium (Xu-Amano et al, J. Exp. Med.. 178. 1309-1320 (1993); M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995); J. L. VanCott et al, J. Immunol. 156. 1504-1514 (1996)). As positive controls, CD4⁺ T cells from nonimmunized mice were stimulated with a solid-phase anti-mouse CD3 mAB (145-2C11). To measure cell proliferation, 0.5 microcuries of [Methyl-³H]-thymidine (Dupont NEN Products, Boston, MA) was added to individual culture wells 18 hours before termination, the cells were harvested and the radioactivity was assessed by liquid scintillation counting after 96 hours of culture. To determine cytokine production by Ag-specific CD4⁺ T cells, the cells were harvested after 48 hours of culture for quantitative reverse transcriptase (RT)-PCR analysis of cytokine- specific mRNA and the supernatants were collected after 96 hours for evaluation of cytokines by ELISA. For IL-2, supernatants from 48 hour cultures were used since this represented the interval for maximal production of this cytokine.

Cytokines in culture supernatants were determined by a modified ELISA (M. Marinaro et al, J. Immunol. 155. 4621-4629 (1995)). Nunc- ImmunoMaxiSorp™ plates were coated with 2.5 microgram/ml of anti-mouse IFN-γ, IL-2, IL-4, IL-5, IL-6 or IL-10 mAB (PharMingen). For secondary Abs and detection enzyme, 0.2 microgram/ml of biotinylated rat anti-mouse cytokine mAb (PharMingen) and 1 :4000 diluted horseradish peroxidase-labeled anti- biotin (Vector Laboratories, Burlingame, CA) were used, respectively.

Quantitative cytokine-specific RT-PCR using rRNA internal standards was conducted as described in M. Yamamoto et al. and T. Hiroi et al. (M. Yamamoto et al, Am. J. Pathol. 148. 331-339 (1996); T. Hiroi et al, Eur. J. Immunol. 25, 2743-2751 (1995). Cytokine-specific rRNA for IFN-γ, 11-2, 11-4, IL-5, IL-6 or IL-10 were used as internal standards. For quantitation, aliquots of total R A were added with a series of diluted rRNA internal standards and standard RT-PCR was performed. Analysis of PCR products were conducted by capillary electrophoresis with a laser induced fluorescence detection system (CE- LIF) (LIF-P/ACE, Beckman Instruments, Fullerton, CA).

OVA- and CT-B-specific CD4⁺ T cell proliferative responses are shown in Figure 3. Groups of C57BL/6 mice were immunized subcutaneously with 100 micrograms of OVA alone (□) or together with 10 micrograms of rCT-B (E0, 1 microgram of nCT (□), or 10 micrograms of mCTs, S61F (D) or El 12K (■), on days 0 and 14. Purified splenic CD4⁺ T cells were cultured at a density of 2 x IO⁶ cells/ml in the presence of 1 mg/ml of OVA or 1 x IO⁷ CT-B-coated beads/ml, T cell depleted, irradiated splenic feeder cells (2.5 x IO⁶ cells/ml) and IL-2 (10 U/ml) in complete medium. Bars represent the mean stimulation index ± SEM in each group. Each group contained 10 mice and are representative of three separate experiments. Figure 4 shows cytokine production from OVA- specific splenic CD4⁺T cells. Molecules of cytokine-specific mRNA were determined by quantitative RT-PCR using rRNA internal standards. Cytokine production was determined by ELISA. The scale of each figure corresponds to mRNA molecules and protein levels produced by nonimmunized CD4⁺ T cells stimulated with anti-CD3 mAb. Bars represent the mean cytokine-profile ± SEM in each group. ND indicates that the molecules were not detected. Each group contained 5 mice and are representative of three separate experiments.

Both mCTs were found to induce OVA-specific CD4⁺ T cell proliferative responses, which were comparable to those seen in mice given nCT as adjuvant. Moreover, splenic OVA-specific CD4⁺ T cells from mice given OVA together with mCTs produced high levels of Th2-type cytokines (IL-4, IL-5, IL-6 and IL- 10), which were comparable to those seen when nCT was used as adjuvant (Fig. 4). On the other hand, CD4⁺ T cells from mice given OVA alone or OVA plus rCT-B did not produce detectable levels of cytokines other than IFN-γ when stimulated with OVA. Abundant Th2-type cytokine-specific mRNA was present in OVA-specific CD4⁺ T cell cultures taken from mice given OVA combined with mCTs or with nCT, but was not detected in CD4⁺ T cells from mice given OVA alone or OVA plus rCT-B (Fig. 4). Further, IFN-γ was detected at low levels in all cultures including OVA-stimulated controls from unimmunized mice. Splenic CT-B-specific CD4⁺ T cells from mice given mCTs or nCT also demonstrated significant proliferation (Fig. 3) and high levels of Th2-type with low but detectable levels of Thl -type cytokines (IFN-γ and IL-2). The quantitative RT-PCR results together with levels of secreted cytokines were consistent with previous studies which showed that nCT induces CD4⁺ Th2-type responses.

The results are expressed as the mean ± SEM. Statistical significance (p < 0.05) was determined by Student's t test and by the Mann- Whitney U test of unpaired samples.

Example 4: mCT S61F as a Mucosal Adjuvant

Purification of mCT and recombinant CT-B (rCT-B E. coli strains containing the plasmids for mCT S61F (Example 1) or rCT-B (M. T. Dertzbaugh et al, Gene. 82, 335-342 (1989)) genes were grown in LB medium (10 g NaCl, 10 g tryptone and 5 g yeast extract/L) with 100 microgram/ml of ampicillin. The mCT and rCT-B were purified using a D-galactose immobilized column (Pierce, Rockford, IL) from a cell suspension prepared by sonication of the bacteria according to the method of Uesaka et al. (Y. Uesaka et al, Microbial

Pathogenesis. 16. 71-76 (1994)). Purity of the molecules was assessed on SDS/PAGE and no contaminating protein bands were noted following silver staining. When ADP-ribosyltransferase activity was examined in the presence of 5 micrograms of ADP-ribosylation factor (ARF) in a total volume of 300 microliters as described in Example 2, no ADP-ribosylation was observed with 4 micrograms of mCT S61F (111 ± 14.5 cpm/50 microliters of reaction mixture) or with PBS (113 ± 6.3 cpm), while the activity of 4 micrograms of nCT (List Biological Laboratories, Campbell, CA) was significantly enhanced with ARF (5101 ± 380 cpm) when compared with nCT only (1735 ± 69 cpm). Immunization and sample collection. C57BL/6 mice were obtained from the Charles River Laboratories (Wilmington, DL) at 5-6 weeks of age and were used at 8-12 weeks of age in this study. Mice were intranasally immunized with a 20 microliter aliquot (10 microliters per nostril) containing 100 micrograms of OVA (Sigma Chemical Co., St. Louis, MO) alone or together with 0.1, 1 or 5 microgram of mCT, 0.1 or 0.5 microgram of nCT, or 5 microgram of rCT-B on days 0, 7 and 14. Further, 25 micrograms of tetanus toxoid (TT) (kindly provided from Dr. Y. Mukai, Osaka University, Biken Foundation, Osaka, Japan) or formalin-treated influenza virus (B. Yamagata) (2 micrograms of hemagglutinin [HA] -equivalent) (kindly provided from Drs. R. B. Couch and I. N. Mbawuike at Baylor College of Medicine, Houston, TX) were immunized intranasally together with 5 micrograms of rCT-B, 0.5 micrograms of nCT or 5 micrograms of mCT S61F using the same protocol as employed for OVA. Nasal and vaginal washes were collected by gently flushing the nasal passage or vaginal canal with 20 microliters of 50 microliters of sterile PBS, respectively. Saliva was obtained following intraperitoneal injection of mice with 100 microliters of 1 mg/ml pilocarpine (Sigma). Fecal extract samples were obtained by adding weighed pellets to PBS containing 0.1% sodium azide (1 ml/ 100 mg fecal sample). The pellet was vortexed, centrifuged, and the supernatants were collected for assay.

Cell isolation. Cervical lymph nodes (CLN) and spleen were aseptically removed and single cell suspensions were obtained. The nasal passage, submandibular gland (SMG), lung or vaginal tissues were carefully excised, teased apart, and dissociated using collagenase type IV (Sigma) in Joklik- modified medium (Life Technologies, Inc., Gaithersburg, MD). After removal of Peyer's patches, the small intestine was stirred in PBS containing 1 mM EDTA at 37° C for 30 minutes and the lamina propria lymphocytes (LPL) were subsequently isolated using collagenase type IV. The mononuclear cells were obtained at the interface of the 40 and 75 % layers of a discontinuous percoll gradient (Pharmacia, Uppsala, Sweden) (K. Fujihash et al, J. Exp. Med.. 183. 1929-1935 (1996)): T. Hiroi et al. Eur. J. Immunol. 25. 2743-2751 (1995)). Detection of Ag-specific Ab titers by ELISA and AFC bv ELISPOT assay. Ab titers in serum and mucosal secretions were determined by ELISA as described in Example 2. Endpoint titers were expressed as the reciprocal log₂ of the last dilution giving an optical density at 450 nm (OD₄₅₀) of ≥ 0.1 units above negative controls. Enumeration of Ag-specific AFC from various tissues was performed by ELISPOT assay. The color reaction was developed with 1.6 mM 3-amino-9-ethyl carbazole in 0.1 M sodium acetate buffer (pH 5.0) containing 0.05% H₂O₂ at room temperature for 1 hour. The plates were washed with water and dried, and AFC were quantitated with the aid of a stereomicroscope.

Figure 5 shows serum OVA- and CT-B-specific IgA, IgM and IgG and IgG subclass responses on day 21 following intranasal immunization with OVA combined with mCT S61F or nCT as adjuvants were determined by endpoint ELISA. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (E23), 0.5 microgram of nCT (HI), or 5 micrograms of mCT, S61F (BB), on days 0, 7 and 14. Serum samples were collected 1 week after the last immunization. Bars represent the mean Ab titer ± 1 SE in each group. Each group consisted of 5 mice and the data are representative of three separate experiments. Neither OVA alone nor admixture of OVA with 5 micrograms of rCT-B elicited detectable serum anti- OVA IgM or IgA Ab responses, although low IgG Ab responses were detected (Fig. 5 A). In preliminary dose response studies, admixture of 0.1 - 1 microgram of mCT S61F or 0.1 microgram of nCT induced OVA-specific IgG Abs in serum and elevated numbers of OVA-specific IgG and IgA AFC in lung tissues and spleen; however, these doses of adjuvants did not elicit optimal OVA-specific IgA Ab responses in saliva, fecal extracts or vaginal washes.

On the other hand, mice intranasally immunized with OVA plus 5 micrograms of mCT S6 IF or 0.5 microgram of nCT showed significantly high serum Ab titers of OVA-specific IgG, IgA, and IgM isotypes (Fig. 5 A) and elevated IgGl and IgG2b subclass responses (Fig. 5B). Assessment of levels and isotype of AFC responses also revealed significant numbers of splenic OVA- specific IgG AFC in mice given OVA with mCT or nCT as mucosal adjuvants, whereas only low numbers of AFC were observed in mice given OVA alone or OVA together with rCT-B (Fig. 6A). Significant OVA-specific IgG and IgA AFC responses were also observed in CLN and in lung tissue cell isolates of mice immunized with OVA and mCT as well as from mice given nCT as mucosal adjuvant (Fig. 6B). Only low AFC responses occurred in mice given OVA alone or OVA together with rCT-B. Figure 6 shows numbers of OVA- and CT-B-specific IgG (A) and IgA(B) AFC in CLN, lung tissues and spleen following intranasal immunization with OVA combined with mCT S61F or nCT as mucosal adjuvants were determined by ELISPOT assay. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (122), 0.5 microgram of nCT (H), or 5 micrograms of mCT, S61F (■) on days 0, 7 and 14. Samples were collected 1 week after the last immunization. Bars represent the mean numbers of AFC ± 1 SE and each group contained 5 mice. The data are representative of three separate experiments.

It should be emphasized that significant OVA-specific IgA Ab responses were seen in multiple mucosal secretions of mice given OVA and mCT as adjuvant. In Figure 7, OVA- and CT-B-specific IgA Ab responses in mucosal secretions were determined by ELISA (A) and numbers of IgA AFC in mucosal tissues by ELISPOT assay (B) following intranasal immunization with OVA combined with mCT S61F or nCT as mucosal adjuvants. Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with 5 micrograms of rCT-B (123), 0.5 microgram of nCT (IH), or 5 micrograms of mCT, S61F (■) on days 0, 7 and 14. Tissue samples and external secretions were taken 1 week after the last immunization. Bars represent the mean Ab titer or numbers of AFC ± 1 SE in each group. Each group contained 5 mice and the data are representative of three separate experiments. Anti-OVA IgA Abs were seen in nasal and vaginal washes, saliva and fecal extracts of mice immunized with OVA and mCT or nCT as adjuvants, while OVA alone or OVA plus rCT-B failed to elicit detectable IgA anti-OVA responses in any mucosal secretion

(Fig. 7A). These results were consistent with AFC analyses which demonstrated significant numbers of OVA-specific IgA AFC in these mucosal effector tissues from mice given OVA and mCT or nCT, whereas AFCs were not seen in mice given OVA alone or OVA together with rCT-B (Fig. 7B). To demonstrate that mCT S61F is an effective adjuvant for more conventional vaccines, groups of mice were also immunized intranasally with tetanus toxoid (TT) or with influenza virus mixed with mCT, nCT or rCT-B. In these studies, the mCT as well as nCT enhanced Ab responses to TT and to influenza virus. Ag-specific IgG, IgA and IgM Ab responses in serum and Ag- specific IgA Ab responses in mucosal secretions were significantly enhanced in mice given mCT or nCT as adjuvants (Table 3). Furthermore, single cell analyses showed that significant numbers of Ag-specific IgG and IgA AFC in spleen, CLN and lung tissues and Ag-specific IgA AFC in mucosal sites of mice immunized with mCT or nCT as adjuvants. On the other hand, rCT-B failed to act as adjuvant for co-administrated TT or influenza virus (Table 3).

Table 3. Antibody Responses in Serum and in Mucosal Secretions Following Intranasal Immunization of Mice with TT or Influenza Virus and mCT S61F or nCT as Mucosal Adjuvants

Serum Isotypes % Mucosal l lgAj (reciprocal log₂ titer) (reciprocal . log₂ titer)

Antigen Adjuvant* IgA IgM IgG Nasal Saliva Fecal Extract Vaginal Used* Wash Wash

None 7 9 10 3 4 <3 <3

TT rCT-B 7 9 11 4 4 <3 <3 nCT 10 14 17 8 7 7 6

S61F 11 13 18 9 8 8 5

None 9 10 13 8 9 7 <3

Influenza rCT-B 10 10 13 8 9 7 <3

Virus nCT 13 12 18 13 10 10 7

S61F 12 13 19 14 11 10 7

*Each group comprised 5 mice and the results are representative of two separate experiments.

⁺ Ag-specific Ab titers in serum and in mucosal secretions on day 21 were determined by endpoint ELISA.

A remarkable characteristic of CT is that in addition to adjuvant properties, it possesses strong immunogenicity, especially to the CT-B component. In this regard, mice immunized with OVA together with mCT or nCT showed high CT-B-specific IgG, IgA and IgM Ab responses (Fig. 5 A), and IgGl and IgG2b subclass anti-CT-B Ab responses in serum (Fig. 5B). Anti- CT-B Ab responses were also seen in mice given rCT-B, but were lower in magnitude when compared with mCT or nCT, even though a higher dose of rCT-B was used. Single cell analyses revealed significant numbers of CT-B- specific IgG and IgA AFC in spleen, CLN and lung tissue cell isolates from mice co-immunized with OVA plus mCT or nCT, with lower numbers of AFC in mice given rCT-B (Fig. 6). CT-B specific IgA Ab responses were also elevated in mucosal secretions, i.e., nasal and vaginal washes, saliva and fecal extracts of mice given mCT or nCT (Fig. 7).

Detection of total and Ag-specific IgE in serum. Two sensitive assays were used to detect increased total serum IgE as well as Ag-specific IgE Abs in mice given mCT or nCT as adjuvants. Total IgE levels were determined by ELISA using mouse IgE mAb (clone 27-74, PharMingen, San Diego, CA) as standard. For primary and secondary Abs, rat anti-mouse IgE mAb (clone R35- 72, PharMingen) and biotinylated rat anti-mouse IgE mAb (clone R35-92, PharMingen) were employed, respectively. Ag-specific serum IgE was detected by a modified IgE-capture luminometric assay as described in Example 2. Light development was carried out in a dynatech ML-3000 luminometer by injection of Ca²⁺ buffer (50 mM Tris, 20 mM calcium acetate, pH 7.5). Endpoint titers were determined as the dilution of each sample showing a 2-fold higher level of luminometric units above background.

IgE levels peaked 2 weeks after the initial immunization. Differences in total and OVA-specific IgE levels were noted between the groups given mCT and nCT as adjuvants, but the differences were not significant. CT-B-specific IgE levels in mice given mCT were significantly lower than seen with nCT (Table 4). Table 4. Serum IgE Responses Induced by Intranasal Immunization with OVA and Either mCT S61F or nCT as Mucosal Adjuvants.

Ag specific-IgE (reciprocal log₂ titer)

Treatment* Group Total IgE^f (ng/ml) OVA CT-B

OVA alone 186 ± 108 3.99 ± 1.25 <3

OVA +rCT-B 159 ± 83 3.63 ± 0.51 <3

OVA + nCT 1094 ± 237 9.14 ± 0.79 6.22 ± 0.84

OVA + S61F 768 ± 218 8.10 ± 1.05 4.24 ± 0.88 *

* Each group contained 5 mice and the results are representative of three separate experiments. t Serum IgE reached maximum levels at two weeks following the initial immunization and were determined by ELISA (for total IgE) or by IgE-capture luminometric assay (for Ag-specific IgE).

% Significantly lower when compared with nCT (p<0.05).

OVA- and CT-B-specific CD4+ T cell responses. Single cell suspensions were isolated from lung tissues and spleen in complete medium (RPMI 1640 supplemented with 10 ml/L of nonessential amino acids solution, 1 mM sodium pyruvate, 10 mM N-[2-hydroxyethyl]piperazine-N'-[2- ethanesulfonic acid] (HEPES), 100 Units/ml penicillin, 100 micrograms/ml streptomycin, 40 micrograms/ml gentamicin and 10% fetal calf serum (FCS). Isolated cells were fractionated on a nylon wool column for 1 hour at 37° C to remove adherent cells. The CD4⁺ T cell subset was the obtained by positive- sorting using a magnetic bead separation system consisting of biotinylated anti- CD4 mAb (clone GKl .5) and streptavidin microbeads (MACS, Miltenyi Biotec Inc., Sunnyvale, CA) (22). Purified splenic CD4⁺ T cells (> 98% purity) were cultured at a density of 2 x IO⁶ cells/ml with OVA (1 mg/ml) or with CT-B- coated beads (IO⁷ beads/ml). The cultures also contained T cell-depleted, irradiated (3000 rads) splenic feeder cells (2.5 x 10⁶ cells/ml) and IL-2 (10 Units/ml) (PharMingen) in complete medium. Purified CD4⁺ cells from nonimmunized mice were stimulated with solid-phase anti-CD3 mAb (145- 2C11) or with the antigens used in the intranasal immunization as positive or negative controls, respectively.

To measure Ag-specific CD4⁺ T cell proliferative responses, 100 microliters of culture in 96-well culture plates (Corning Glass Works, Corning, NY) were incubated at 37° C in 5% CO₂ and 10 microliters of 50 microCurie/ml [methyl-³H]-thymidine was added to each well 18 hours before termination. Cells were harvested onto glass fiber filters for measurement of radioactivity by liquid scintillation counting after 96 hours of culture. To determine cytokine production by Ag-specific CD4⁺ T cells, 1 ml of culture was incubated in 24-well cell culture plates (Coster Corp., Cambridge, MA) at 37° C in 5% CO₂ and cells were harvested after 48 hours of culture for quantitative reverse transcriptase-PCR (RT-PCR) analysis of cytokine-specific mRNA. Supernatants were collected after 96 hours for evaluation of cytokines by ELISA. For IL-2 analysis by ELISA, supernatants from 48 hour cultures were used. Detection of cytokines by ELISA. Cytokines in culture supernatants were determined by ELISA (6, 7). Briefly, Nunc-ImmunoMaxiSorp™ plates were coated with 2.5 micrograms/ml of rat anti-mouse IFN-γ, IL-2, IL-4, IL-5, IL-6 and IL-10 mAb (PharMingen) as in Example 3. Serial dilutions of culture supernatants or standard cytokines (PharMingen) were added in duplicate. For secondary Ab and detection enzymes, 0.2 micrograms/ml of biotinylated rat anti- mouse cytokine mAb (PharMingen) and 1.40000 diluted horseradish peroxidase- labeled anti-biotin (Vector Laboratories, Burlingame, CA) were employed as in Example 3. The ELISA assays were capable of detecting 20 picograms/ml for IFN-γ, 0.1 Unit ml for IL-2, 10 picograms/ml for IL-4, 2 Unit/ml for IL-5, 1 nanogram/ml for IL-6 and 0.5 nanogram/ml for IL-10.

Quantitative RT-PCR analysis of cytokine-specific mRNA. Cytokine- specific RT-PCR and quantitative analysis of RT-PCR products by capillary electrophoresis were done as described in Example 3 with minor modifications. Briefly, total RNA was isolated by the acid guanidinium thiocyanate phenol chloroform extraction procedure and subjected to standard RT-PCR. RT products with a series of diluted recombinant DNA internal standards were amplified by PCR and quantitative analysis of RT-PCR products was conducted by capillary electrophoresis with a laser-induced fluorescence detection system (LIF-P/ACE, Beckman Instruments).

Figure 8 shows OVA- and CT-B-specific CD4⁺ T cell proliferative responses isolated from lung (A) and spleen (B). Groups of C57BL/6 mice were immunized with 100 micrograms of OVA alone (□) or together with

10 micrograms of rCT-B (EJ), 0.5 microgram of nCT (H), or 5 micrograms of mCT, S61F (■) on days 0, 7 and 14. Purified CD4⁺ cells were cultured at a density of 2 ^χl0⁶ cells/ml in the presence of 1 mg/ml of OVA or 10⁷/ml of CT- B-coated beads, T cell depleted, irradiated splenic feeder cells (2.5 x IO⁶ cells/ml) and IL-2 (10 U/ml). Bars represent the mean stimulation index ± 1 SE and each group contained 5 mice. The data were similar and are representative of four separate experiments. Culture of CD4⁺ T cells from lung tissues or spleen with either OVA or CT-B resulted in significant proliferative responses clearly indicating the presence of both OVA-specific and CT-B- specific CD4⁺ Th cells in mice which had received intranasal administration of either mCT or nCT (Fig. 8).

Figure 9 shows the results of assessment of cytokine production form OVA-specific CD4⁺ T cells isolated from lung tissue. Molecules of cytokine- specific mRNA were determined by quantitative RT-PCR using recombinant internal standards. Cytokine protein production was determined by ELISA. The scale of each figure corresponds to mRNA molecules and protein levels produced by nonimmunized CD4⁺ T cells stimulated with anti-CD3 mAb. ND indicates not detected. Bars represent the mean cytokine profile ± 1 SE in each group. The data are representative of four separate experiments. It is apparent that OVA-specific CD4⁺ T cells from lung tissues of mice given OVA and mCT released high levels of Th2-type cytokines (IL-4, IL-5, IL-6 and IL10) into the culture. These responses were comparable to those obtained when nCT was used as adjuvant; however, cytokine responses produced by CD4⁺ T cells from mice given OVA alone or OVA plus rCT-B were detectable but considerably lower than observed with mCT or nCT. Cytokine-specific mRNA was examined by quantitative RT-PCR in RNA extracts from OVA-specific CD4⁺ T cell cultures. Again, Th2-type cytokine-specific mRNA was readily detected in OVA-specific CD4⁺ T cells. Again, Th2-type cytokine-specific mRNA was readily detected in OVA-specific CD4⁺ T cells from lung tissues of mice given OVA with mCT or nCT as mucosal adjuvants. Much lower levels of these cytokines were noted in cultures from mice given OVA alone or OVA plus rCT-B. On the other hand, Thl -type cytokines (IFN-γ and IL-2) were detectable at low levels in all samples including controls from nonimmunized mice. CT-B-specific CD4⁺ T cells from lung tissues of mice given OVA plus mCT or nCT also exhibited high levels of Th2-type and low levels of Thl -type cytokines. Splenic OVA- or CT-B-specific CD4⁺ T cells from mice given mCT or nCT also showed high levels of Th2-type cytokines by both ELISA and quantitative RT-PCR.

Results are reported as mean ± one standard error (SE). Statistical significance (p < 0.05) was determined by Student's t test and by the Mann- Whitney U test of unpaired samples.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The UAB Research Foundation

(ii) TITLE OF INVENTION: USE OF CHOLERA TOXIN MUTANTS AS MUCOSAL ADJUVANTS

(iii) NUMBER OF SEQUENCES: 7

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Schwegman, Lundberg, Woessner & Kluth, P. A.

(B) STREET: 121 South Eigth Street, 1600 TCF Tower

(C) CITY: Minneapolis

(D) STATE: MN

(E) COUNTRY: USA

(F) ZIP: 55402

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: Windows 95

(D) SOFTWARE: FastSEQ for Windows Version 2.0b

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unknown

(B) FILING DATE: 03-APR-1998

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/043410

(B) FILING DATE: 04-APR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Embretson, Janet E

(B) REGISTRATION NUMBER: 39,665

(C) REFERENCE/DOCKET NUMBER: 557.004WO1

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 612-373-6959

(B) TELEFAX: 612-339-3061

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 240 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Asn Asp Asp Lys Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp Glu lie

1 5 10 15

Lys Gin Ser Gly Gly Leu Met Pro Arg Gly Gin Ser Glu Tyr Phe Asp

20 25 30

Arg Gly Thr Gin Met Asn lie Asn Leu Tyr Asp His Ala Arg Gly Thr

35 40 45

Gin Thr Gly Phe Val Arg His Asp Asp Gly Tyr Val Ser Thr Ser lie

50 55 60

Ser Leu Arg Ser Ala His Leu Val Gly Gin Thr lie Leu Ser Gly His 65 70 75 80

Ser Thr Tyr Tyr lie Tyr Val lie Ala Thr Ala Pro Asn Met Phe Asn

85 90 95

Val Asn Asp Val Leu Gly Ala Tyr Ser Pro His Pro Asp Glu Gin Glu

100 105 110

Val Ser Ala Leu Gly Gly lie Pro Tyr Ser Gin lie Tyr Gly Trp Tyr

115 120 125

Arg Val His Phe Gly Val Leu Asp Glu Gin Leu His Arg Asn Arg Gly

130 135 140

Tyr Arg Asp Arg Tyr Tyr Ser Asn Leu Asp lie Ala Pro Ala Ala Asp 145 150 155 160

Gly Tyr Gly Leu Ala Gly Phe Pro Pro Glu His Arg Ala Trp Arg Glu

165 170 175

Glu Pro Trp lie His His Ala Pro Pro Gly Cys Gly Asn Ala Pro Arg

180 185 190

Ser Ser Met Ser Asn Thr Cys Asp Glu Lys Thr Gin Ser Leu Gly Val

195 200 205

Lys Phe Leu Asp Glu Tyr Gin Ser Lys Val Lys Arg Gin lie Phe Ser

210 215 220

Gly Tyr Gin Ser Asp lie Asp Thr His Asn Arg lie Lys Asp Glu Leu 225 230 235 240

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 240 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Asn Gly Asp Arg Leu Tyr Arg Ala Asp Ser Arg Pro Pro Asp Glu lie

1 5 10 15

Lys Arg Ser Gly Gly Leu Met Pro Arg Gly His Asn Glu Tyr Phe Asp

20 25 30

Arg Gly Thr Gin Met Asn lie Asn Leu Tyr Asp His Ala Arg Gly Thr

35 40 45

Gin Thr Gly Phe Val Arg Tyr Asp Asp Gly Tyr Val Ser Thr Ser Leu

50 55 60

Ser Leu Arg Ser Ala His Leu Ala Gly Gin Ser lie Leu Ser Gly Tyr 65 70 75 80

Ser Thr Tyr Tyr lie Tyr Val lie Ala Thr Ala Pro Asn Met Phe Asn 85 90 95

Val Asn Asp Val Leu Gly Val Tyr Ser Pro His Pro Tyr Glu Gin Glu

100 105 110

Val Ser Ala Leu Gly Gly lie Pro Tyr Ser Gin lie Tyr Gly Trp Tyr

115 120 125

Arg Val Asn Phe Gly Val lie Asp Glu Arg Leu His Arg Asn Arg Glu

130 135 140

Tyr Arg Asp Arg Tyr Tyr Arg Asn Leu Asn lie Ala Pro Ala Glu Asp 145 150 155 160

Gly Tyr Arg Leu Ala Gly Phe Pro Pro Glu His Gin Ala Trp Arg Glu

165 170 175

Glu Pro Trp lie His His Ala Pro Asn Gly Cys Gly Asn Ser Ser Arg

180 185 190

Thr lie Thr Gly Asp Thr Cys Asn Glu Glu Thr Gin Asn Leu Ser Thr

195 200 205 lie Tyr Leu Arg Glu Tyr Gin Ser Lys Val Lys Arg Gin lie Phe Ser

210 215 220

Asp Tyr Gin Ser Glu Val Asp lie Tyr Asn Arg lie Arg Asp Glu Leu 225 230 235 240

(2) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 103 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Thr Pro Gin Asn lie Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gin

1 5 10 15 lie His Thr Leu Asn Asp Lys lie Phe Ser Tyr Thr Glu Ser Leu Ala

20 25 30

Gly Lys Arg Glu Met Ala lie lie Thr Phe Lys Asn Gly Ala Thr Phe

35 40 45

Gin Val Glu Val Pro Gly Ser Gin His lie Asp Ser Gin Lys Lys Ala

50 55 60 lie Glu Arg Met Lys Asp Thr Leu Arg lie Ala Tyr Leu Thr Glu Ala 65 70 75 80

Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala lie

85 90 95

Ala Ala lie Ser Met Ala Asn 100

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 103 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Ala Pro Gin Thr He Thr Glu Leu Cys Ser Glu Tyr His Arg Thr Gin

1 5 10 15

He Tyr Thr He Asn Asp Lys He Leu Ser Tyr Thr Glu Ser Met Ala

20 25 30

Gly Lys Arg Glu Met Val He He Glu He Lys Ser Gly Glu Thr Phe

35 40 45

Gin Val Glu Val Pro Gly Ser Gin His He Asp Ser Gin Lys Lys Ala

50 55 60

He Glu Arg Met Lys Asp Thr Leu Arg He Thr Tyr Leu Thr Glu Thr 65 70 75 80

Lys He Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn Ser He

85 90 95

Ala Ala He Ser Met Lys Asn 100

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 103 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Pro Gin Ser He Thr Glu Leu Cys Ser Glu Tyr His His Thr Gin

1 5 10 15

He Tyr Thr He Asn Asp Lys He Leu Ser Tyr Thr Glu Ser Met Ala

20 25 30

Gly Lys Arg Glu Met Val He He Thr He Lys Ser Gly Ala Thr Phe

35 40 45

Gin Val Glu Val Pro Gly Ser Gin His He Asp Ser Gin Lys Lys Ala

50 55 60

He Glu Arg Met Lys Asp Thr Leu Arg He Thr Tyr Leu Thr Glu Thr 65 70 75 80

Lys He Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn Ser He

85 90 95

Ala Ala He Ser Met Glu Asn 100

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : GGATATGTTT TTACCTCAAT T 21 (2) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Other

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : GATGAACAAA AAGTTTCTGC T 21

Claims

WHAT IS CLAIMED IS:

1. A mutant cholera toxin selected from the group consisting of S61 F,

El 12K, a nontoxic subunit of S61F, a nontoxic subunit of El 12K, a nontoxic derivative of S61 F and a nontoxic derivative of E 112K, wherein the nontoxic subunit or derivative is effective as an adjuvant when co- administered to a bird or mammal in combination with an immunogenic amount of antigen.

2. The mutant cholera toxin of claim 1 which is S61F or El 12K.

3. The mutant cholera toxin of claim 1 which is S61F.

4. The mutant cholera toxin of claim 1 which is El 12K.

5. A vaccine comprising (i) the mutant cholera toxin of claim 1 or 2, (ii) an immunogenic amount of an antigen, and (iii) a pharmaceutically acceptable carrier.

6. The vaccine of claim 5 formulated for mucosal administration.

7. The vaccine of claim 6 formulated for intranasal or oral administration.

8. The vaccine of claim 5 formulated for human administration.

9. A method comprising administering to a human a mucosal adjuvant comprising the mutant cholera toxin of claim 1 or 2.

10. A method comprising administering to a human the vaccine of claim 5 or 6.