WO2010090873A2 - Polypeptides and their use as adjuvants - Google Patents

Abstract

This document provides methods and materials relating to a B subunit polypeptide from a Type II heat-labile enterotoxin that contain at least one amino acid modifications. Methods and materials for stimulating the immune system of a mammal are also provided.

Description

POLYPEPTIDES AND THEIR USE AS ADJUVANTS

CROSS-REFERENCE TO RELATED APPLICATION

This application is based on, and claims the benefit of, U.S. Provisional Application No. 61/146,088 filed on January 21, 2009, which is incorporated by reference herein in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The U.S. Government has certain rights in this invention pursuant to Grant Nos. DE017138 and DE13833 awarded by the National Institutes of Health.

BACKGROUND

1. Technical Field

This document provides methods and materials relating to a B subunit polypeptide from a Type II heat-labile enterotoxin that contains one or more amino acid modifications.

2. Background Information

Heat-labile enterotoxins such as cholera toxin from Vibrio cholerae and Type I and Type II toxins from Escherichia coli have been studied for their immunostimulatory properties and for their potential to function as adjuvants in vaccines. Each of these heat-labile enterotoxins displays AB5 oligomeric structure, in which an enzymatically toxic subunit (A) is linked to a pentameric ganglioside- binding subunit (B₅). The catalytic moiety is at the N-terminal segment (Al), whereas the C-terminal segment (A2) acts as a non-covalent linker to the central pore of the doughnut-shaped pentamer of B polypeptides. Although enterotoxins such as cholera toxin and the Type II E. coli toxin are potent mucosal adjuvants, their intrinsic enterotoxicity has precluded their use in therapeutic applications.

U.S. Publication No. 2006/0182765 describes the adjuvant activity of B subunit pentamers. The present disclosure identifies a number of amino acid modifications within B subunit polypeptides from Type II heat-labile enterotoxins and describes the adjuvant activities of such polypeptides. SUMMARY

This document describes methods and materials for stimulating an immune response in a mammal. This document is based, in part, on the discovery of the immunostimulatory properties of isolated B subunit polypeptides of Type II heat- labile enterotoxins having one or more amino acid modifications. The modified B subunit polypeptides provided herein can be used as an adjuvant to stimulate a mammal's immune response to an antigen.

In one aspect, a purified B subunit polypeptide from a type II heat-labile enterotoxin is provided that has at least one amino acid modifications. The amino acid modification can be at residue 69, 70, 73, and 74 numbered relative to residues 24-123 of SEQ ID NO: 1. In various embodiments, a Met at position 69 can be replaced with a residue having greater hydrophobicity than Met, an Ala at position 70 can be replaced with a residue having greater hydrophobicity than Ala, a Leu at position 73 can be replaced with a residue having greater hydrophobicity than Leu, or a Ser at position 74 can be replaced with a residue having greater hydrophobicity than Ser. For example, the Met at position 69 can be replaced with a VaI, He, Leu, Phe, Trp, or Cys; the Ala at position 70 can be replaced with an He, Leu, Phe, Trp, or Cys; the Leu at position 73 can be replaced with a VaI, He, Met, Phe, Trp, or Cys; or the Ser at position 74 can be replaced with an Ala, He, VaI, Leu, Met, Phe, Trp, or Cys.

The modified B subunit polypeptides described herein further can include an amino acid modification at position 22, 75, and/or 99 numbered relative to residues 24-123 of SEQ ID NO: 1. For example, the GIu at position 22 can be replaced with a VaI, He, Leu, Phe, Trp, or Cys, the GIy at position 75 can be replaced with an Ala, He, VaI, Leu, Met, Phe, Trp, or Cys; and/or the GIu at position 99 can be replaced with a VaI, He, Leu, Phe, Trp, and Cys. Representative modified B subunit polypeptides can have the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

In another aspect, isolated nucleic acids encoding the modified B subunit polypeptides described herein are provided.

In another aspect, a pentamer of B subunit polypeptides from a type II heat- labile enterotoxin is provided where at least one of the polypeptides in the pentamer is a modified B subunit polypeptide as described herein. Such a pentamer can exhibit immunostimulatory activity and can be used as an adjuvant. In another aspect, a pharmaceutical composition is provided that comprises, or consists essentially of, a pharmaceutically acceptable carrier and a modified B subunit polypeptide as described herein or a pentamer containing at least one of the modified B subunit polypeptide as described herein. The pharmaceutical composition can further include an antigen. Representative antigens include, for example, Agl/II adhesion protein of Streptococcus mutans; saliva-binding region of Agl/II adhesion protein of Streptococcus mutans; PspA and PspC virulence proteins of Streptococcus pneumoniae; fimbriae of P orphyromonas gingivalis; lectin antigen of Entamoeba histolytica; P6 surface protein of nontypeable Haemophilus influenza; HBsAg of Hepatitis B virus; and TbpA and TbpB transferrin-binding proteins of ^'Neisseria gonorrhoeae.

In another aspect, methods of stimulating a mammal's immune response to an antigen are provided. Such methods can include administering, to the mammal, a) a modified B subunit polypeptide as described herein with an antigen, b) a pentamer of B subunit polypeptides including at least one modified B subunit polypeptide as described herein with an antigen, c) or a pharmaceutical composition as described herein. The method can comprise administration to a mucosal surface. Representative mucosal surfaces include, without limitation, intranasal, oral, parenteral, rectal, or vaginal. A representative mammal is a human. The polypeptide, the pentamer or the pharmaceutical composition can be administered simultaneously with the antigen or sequentially with the antigen. In certain embodiments, the polypeptide, the pentamer or the pharmaceutical composition and the antigen are administered together in the same composition. Administration can be by a route such as intranasal, oral, gastrointestinal, rectal, vaginal, and genitourinary tract.

In another aspect, an article of manufacture is provided. The article of manufacture can include, or consist essentially of, one or more of the modified B subunit polypeptides described herein, a pentamer including at least one of the modified B subunit polypeptides described herein, or a pharmaceutical composition described herein, along with packaging material and written instructions for stimulating the immune response of a mammal.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

Figure 1 is graphs showing the binding activity of wild-type LT-IIb-Bs and upper region hydrophilic point mutants. Binding of wild type (WT) LT-IIb-Bs or upper-region point mutants (M69E, A70D, L73E, and S74D) was determined on microtiter wells coated with mouse or human TLRs as indicated (A,B and D,E) or with (C) GDIa ganglioside. The LT-IIb holotoxin and a lower-region point mutant of LT-IIb-Bs (T 131) were used as negative controls in the TLR (A,B and D,E) and GDIa (C) binding assays, respectively. All ligands were used at 10 μg/ml, and bound protein was detected colorimetrically after probing with anti-LT-IIb antibody, followed by addition of peroxidase-conjugated secondary antibody. Data are shown as means ± standard deviations (SD) (n = 3) from one of three independent sets of experiments that yielded similar results. Asterisks denote statistically significant (p < 0.05) binding compared to negative controls, and black circles in (D) and (E) indicate significantly higher (p < 0.05) binding by WT compared to all upper-region point mutants.

Figure 2 is graphs showing NF-κB activation by wild-type LT-IIb-Bs and upper region hydrophilic point mutants. Wild-type (WT) LT-IIb-Bs and indicated point mutants (all at 10 μg/mL) were tested for their capacity to activate NF -KB in reporter THP-I -BLUE™ cells in a TLR2- or TLRl -dependent way (A and B, respectively). Prior to stimulation, the cells were pretreated for 30 minutes with (A) anti-TLR2 or (B) anti-TLRl (or other anti-TLR antibodies, as indicated, for control purposes). Activation was determined colorimetrically by measuring the activity of NF-κB-inducible alkaline phosphatase secreted in the culture supernatants upon 24- hour incubation. PaHi₃CSK₄, LPS, and FSL-I (which activate TLR2/1, TLR4, and TLR2/6, respectively) were used for monitoring the specificity of the blocking antibodies. Results are presented as means ± SD (n = 3) from one of two independent sets of experiments yielding similar findings. Asterisks indicate statistically significant (p < 0.05) activation of NF-κB compared to no-agonist control and black circles show significant (p < 0.05) inhibition of activation.

Figure 3 is graphs showing antigen-presenting cell activation by wild-type LT- IIb-B5 and upper region hydrophilic point mutants. Mouse peritoneal macrophages (A-C) or BMDC (D-I) were stimulated for 18-20 hours with wild-type (WT) LT-IIb- B5 or the indicated point mutants (10 μg/mL). Induction of release of the indicated cytokines (A-F) in culture supernatants was measured by ELISA. Data are means ± SD (n = 3) from one of three independent sets of experiments yielding similar findings, and asterisks indicate significantly (p < 0.05) higher cytokine production compared to medium-only control. Up-regulation of the indicated co-stimulatory molecules was assayed by FACS and is reported as mean fluorescent intensity (MFI) (G-I); the data are representative of five independent experiments yielding similar results. Figure 4 is a graph showing the effect of TLRl alterations on cooperative

TLR2-dependent cell activation by LT-IIb-B₅. Replacement of up to the first eight N- terminal LRRs (construct [T6(l-8)/Tl]/T2) had a relatively minor effect, but the additional replacement of the subsequent four LRRs (construct [T6(1-12)/T1]/T2) completely abrogated the response. Replacement of up to the first eight N-terminal LRRs of TLR6 with those of TLRl (construct [T 1 (l-8)/T6]/T2) was incapable to rescue LT-IIb-B₅-induced cell activation.

Figure 5 is graphs showing the effect of TLRl point mutations on cell activation by LT-IIb-B₅. SW620 cells were co-transfected with human TLR2 and TLRl (the latter in wild type or point mutant versions, as indicated), as well as with a firefly luciferase reporter gene and a Renilla transfection control. After 48 hours, the cells were stimulated for 6 hours with (A) LT-IIb-B₅ (2 μg/mL) or (B) Pam3CSK4 (20 ng/mL). Cellular activation is reported as relative luciferase activity, normalized to that of cells transfected with reporter and empty vectors. Results are presented as means ± SD (n = 3) and asterisks indicate activities that are significantly (p < 0.05) lower compared to transfectants that received wild-type (WT) TLRl .

Figure 6 is graphs showing agonist dose- and cell dose-dependent cytokine induction by LT-IIb-B₅ in bone marrow-derived dendritic cells ("BMDC"). BMDC were stimulated with LT-IIb-B₅ (A-D) or a TLR2-nonbinding mutant (S74D) (C-D). Pam3Cys (100 ng/niL) and LPS (100 ng/mL) were used as positive controls (C-D). Induction of TNF-α (A,C) or IL-6 (B,D) production in culture supernatants was measured by ELISA. (A) and (B) show agonist dose-dependent cytokine induction, whereas (C) and (D) show BMDC dose-dependent cytokine induction. Asterisks indicate significantly (p < 0.05) higher cytokine production by stimulation compared to no agonist stimulation. Data are means ± standard deviations (n = 3) from one of three independent sets of experiments yielding similar results. Asterisks indicate significantly (p < 0.05) enhanced cytokine production compared to medium-only stimulation. Figure 7 is graphs showing the mucosal adjuvanticity of the B pentameric subunit of the holotoxin (LT-IIb-Bs). Groups of BALB/c mice were intranasally immunized with S. mutans Agl/II in the absence or presence OfLT-IIb-B₅ or intact LT-IIb adjuvant at days 1, 15, and 29. Antibody responses (salivary IgA (A), vaginal IgA (B), serum IgA (C), and serum IgG (D)) were monitored at days 22, 36, and 50. Data are means ± standard deviations (n = 6). Black circles indicate statistically significant (p < 0.05) enhancement of antibody responses compared to sham immunization, whereas asterisks show significant (p < 0.05) enhancement of antibody responses compared to Agl/II ("Ag") alone.

Figure 8 is a graph showing competitive inhibition of LT-IIb-B₅ binding to TLR2 by PaHi₃CSK₄. Binding of biotinylated LT-IIb-B₅ (0.16 μM) to plate-bound TLR2 in the presence of increasing concentrations of unlabeled LT-IIb-B₅ or Pani3CSK4. Bound protein was probed with peroxidase-conjugated streptavidin and binding was determined colorimetrically. The data (means ± SD; n = 3) were normalized to the binding activity of labeled LT-IIb-B₅ in the presence of 10 mg/mL BSA only (uninhibited control), the mean activity of which was taken as 100.

Asterisks indicate significant inhibition of binding (p < 0.05), and black circles denote significant differences between the unlabeled competitors (p < 0.05).

Figure 9 is a graph showing the effect of hydrophobic point mutants of LT- Hb-B₅. Binding of wild-type (WT) LT-IIb-B₅ or upper-region point "enhanced hydrophobic" mutants (S74I and S74A) was determined on microtiter wells coated with the indicated TLRs. The LT-IIb holotoxin and medium only ("no-agonist") were used as negative controls (binding below the discontinuous horizontal line is considered non-specific). All ligands were used at 10 μg/mL, and bound protein was detected colorimetrically after probing with anti-LT-IIb antibody, followed by the addition of a peroxidase-conjugated secondary antibody. Data are shown as means ± standard deviations (n = 3).

Figure 10 is a sequence alignment of the LT-IIa, LT-IIb, and LT-IIc B subunit polypeptides (including signal peptides). The sequences of the B subunit polypeptides of LT-IIa, LT-IIb, and LT-IIc, which are set forth in SEQ ID NO:4, SEQ ID NO:1, and SEQ ID NO:5, respectively, were aligned using the CLUSTAL 2.0.10 multiple sequence alignment program. Amino acids which are conserved in all three B polypeptides are denoted by "*". The amino acids at positions 13, 14, 34, and 92 (numbered relative to the mature B polypeptide (i.e., the mature polypeptide corresponds to residues 24-123 of SEQ ID NO: I)) which, in LT-IIa-B and LT-IIb-B, are essential for ganglioside receptor binding, are noted with "+". The domain which, in LT-IIa was genetically shown to be essential for interacting with TLR2 and which is conserved in LT-IIb and LT-IIc, is indicated with a line above the sequence. The likely cleavage site for signal peptidase I in all three B polypeptides is noted with a "/". LT-IIc-B shows less overall homology to either LT-IIa-B or LT-IIb-B, but LT- Hc-B has certain regions that are highly conserved and include residues important to ganglioside binding and TLR2 association.

Figure 11 is a graph showing enhanced binding of hydrophobic point mutants OfLT-IIb-B₅ to TLR2 and TLRl . Binding of wild-type (WT) LT-IIb-B₅ or upper- region hydrophobic point mutants (S74A and S74I) was determined on microtiter wells coated with TLR2 or TLRl . The LT-IIb holotoxin was used as a negative control. All ligands were used at 10 μg/mL, and bound protein was detected colorimetrically after probing with anti-LT-IIb antibody. Data are shown as means ± SD. * = statistically significant (p < 0.01) enhancement of binding compared to WT.

DETAILED DESCRIPTION

This document describes methods and materials relating to B subunit polypeptides from Type II heat-labile enterotoxins having at least one amino acid modification. A pentamer that contains at least one such modified B subunit polypeptide typically exhibits increased immunostimulatory activity compared to a pentamer of wild type B subunit polypeptides. Therefore, B subunit polypeptides of a heat-labile enterotoxin having at least one amino acid modification are provided, as are pentamers that contain at least one of such polypeptides and methods of making and using such polypeptides and pentamers. In addition, this document describes administering such polypeptides or pentamers to a mammal to stimulate the mammal's immune response to an antigen.

Polypeptides and Nucleic Acids

The B subunit polypeptides of the Type II family of E. coli enterotoxins are classified into Type Ha ("LT-IIa-B"), Type lib ("LT-IIb-B"), and Type Hc ("LT-IIc- B") B polypeptides. See, for example, Nawar et at., 2007, Infect. Immun., 75:621-33; and U.S.2006/0182765. This document describes a B subunit polypeptide of a Type II heat-labile enterotoxin in which at least one amino acid residue has been modified relative to, for example, a wild type B subunit polypeptide. As used herein, a "wild type" polypeptide refers to a polypeptide having an amino acid sequence that is unmodified relative to a native amino acid sequence (e.g., the amino acid sequence encoding the mature B subunit polypeptide of Escherichia coli). Representative wild- type B subunit polypeptides have the sequence set forth in SEQ ID NO: 1 (LT-IIb-B), SEQ ID NO:4 (LT-IIa-B), and SEQ ID NO:5 (LT-IIc-B). The term "polypeptide" as used herein refers to a polymer of three or more amino acids covalently linked by amide bonds. As described herein, an amino acid modification generally refers to a replacement of the amino acid at the target position with an amino acid that has greater hydrophobicity, although in some instances, an amino acid at the target position may be replaced with an amino acid that has less hydrophobicity (i.e., is more hydrophilic).

Hydrophobicity is a measure of the solubility of each amino acid in water and can be represented using a hydrophobicity index, in which glycine is considered neutral and given a value of 0 and phenylalanine is considered the most hydrophobic and given a value of 100, while aspartic acid is considered the least hydrophobic (i.e., the most hydrophilic) and given a value of -55. Table 1 shows the hydrophobicity index of each amino acid (adapted from Monera et al, J. Protein Sci., 1 :319-329 (1995); the scale was extrapolated for residues that are less hydrophobic than glycine (i.e., hydrophilic residues)). Table 1. Hydrophobicity Index for Amino Acid Residues

The modified B subunit polypeptides provided herein typically contain at least one amino acid modification at position 69, 70, 73, or 74 (relative to the mature B subunit polypeptide (i.e., corresponding to residues 24-123 of SEQ ID NO: I)) that replaces the wild type amino acid with an amino acid that has greater hydrophobicity. For example, wild type mature B subunit polypeptides have a Met, Ala, Leu, and Ser at position 69, 70, 73, and 74, respectively. Therefore, modified B subunit polypeptides are provided that contain an amino acid at position 69 that has greater hydrophobicity than Met. According to Table 1, amino acids that have a greater hydrophobicity than Met include, for example, VaI, Leu, Trp, He, and Phe. Similarly, modified B subunit polypeptides are provided that contain an amino acid at position 70 that has greater hydrophobicity than Ala (e.g., Cys, Leu, Trp, He, and Phe); an amino acid at position 73 that has greater hydrophobicity than Leu (e.g., Trp, He, and Phe); an amino acid at position 74 that has greater hydrophobicity than Ser (e.g., Ala, Cys, Met, VaI, Leu, Trp, He, or Phe); or any combination of modifications thereof.

In addition to one or more modifications at residues 69, 70, 73, or 74, the modified B subunit polypeptides provided herein also can contain an amino acid modification at position 22, 75, or 99 (relative to the mature B subunit polypeptide (e.g., corresponding to residues 24-123 of SEQ ID NO: I)) that replaces the wild type amino acid with an amino acid that has greater hydrophobicity. For example, wild type LT-IIb-B has a GIu at position 22 while wild type LT-IIa-B and LT-IIc-B have a GIn at position 22; wild type LT-IIb-B has a GIy at position 75, wild type LT-IIa-B has a Asp at position 75, and wild type LT-IIc-B has a Asn at position 75; and wild type LT-IIb-B and wild type LT-IIa-B have a GIu at position 99. Wild type B polypeptides can be modified by replacing any of these residues with a residue that has greater hydrophobicity according to Table 1. It would be understood by those skilled in the art that, in addition to hydrophobicity, the size of the amino acid side chain needs to be considered when making any modification to avoid improper or lack of folding due to, for example, steric hindrance. Representative B subunit polypeptides having a modified amino acid sequence are shown, for example, in SEQ ID NO:2 and SEQ ID NO:3.

Amino acid residue modification generally occurs at the nucleic acid level. Modifications to a nucleic acid molecule include, without limitation, single or multiple nucleotide transitions (purine to purine or pyrimidine to pyrimidine) or transversions (purine to pyrimidine or vice versa) and single- or multiple-nucleotide deletions or insertions. Modifications can be generated in an isolated nucleic acid of the target polypeptide using any number of methods known in the art. For example, site-directed mutagenesis can be used to modify a nucleic acid sequence encoding a B subunit polypeptide from a Type II heat-labile enterotoxin. One of the most common methods of site-directed mutagenesis is oligonucleotide-directed mutagenesis. In oligonucleotide-directed mutagenesis, an oligonucleotide encoding the desired change(s) in sequence is annealed to one strand of the DNA of interest and serves as a primer for initiation of DNA synthesis. In this manner, the oligonucleotide containing the sequence change is incorporated into the newly synthesized strand. See, for example, Kunkel, Proc. Natl. Acad. ScL USA 82:488 (1985); Kunkel et al, Meth. Enzymol. 154:367 (1987); Lewis & Thompson, Nucl. Acids Res. 18:3439 (1990); Bohnsack, Meth. MoI. Biol. 57:1 (1996); Deng & Nickoloff, Anal. Biochem. 200:81 (1992); and Shimada, Meth. MoI. Biol. 57:157 (1996). In some cases, a polypeptide provided herein can be a substantially pure polypeptide. The term "substantially pure" as used herein with reference to a polypeptide means the polypeptide is substantially free of other polypeptides, lipids, carbohydrates, and nucleic acid with which it is naturally associated. Thus, a substantially pure polypeptide is any polypeptide that is removed from its natural environment and is at least 60 percent pure. A substantially pure polypeptide can be at least about 65, 70, 75, 80, 85, 90, 95, or 99 percent pure. Typically, a substantially pure polypeptide will yield a single major band on a non-reducing polyacrylamide gel. In some cases, purified preparations of B pentamers can contain negligible endotoxic activity (< 0.007 ng LPS/μg protein) according to quantitative Limulus amebocyte lysate assay kits (BioWhittaker, Walkersville, MD; or Charles River Endosafe, Charleston, SC).

Polypeptides provided herein can be produced by any number of methods well known in the art. By way of example and without limitation, a polypeptide can be obtained by expression of a recombinant nucleic acid encoding the polypeptide or by chemical synthesis (e.g., by solid-phase synthesis or other methods well known in the art, including synthesis with an ABI peptide synthesizer; Applied Biosystems, Foster City, CA). In some cases, expression vectors that encode polypeptides provided herein can be used to produce a polypeptide. For example, standard recombinant technology using expression vectors encoding a polypeptide provided herein can be used. Expression systems that can be used for small or large-scale production of the polypeptides provided herein include, without limitation, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the polypeptides provided herein. The resulting polypeptides can be purified. In some cases, suitable methods for purifying the polypeptides of the invention can include, for example, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography. The extent of purification can be measured by any appropriate method, including but not limited to: column chromatography, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. A polypeptide provided herein can be designed or engineered to contain a tag sequence that allows the polypeptide to be purified (e.g., captured onto an affinity matrix). For example, a tag such as c-myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino termini. Other fusions that can be used include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. This document also provides methods and materials related to isolated nucleic acid molecules encoding the polypeptides described herein. The term "nucleic acid" as used herein encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA. The nucleic acid can be double- stranded or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear.

The term "isolated" as used herein with reference to nucleic acid refers to a naturally-occurring nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5 ' end and one on the 3' end) in the naturally-occurring genome of the organism from which it is derived. For example, an isolated nucleic acid can be, without limitation, a recombinant DNA molecule of any length, provided one of the nucleic acid sequences normally found immediately flanking that recombinant DNA molecule in a naturally-occurring genome is removed or absent. Thus, an isolated nucleic acid includes, without limitation, a recombinant DNA that exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote. In addition, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

The term "isolated" as used herein with reference to nucleic acid also includes any non-naturally-occurring nucleic acid since non-naturally-occurring nucleic acid sequences are not found in nature and do not have immediately contiguous sequences in a naturally-occurring genome. For example, non-naturally-occurring nucleic acid such as an engineered nucleic acid is considered to be isolated nucleic acid. Engineered nucleic acid can be made using common molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally-occurring nucleic acid can be independent of other sequences, or incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), or the genomic DNA of a prokaryote or eukaryote. In addition, a non-naturally-occurring nucleic acid can include a nucleic acid molecule that is part of a hybrid or fusion nucleic acid sequence. It will be apparent to those of skill in the art that a nucleic acid existing among hundreds to millions of other nucleic acid molecules within, for example, cDNA or genomic libraries, or gel slices containing a genomic DNA restriction digest is not to be considered an isolated nucleic acid.

Any appropriate method including, without limitation, common molecular cloning and chemical nucleic acid synthesis techniques can be used to obtain isolated nucleic acid molecules. For example, isolated nucleic acids provided herein can be obtained using the polymerase chain reaction (PCR) and/or recombinant nucleic acid technology. PCR refers to a procedure or technique in which target nucleic acid is amplified in a manner similar to that described in U.S. Patent No. 4,683,195, and subsequent modifications of the procedure described therein. General PCR techniques are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond are used to design oligonucleotide primers that are identical or similar in sequence to opposite strands of a potential template to be amplified. Any appropriate oligonucleotide primer can be used.

In some cases, isolated nucleic acid molecules can be obtained by recombinant nucleic acid technology. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid molecule of the invention. Isolated nucleic acids of the invention also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides .

Methods well known to those skilled in the art may be used to subclone isolated nucleic acid molecules encoding B subunit polypeptides of interest into expression vectors containing relevant coding sequences and appropriate transcriptional/translational control signals. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, New York (1989). Expression vectors of the invention can be used in a variety of systems (e.g., bacteria, yeast, insect cells, and mammalian cells), as described herein. Examples of suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, herpes viruses, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. A wide variety of suitable expression vectors and systems are commercially available, including the pET series of bacterial expression vectors (Novagen, Madison, WI), the Adeno-X expression system (Clontech), the Baculogold baculovirus expression system (BD Biosciences Pharmingen, San Diego, CA), and the pCMV-Tag vectors (Stratagene, La Jolla, CA).

Adjuvants and Methods of Using

The modified B subunit polypeptides described herein and pentamers containing at least one such polypeptide can be used to generate immunostimulatory compositions. A composition described herein generally contains at least one modified B subunit polypeptide of a Type II heat-labile enterotoxin as described herein. In some cases, the composition can contain a pentamer of B subunit polypeptides in which at least one of the B subunit polypeptides in the pentamer is a modified B subunit polypeptide as described herein. For example, a pentamer can include a single modified B subunit polypeptide with the remainder of the polypeptides being wild-type (e.g., having the sequence shown in SEQ ID NOs: 1, 4, or 5) or a pentamer can include multiple (e.g., 2, 3, 4 or 5) modified B subunit polypeptides. If multiple modified B subunit polypeptides are present in a pentamer, such modified polypeptides can have the same modification(s) or can have different modifications.

A composition (e.g., a modified B subunit polypeptide or a pentamer that includes at least one modified B subunit polypeptides) can be used as an adjuvant to stimulate a mammal's immune response, typically against an antigen. As used herein, mammals include, without limitation, humans, monkeys, horses, cows, goats, dogs, cats, rabbits, rats, and mice. An adjuvant as described herein can be particularly useful when administering to the mucosal surfaces. Mucosal surfaces include, for example, intranasal, oral, parenteral, rectal, and vaginal surfaces and, accordingly, compositions can be administered by a route including intranasally, orally, gastrointestinally, rectally, vaginally, or via the genitourinary tract. Adjuvants described herein can be formulated using techniques and procedures well known in the art (see, e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition 1985, 126). Any antigen can be used with an adjuvant described herein, but representative antigens include, for example, the Agl/II adhesion protein of Streptococcus mutans; saliva-binding region of the Agl/II adhesion protein of Streptococcus mutans; PspA and PspC virulence proteins of Streptococcus pneumoniae; fimbriae of Porphyromonas gingivalis; lectin antigen of Entamoeba histolytica; P6 surface protein of nontypeable Haemophilus influenza; HBsAg of Hepatitis B virus; and TbpA and TbpB transferrin-binding proteins of Neisseria gonorrhoeae. Any appropriate ratio of adjuvant to antigen can be used. For example, the adjuvantantigen ratio can be 50:50 (vohvol). Alternatively, the adjuvantantigen ratio can be, without limitation, 90:10, 80:20, 70:30, 64:36, 60:40, 55:45, 40:60, 30:70, 20:80, or 90:10.

An adjuvant composition as described herein can be administered to a mammal at the same time (i.e., simultaneously) as the antigen is administered, or the adjuvant and an antigen can be administered to a mammal sequentially. When an adjuvant and an antigen are administered simultaneously, they can be administered as separate compositions or together in the same composition. In some cases, a composition (that may or may not include an antigen) can be administered with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with the intended route of administration. Pharmaceutical carriers suitable for administration of the compositions provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. For example, a pharmaceutically acceptable carrier can include a solid, semi-solid, or liquid material that acts as a vehicle, carrier, or medium for a modified B subunit polypeptide of a heat-labile enterotoxin. The use of such pharmaceutically acceptable carriers with compositions is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier is incompatible with the active compound, use thereof in any of the compositions described herein is contemplated.

Typically, an adjuvant is administered to a mammal such that the mammal produces a greater immune response toward an antigen compared to the immune response produced toward the antigen in the absence of the adjuvant. A suitable amount of an adjuvant is one that increases an immune response in a mammal but does not result in significant toxicity. A suitable amount of an adjuvant can depend on factors such as, without limitation, the route of administration; the nature of the composition; the weight of the mammal; the particular antigen, and the concurrent administration of other vaccines or drugs. A suitable amount of an adjuvant can be established by one of ordinary skill in the art through routine trials establishing dose response curves.

Any appropriate method can be used to evaluate a mammal's immune response following administration of an adjuvant containing at least one modified B subunit polypeptide as described herein. For example, a biological sample from a mammal can be examined to evaluate the immune response in vitro. The biological sample can be blood (e.g., serum) or a mucosal sample (e.g., saliva or gastric and bronchoalveolar lavages). In some cases, biological samples are collected prior to and after administration of an adjuvant as described herein. In some cases, an immune response can be evaluated by detecting immunostimulatory or inflammatory cytokine release by dendritic cells or macrophages. Secretion of cytokines such as IL-12, IL-6, IL-Ib, IL-IO, and TNF-α, can be measured, for example, by an enzyme linked immunosorbent assay (ELISA; see, for example, Example 1 below). Activation of naϊve T-cells can be assayed by, for example, measuring the incorporation of H- thymidine into newly synthesized DNA in proliferating cells, by measuring induction of cytolytic T-cell activity, or by detecting T-cell activation markers such as CD44 and/or CD69. Another method of assaying for the activation of naϊve T-cells is by indirectly measuring T-cell division by monitoring the fluorescence intensity of carboxyfluorescein succinimidyl ester (CFSE)-stained T-cells in vivo or in vitro. As T-cell proliferation increases, the intensity of detectable CFSE decreases. In some cases, induction of co-stimulatory molecule expression (e.g., CD40, CD80, CD86) can be detected in dendritic cells. In some cases, expression or translocation of NF- KB can be measured by, for example, cell staining with a commercially available antibody against NF-κB (available from, for example, Cell Signaling Technologies, Inc. (Beverly, MA)). In some cases, expression of co-receptor Cdl4 and/or immunostimulatory genes such as the growth factor G-CSF (CsO), cyclooxygenase-2 (Ptgs2), and the macrophage-inducible C-type lectin (Clec4e) can be evaluated. See Liang et al, Vaccine 27: 4302-08 (2009).

In some cases, an adjuvant as described herein can be administered with one or more additional components. For example, an adjuvant as described herein can be administered with a penetration enhancer to promote the efficient delivery of an adjuvant provided herein to a mucosal surface. In some cases, a penetration enhancer can be a surfactant (e.g., sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, etherdimethylsulfoxide, and decylmethylsulfoxide); a fatty acid (e.g., oleic acid, lauric acid, myristic acid, palmitic acid, and stearic acid); a bile salt (e.g., cholic acid, dehydrocholic acid, and deoxycholic acid); a chelating agent (e.g., disodium ethylenediamine tetraacetate, citric acid, and salicylates); or a non-chelating non-surfactant (e.g., unsaturated cyclic urea).

This document also provides articles of manufacture that can include any of the compositions described herein (e.g., an adjuvant (e.g., a modified B subunit polypeptide or a pentamer that includes at least one of such modified B subunit polypeptides) with or without an antigen and with or without a pharmaceutically acceptable carrier). For example, any of the compositions described herein can be combined with packaging material to generate a kit. Components and methods for producing articles of manufacture and kits are well known. In addition to a modified B subunit polypeptide of a Type II heat-labile enterotoxin, or pentamers containing one or more of such modified polypeptides, an article of manufacture or kit further can include, for example, one or more antigens, sterile water, pharmaceutical carriers, buffers, antibodies, indicator molecules, and/or other reagents for stimulating or evaluating a mammal's immune response. In addition, an article of manufacture or kit can include information regarding the potential benefits associated with administration of a composition as described above. For example, printed instructions describing how the composition contained therein can be used to stimulate a mammal's immune response can be included in such articles of manufacture or kits. The components in an article of manufacture can be packaged in a variety of suitable containers. The container can be any vessel or other sealed or sealable apparatus that can hold, for example, a pharmaceutical composition. The container employed can depend on the exact dosage form involved. In some cases, more than one container can be used together in a single package. For example, a liquid composition can be contained in an ampoule, which is, in turn, contained within a box or sealed pouch. In some cases, an article of manufacture can include a composition as described herein in a pre-packaged form in quantities sufficient for a single administration or for multiple administrations in, for example, sealed ampoules, capsules, or cartridges. Such containers can be air tight and/or waterproof, and can be labeled for appropriate use.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES Example 1 - Methods Microbial Molecules

The construction of recombinant plasmids encoding His-tagged LT-IIb holotoxin or its B pentamer (LT-IIb-Bs), in wild-type or GDla-nonbinding version (T13I), was performed as previously described (Hajishengallis et al., Infect. Immun. 73:1343-1349 (2005); Hajishengallis et al, Infect. Immun. 72:6351-8 (2004)). Single- point substitution mutants of LT-IIb-Bs were engineered by means of site-directed mutagenesis (QUIKCH ANGE® kit; Stratagene). The proteins were extracted from the periplasmic space of transformed E. coli DH5αF'Kan using polymyxin B treatment, and were purified using ammonium sulfate precipitation, followed by nickel affinity chromatography and size-exclusion chromatography using a Sephacryl- 100 column and an AKTA-FPLC system (GE Healthcare). Identity and purity were confirmed by SDS-PAGE, immunoblotting with specific rabbit IgG antibodies, and by quantitative Limulus amebocyte lysate assay kits (BioWhittaker or Charles River Endosafe) which determined negligible endotoxic activity (< 0.007 ng/μg protein). Further evidence against contamination with LPS or other heat-stable contaminants was obtained upon LT-IIb-Bs or holotoxin boiling, which destroys their biological activities. Control TLR agonists, including E. coli LPS (ultrapure grade), PaHi₃CSK₄ lipopeptide, and FSL-I lipopeptide were purchased from InvivoGen. The reagents were used at effective concentrations as previously determined. See Hajishengallis et al., Infect. Immun. 73:1343-1349 (2005); Hajishengallis et al., Infect. Immun. 72:6351-8 (2004).

FACS Analysis and Antibodies

Flow cytometry was used for phenotypic characterization of the generated BMDC, as well as for determining co-stimulatory molecule up-regulation in activated BMDC. Briefly, BMDC were analyzed using the BD FACSCalibur and the CellQuest software after staining with the following fluorescently-labeled antibodies to cell surface markers or with appropriate isotype controls: CDl Ic (clone HL3); CDl Ib (Ml/70); CD40 (HM40-3); CD54 (YNl/1.7.4); CD80 (16-10A1); CD86 (GLl); I-A/I-E (M5/114.15.2); GrI (RB6-8C5) (all reagents were from eBioscience, Inc. except for the anti-CD 1 Ic antibody, which was from BD Pharmingen). The analysis showed that the harvested BMDC contained < 1% macrophages and < 5% granulocytes.

Binding Assays

Binding of ligands to plate -immobilized GDIa or TLRs was assessed as previously described (Liang et al., J. Immunol. 178:4811-4819 (2007); Nawar et al., Infect. Immun. 73, 1330-42 (2005)). Briefly, 96-well microtiter wells were coated overnight at 4°C with GDIa (Matreya LLC), or with recombinant mouse or human TLR2, TLRl, or TLR6 (R&D systems). After blocking non-specific binding sites with 5% (w/v) BSA, wild-type or mutant LT-IIb-Bs was incubated in PBS containing 10 mg/ml BSA. Bound protein was detected colorimetrically using rabbit IgG anti- LT-IIb antibody followed by peroxidase-conjugated goat anti-rabbit IgG (adsorbed against human or mouse IgG). In competitive inhibition assays, LT-IIb-Bs was used in biotinylated form and bound protein was probed with peroxidase-conjugated streptavidin.

Cellular Activation Assays

Cytokine induction in stimulated macrophage or BMDC culture supernatants was measured by ELISA, using kits from eBioscience Inc. (San Diego, CA). THPl- BLUE™ cells (InvivoGen), stably transfected with NF-κB-inducible reporter system, were used for colorimetric determination of NF-κB activation. This involved measuring the activity of NF-κB-inducible alkaline phosphatase secreted in stimulated culture supernatants, using a Synergy HT multi-mode microplate reader (Bio-Tek). In certain experiments, the cells were pretreated for 30 minutes with blocking antibodies to TLR2 (clone TL2.1), TLRl (polyclonal), TLR6 (polyclonal) or TLR4 (clone

HTA125) (InvivoGen). The monoclonal antibodies were used at 0.25 μg/mL and the polyclonal at 1 μg/mL.

TLRl Mutants and Reporter Gene Assays A common variant of human TLRl , represented by the NCBI accession no.

AAI09095 and referred to as "wild-type" TLRl, was used as the basis for generating various mutants and variants. TLRl point mutants (F314D, Q316K, Y320N, E321V, I328N, R337G, M338W, V339S, H340G) were generated by random mutagenesis using error-prone PCR {see Cirino et al., Methods in MoI. Biol. 231 :3-9 (2003)) followed by sequencing. A polymorphic variant (P315L) of TLRl was generated as previously described (Omueti et al., J. Immunol. 178:6387-6394 (2007)) based on the technique of overlap extension PCR. The mutants/variants were generated as N- terminal FLAG™-tagged constructs within pFLAG™-CMV (Sigma- Aldrich) and were verified by sequencing. Wild-type or genetically altered TLRl or TLR6 were used as TLR2 signaling partners in reporter assays of inducible luciferase activity. Briefly, SW620 cells (which are deficient in TLR-I, -2, and -6 expression) were co- transfected with various combinations of wild-type, point mutants, or chimeric TLRs, along with a firefly luciferase reporter gene and a Renilla luciferase transfection control, as previously described (Omueti et al., J. Biol. Chem. 280:36616-36625 (2005)). Two days post-transfection, the cells were stimulated for 6 hours and the Renilla and firefly luciferase activities were measured in cell lysates using the Promega luciferase reporter assay system. Luciferase activity was calculated as a ratio of firefly luciferase activity to Renilla luciferase activity, to correct for transfection efficiency.

Protein-Protein Docking Analysis

The crystal structures of human TLR2/TLRl-lipopeptide complex (Jin et al., Cell 130:1071-82 (2007)) and of LT-IIb holotoxin (Van Den Akker et al., Structure 4:665-678 (1996)) were determined at 2.1 and 1.9 A resolution, respectively. The TLR2/TLR1 and LT-IIb-Bs structures were submitted as receptor and ligand, respectively, to public protein-protein docking servers GRAMM-X (vakser.bioinformatics.ku.edu/resources/gramm/grammx on the World Wide Web), ZDOCK (zdock.bu.edu/db insert.php on the World Wide Web), and ClusPro

(nrc.bu.edu/cluster on the World Wide Web) for complex prediction. The analysis was carried out using default parameter, except that the DOT docking program was selected in the ClusPro server. The Firedock Server (bioinfo3d.cs.tau.ac.il/FireDock/index on the World Wide Web) was used for further refinement and scoring for global energy values and the structure with the lowest energy was selected for further structure analysis. The LigPlot software (csb.yale.edu/userguides/graphics/ligplot/ligplo^descrip on the World Wide Web) was used for plotting intermolecular interactions, and the Crystallography & NMR System (CNS) program (cns.csb.yale.edu/vl.l on the World Wide Web) was used for buried surface calculation. Chains D-H of the original LT-IIb-Bs structure file in PDB were renamed B 1-5, respectively, for convenience and clarity.

Cell Isolation and Culture

The mice used for generating BMDC included wild-type BALB/c or C57BL/6 (The Jackson Laboratory, Bar Harbor, ME). All animal procedures associated with tissue harvesting were approved by the institutional animal care and use committee, in compliance with established federal and state policies. Bone marrow-derived dendritic cells (BMDC) were generated as described by Lutz et al., J. Immunol. Methods 223(l):77-92 (1999). Briefly, bone marrow cells from femurs and tibia of 8-12-week- old mice were plated at 2xlO⁵ cells/mL and cultured at 37°C and 5% CO₂ atmosphere, in complete RPMI (RPMI 1640 containing 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 100 units/mL penicillin G, 100 μg/mL streptomycin, and 0.05 mM 2-mercaptoethanol; InVitrogen, Carlsbad, CA) supplemented with 20 ng/mL recombinant murine GM-CSF (Peprotech, Rocky Hill, NJ). The non-adherent cells were harvested on day 8 and were phenotypically characterized by flow cytometry. The generated BMDC were cultured in complete RMPI. Cell viability was monitored using the CELLTITER-BLUE™ assay kit (Promega, Madison, WI). None of the experimental treatments affected cell viability compared to medium-only control treatments. Thioglycollate-elicited macrophages were isolated from the peritoneal cavity of BALB/c mice, as previously described in Hajishengallis et al., Infect. Immun. 73: 1343-1349 (2005), and were cultured in complete RPMI. Human monocytic THPl -BLUETM cells (InvivoGen) were maintained in complete RPMI. Cell viability was monitored using the CELLTITER- BLUE™ assay kit (Promega). None of the experimental treatments affected cell viability as compared to medium-only control treatments.

Quantitative Real-time PCR Arrays Gene expression was quantified using a TLR signaling pathway-focused array following the manufacturer's protocol (RT2 profiler arrays; SuperArray Bioscience, Frederick, MD). Briefly, BMDC were stimulated with LT-IIb-B₅ and RNA was extracted from cell lysates using the PerfectPure RNA cell kit (5 Prime, Fisherscience, Pittsburg, PA). The RNA was reverse transcribed and quantitative real-time PCR with cDNA was performed using an ABI 7500 system (Applied Biosystem, Foster

City, CA). BMDC without stimulation were used as control. The data were analyzed using a Web-based PCR Array Analysis software by SuperArray Bioscience.

Detecting Immunostimulation Phenotypic characterization of the generated BMDC and determination of co- stimulatory molecule up-regulation in activated BMDC were performed by flow cytometric analysis, using the FACSCalibur and the CellQuest software (Becton- Dickinson). Fluorescently labeled monoclonal antibodies to the following cell surface markers were used: CDl Ic (clone HL3); CDl Ib (Ml/70); CD40 (HM40-3); CD54 (YNl/1.7.4); CD80 (16-10A1); CD86 (GLl); I-A/I-E (M5/114.15.2); GrI (RB6-8C5); and F4/80 (BM8). All antibodies and isotype controls were from eBioscience except for anti-CD 1 Ic from BD Pharmingen. The cells were pretreated with Fc Block CD 16/32 (clone 93) and subsequently labeled with antibodies in "staining" buffer (Dulbecco's PBS containing 0.1% BSA and 0.01% azide), followed by washing and flow cytometry. The phenotypic analysis of the harvested BMDC revealed the presence of a highly pure CDl lc+ population with <1% macrophages and <5% granulocytes. To assay for T cell proliferation, CD4+ helper T cells were purified from splenocytes of 6-8 week-old BALB/c or C57BL/6 mice using the autoMACS™ separator and anti-CD4+ beads (Miltenyi Biotec, Auburn, CA). T cell proliferation was assessed by flow cytometry (BD FACSCalibur) after a two-day co-culture of carboxyfluorescein succinimidyl ester (CFSE)-stained T cells with LT-IIb-B₅- stimulated or un- stimulated BMDC, in the presence of suboptimal concentration (30 ng/mL) of anti-CD3 (145-2C11; BD Biosciences). To accurately evaluate the effects OfLT-IIb-B₅ on BMDC-mediated proliferation of helper T cells, the BMDC used in the co-culture system were previously irradiated to prevent their proliferation and were extensively washed to remove residual LT-IIb-B₅. In some experiments, T cell proliferation was determined using the BrdU cell proliferation assay kit, as recommended by the manufacturer (Calbiochem, San Diego, CA). Briefly, stimulated or un-stimulated cells were irradiated and co-cultured with CD4+ helper T cells and BrdU label. Twenty-four hours later, BrdU incorporation was assessed by intracellular staining with anti-BrdU antibody, followed by peroxidase-conjugated secondary antibody. The peroxidase reaction was performed using tetramethyl benzidine chromogenic substrate and the optical density signal at 450 nm was read on a microplate reader (Bio-Tek Instruments, Winooski, VT).

Mouse Model of Mucosal Immunization

The use of animals was reviewed and approved by the Institutional Animal Care and Use Committee, in compliance with established Federal and State guidelines. Groups of 6 female BALB/c mice, 10 to 12 weeks old, were used for intranasal immunization, essentially as previously described. See Nawar et al., Infection and Immunity 73(3): 1330-42 (2005); Hajishengallis et al., J. Immunol.

154(9):4322-32 (1995). Agl/II, a protein adhesin from Streptococcus mutans, was used as the immunogen, either alone or admixed with LT-IIb-B₅ or LT-IIb holotoxin (positive control adjuvant). The mice were administered three doses of Agl/II (10 μg) with or without LT-IIb-B₅ or LT-IIb (both at 1 μg), or buffer only (sham immunized) at 14-day intervals, i.e., at days 1, 15, and 29. The immunogen/adjuvant mixture was slowly administered in a standardized volume (15 μL) to the external nares by means of a micropipettor. Serum was obtained by centrifugation of blood samples collected from the tail vein. Saliva samples were collected by means of a pipettor fitted with a plastic tip after stimulation of the salivary flow by intraperitoneal injection of 5 μg carbachol. Vaginal wash samples were collected by instilling 75 μL of sterile PBS with a pipettor and tip, and flushing three times. Pre-immune samples were obtained one day before the immunizations (and confirmed the lack of Agl/II-specific antibodies) and post-immunization collections were made one week after the second and third immunization, as well as two weeks after the third immunization (i.e., at days 22, 36, and 50). Serum and secretions were stored at -80⁰C until assayed. The levels of isotype-specific anti-Agl/II antibodies from serum and secretions were determined by ELISA on microtiter plates coated with 1 μg/mL Agl/II. Total S-IgA was determined on plates coated with goat anti-mouse IgA. The plates were developed with the appropriate peroxidase-conjugated goat anti-mouse Ig isotype (Southern Biotechnology Associates, Inc., Birmingham, AL) and tetramethyl benzidine chromogenic substrate. Optical density values were measured in an ELISA plate reader. The assay was calibrated by means of a serially diluted standard (Mouse Ig Reference Serum; ICN, Costa Mesa, CA) and a standard curve was generated by a computer program based on four parameter logistic algorithms. Antibody data were expressed in μg/mL (serum) or % specific antibody/total IgA (secretions).

Statistical Analysis Data were evaluated by ANOVA and the Dunnett multiple-comparison test using the InStat program (GraphPad Software). Where appropriate (comparison of two groups only), unpaired two-tailed t tests were performed. Statistical significance was defined as/? < 0.05. All experiments were performed at least twice for verification.

Example 2 - Hydrophilic Modifications of LT-IIb-B

Critical Hydrophilic Residues in the "Upper Region " of LT-IIb-B ₅ Involved in TLR

Binding

To test the hypothesis that the solvent-accessible hydrophobic surface in the "upper region" of LT-IIb-Bs is critical for TLR2 binding, since this interaction is abrogated when this surface is buried by the A subunit in the LT-IIb holotoxin, point- substitution mutations rendering the involved hydrophobic residues hydrophilic (M69E, A70D, L73E, and S74D) were made as described herein. It was reasoned that the hydrophilic/charged nature of the mutated residues would abrogate the interaction of the modified LT-IIb-Bs molecules with TLR2. The hypothesis was supported by the findings from TLR2 binding assays comparing the mutants with wild-type LT-IIb- B₅. Indeed, all mutants lost the ability to bind mouse or human TLR2, as seen with the LT-IIb holotoxin which served as negative control (Figure 1 A, B). In stark contrast, the mutants retained the capacity to bind GDIa (Figure 1C), an interaction that is mediated by the "lower region" of the doughnut-shaped B pentamer. For control purposes, a lower-region point mutant (T 131) OfLT-IIb-B₅ was tested and, as expected, did not bind GDIa (Figure 1C), although it could bind TLR2 similarly to the wild-type. These findings suggested that the upper-region point mutations

(M69E, A70D, L73E, and S74D) did not cause global alterations in the B pentamer, since successful assembly of the B pentamer is a requirement for GDIa binding.

By analogy to Pani₃CSK₄, agonists that activate the TLR2/1 heterodimer may be expected to contact both TLR components. Previous studies identified TLRl as the signaling partner of TLR2 in response to LT-IIb-B₅, although the possibility for a direct LT-IIb-B₅/TLRl interaction was not addressed. See Liang, S. et al., J. Biol. Chem. 282, 7532-7542 (2007); Liang et al., J. Immunol. 178:4811-4819 (2007). To determine whether LT-IIb-B₅ binds TLRl and, if so, to investigate the role of the upper region hydrophobic surface in this interaction, the LT-IIb holotoxin, which does not activate TLR2/1, was used as a negative control. Although LT-IIb-B₅ displayed statistically significant binding to mouse or human TLRl (p < 0.05 vs. LT-IIb negative control; Figure 1 D, E), this was not as pronounced as binding to TLR2 (contrast with Figure 1 A,B). The mutants showed low-level, intermediate TLRl binding between that of the LT-IIb holotoxin and the wild-type B pentamer (Figure 1 D, E). As expected, none of the ligands, wild-type or mutant, could bind TLR6. Data presented in Figure 1 collectively indicated that LT-IIb-B₅ interacts with both TLR2 and TLRl and, moreover, a defined hydrophobic site in its upper region plays a critical role in TLR2 binding but a relatively modest role in TLRl binding.

Hydrophilic Point Mutations in the LT-IIb-Bs Upper Region Abrogate TLR2/I- dependent Immunostimulation

It was next determined whether the upper-region point mutants also lost the ability to induce TLR2/1 -dependent immunostimulation. Moreover, since these mutants retained GD la-binding activity, they could be appropriately used as tools for investigating whether LT-IIb-Bs exhibits GD la-dependent immunostimulatory properties that are independent of TLR2/1 activation. All four hydrophilic mutants (M69E, A70D, L73E, and S74D) failed to induce NF-κB activation in reporter THPl- Blue cells, in contrast to wild-type LT-IIb-Bs which readily activated NF-κB in a

TLR2-dependent way (Figure 2A). Consistent with the notion that LT-IIb-Bs binds TLRl but not TLR6, LT-IIb-Bs-induced NF-κB activation was inhibitable by anti- TLRl but not by anti-TLR6 (Figure 2B). The specificity of anti-TLR blocking antibodies was confirmed by including appropriate assay controls, i.e., PamsCSIQ (TLR2/1 agonist), FSL-I (TLR2/6 agonist), and LPS (TLR4 agonist) (Figure 2 A, B). Moreover, all four hydrophilic point mutations inhibited the ability of LT-IIb-Bs to induce cytokine production in mouse peritoneal macrophages (Figure 3 A-C) or bone marrow-derived dendritic cells (BMDC) (Figure 3 D-F), suggesting that GDIa binding is not sufficient per se for these activities. Similarly, wild-type LT-IIb-Bs upregulated expression of co-stimulatory molecules (CD40, CD80, and CD86), although the mutants showed little or no activity compared to no-agonist controls (Figure 3 G-I). Thus, the hydrophobic upper region Of LT-IIb-B₅, defined by M69, A70, L73, and the Cβ of S74, is critical for TLR2/1 -dependent immunostimulation, in contrast to the GD la-binding site in the lower region of the molecule. Intriguingly, M69, A70, L73, and S74 are shared by LT-IIa-B₅, the only other known enterotoxin B pentamer that activates TLR2, but not by the Type I B pentamers, LT-I-B₅ and CT-B₅ (see Van Den Akker et al., Structure 4:665-678 (1996)).

Effects of TLRl point mutations on LT-IIb-B 5-induced cell activation The ectodomain of both TLR2 and TLRl comprises 19 leucine-rich repeat

(LRR) modules. See Jin et al, Cell 130:1071-1082 (2007). Using TLR1/6 chimeric receptors, constructed by reciprocally exchanging increasing segments of N-terminal LRR modules between these two highly homologous receptors, it was determined which segment(s) of TLRl were required for cooperative TLR2-induced cell activation in response to LT-IIb-B₅. It was determined that, as an increasing number of N-terminal LRRs of TLRl were replaced by corresponding TLR6 LRRs, the cells progressively lost their ability for TLR2-dependent activation in response to LT-IIb- B₅ (Figure 4). Specifically, although replacement of up to the first eight N-terminal LRRs (construct [T6(l-8)/Tl]/T2) had a relatively minor effect, the additional replacement of the subsequent four LRRs (construct [T6(1-12)/T1]/T2) completely abrogated the response (Figure 4). The testing of the reverse chimeric constructs showed that replacement of up to the first eight N-terminal LRRs of TLR6 with those of TLRl (construct [Tl(l-8)/T6]/T2) was incapable to rescue LT-IIb-B₅-induced cell activation (Figure 4). This required the replacement of LRR1-12 of TLR6 by the corresponding TLRl LRRs (construct [Tl(l-12)/T6]/T2) and the response was completely restored by replacement of LRRl-17 of TLR6 with those of TLRl (Figure 4). Taken together, these data indicate that the LRR9-12 region of TLRl is critical for cooperative TLR2-induced cell activation by LT-IIb-Bs, whereas regions above or below LRR9-12 play a relatively minor role.

The crystallographic structure of the PamsCSIQ-induced active conformation of the TLR2/1 heterodimer revealed a number of residues involved in the dimerization interface and/or ligand binding. These included P315 of TLRl in the hydrophobic core of the dimer interface, which when mutated to L (to mimic the P315L natural polymorphism) leads to inhibition of PamsCSIQ-induced signaling. See Omueti et al, J. Immunol. 178, 6387-6394 (2007). The P315L variant, as well as a number of other TLRl point mutations in the dimer interface, inhibited TLR2-dependent activation of transfected SW620 cells by LT-IIb-Bs (Figure 5A). In contrast, mutation of a residue (I328N) that is not involved in dimer interface (or ligand binding) had no effect on the ability of LT-IIb-Bs to activate the transfectants (Figure 5A). The effects of the same TLRl point mutations on PamsCSIQ-induced cell activation followed a similar pattern (Figure 5B), except for R337G which had no significant influence on cell activation by PaHi₃CSK₄, although it modestly inhibited activation by LT-IIb-B₅ (Figure 5). A mutated residue (H340G) that was not predicted to be involved in dimer interface or ligand binding inhibited both LT-IIb-B₅- and PamsCSIQ-induced cell activation (Figure 5). Since most of the TLRl interface residues influenced the ability of both PaIn₃CSK₄ and LT-IIb-B₅ to induce cell activation, it appears that the reported Pani3CSK4-induced "m"-shaped TLR2/1 structure may constitute a predominant activating conformation, although subtle variations are possible depending on the specific TLR2/1 agonist. Thus, the Pani3CSK4-induced active conformation of TLR2/1 could be used to model the interaction of this structure with LT-IIb-B₅. In Vivo and In Vitro Adjuvant Activities

The capacity of LT-IIb-Bs to stimulate cytokine production in BMDC was tested. Stimulation of BMDC with LT-IIb-Bs resulted in production of TNF-α and IL-6 in both agonist dose-dependent and cell dose-dependent manner (Figure 6 A-B and C-D, respectively). In contrast to LT-IIb-Bs or prototypical TLR2 (Pam3Cys) or TLR4 (LPS) agonists, the hydrophilic TLR2-nonbinding point mutant (S74D) of LT- IIb-B5 failed to induce cytokine production. The ability of LT-IIb-Bs to up-regulate expression of class II MHC and co-stimulatory molecules was also tested. For this purpose, BMDC were harvested as early as 7 days after incubation with GM-CSF in order to obtain immature dendritic cells and prevent further maturation. LT-IIb-Bs (as well as LPS; positive control) enhanced the expression of class II MHC as well as of CD80 and CD86, which are necessary for induction of T cell proliferation through CD28 signaling. CD40, which is important for DC maturation and induction of adaptive immunity, was also up-regulated and so was CD54, which contributes to optimal T cell activation through LFA-I interaction. Moreover, LT-IIb-Bs caused moderate up-regulation of inducible T-cell co-stimulator-ligand (ICOSL; CD275), which is known to contribute to Th2 cell development by interacting with ICOS, a CD28-related molecule on T cells.

It was determined whether up-regulation of co-stimulatory molecule expression on BMDC by LT-IIb-B₅ results in functional co-stimulation of co-cultured CD4+ T cells. In these experiments, BMDC were treated or not with wild-type LT- Hb-B₅ or the S74D mutant (negative control) prior to co-culture with CFSE-stained CD4+ T cells in the presence of sub-optimal concentration of anti-CD3. FACS analysis of CD4+ T cell division demonstrated that LT-IIb-B₅-treated BMDC promoted T cell proliferation, in contrast to the S74D mutant, the effect of which was indistinguishable from the medium-only control. The ability OfLT-IIb-B₅ to activate BMDC for inducing CD4+ T cell proliferation was also tested using an independent method (BrdU incorporation). Again, in contrast to the S74D hydrophilic mutant, wild-type LT-IIb-B₅ (as well as Pam3Cys or LPS; positive controls) promoted BMDC-induced proliferation of CD4+ T cells.

To determine whether LT-IIb-B₅ displayed mucosal adjuvant capacity, groups of mice were immunized intranasally with Streptococcus mutans protein Agl/II (10 μg), in the absence or presence OfLT-IIb-B₅ or LT-IIb holotoxin (both at 1 μg). In general, mice given Agl/II with LT-IIb-Bs or LT-IIb elicited significantly (p < 0.05) higher mucosal and systemic Agl/II-specific antibody responses than mice immunized with Agl/II alone (Figure 7). Interestingly, the capacity of LT-IIb-Bs to stimulate the salivary IgA response to Agl/II was comparable to that of LT-IIb (Figure 7 A; days 36 and 50), although the holotoxin was the most potent adjuvant in augmenting vaginal IgA (Figure 7B) or serum IgG (Figure 7D) responses. Moreover, LT-IIb-Bs and LT- Hb displayed similar abilities in promoting the serum IgA antibody responses at the earlier time points examined (Figure 7C; days 22 and 36). These data clearly demonstrate that LT-IIb-Bs can potentiate specific antibody responses to a mucosally co-administered protein antigen.

Example 3 - Hydrophobic Modifications of LT-IIb-Bs Competitive Inhibition OfLT-IIb-B₅ Binding to TLR2 by Pam₃CSK₄

The interaction of LT-IIb-Bs with TLR2/1 was modeled to understand the role of the heterodimer's hydrophobic pockets. GRAMM-X, ZDOCK, and ClusPro protein-protein docking analysis suggested additional models of the TLR2/1 -LT-IIb- B5 interaction, which were assigned into four groups on the basis of the TLR2 or TLRl sites involved. Groups 1 and 2 predicted LT-IIb-Bs to bind to TLRl only or TLR2 only, respectively. In Group 3, LT-IIb-B₅ was predicted to bind to the C- terminus of both TLR2 and TLRl . Group 4 predicted that LT-IIb-B₅ binds the convex surface of the central domains of the TLR2/1 heterodimer and that LT-IIb-B₅ interacts primarily with the TLR2 component. Putative contact points were located in regions that extend from leucine-rich repeat (LRR)5 to LRRlO, thus partially overlapping with the PamsCSIQ-binding site. If true, then PaHi₃CSK₄ should be able to compete with LT-IIb-B₅ for TLR2 binding. As demonstrated in Figure 8, the binding of biotinylated LT-IIb-B₅ was indeed inhibited by PamsCSIQ in a dose- dependent manner, although unlabeled LT-IIb-B₅ exhibited stronger inhibitory activity (Figure 8).

Point-substitution mutations were made to render the upper-region hydrophobic residues more hydrophobic. For example, point-substitution mutations rendering the S74 residue more hydrophobic (S74I and S74A) were made as described herein. Binding assays were performed to determine the effect of upper- region hydrophobic point mutants OfLT-IIb-B₅ on the binding of toll-like receptors TLRl, TLR2, and TLR6 relative to wild-type LT-IIb-Bs and negative controls. As demonstrated in Figures 9 and 11 , enhanced binding to all three TLRs was observed for the hydrophobic point mutant S 74 A, whereas enhanced binding to TLR2 and TLR6 (Figure 9) was observed for the hydrophobic point mutant S74I, as compared to the controls including the wild-type (parent) molecule. Strikingly, S74A displayed six-fold stronger binding to TLR2 than the wild-type molecule (Figure 11). Moreover, S74A showed enhanced binding to TLRl relative to the wild-type molecule. The other hydrophobic point mutant, S74I, displayed modestly improved TLR2 binding compared to the wild-type parent molecule (Figure 11). These results were consistent with previous data suggesting that the hydrophobic upper region of LT-IIb-B5 is critical for interactions with the toll-like receptors and for TLR2/1- dependent immunostimulation. These preliminary findings also underscore the feasibility of generating LT-IIb-B5 hydrophobic mutants with enhanced function over the wild-type molecules. Moreover, the ability of the hydrophobic point mutants S74A and S74I to bind TLR6 (which is not bound by the parent molecule) suggests that the mutants may be able to activate the TLR2/6 heterodimer in addition to the TLR2/1 heterodimer.

Based on this analysis, a model of the predicted intermolecular interactions of LT-IIb-B5 with TLR2 was generated. The model predicts that LT-IIb-Bs binds to TLR2 mainly through hydrophobic interactions, as well as through a possible hydrogen bond between residue V72 of the B2 chain of LT-IIb-Bs and residue Q 187 of TLR2. The predicted hydrophobic interactions include residues E22, V72, L73, S74, G75, and E99 OfLT-IIb-B₅ and residues T161, K164, Q187, T236, and N290 of TLR2. Therefore, point-substitution mutations rendering the involved residues more hydrophobic (e.g., M69[V/I/L/F/W/C], A70[I/L/F/W/C], L73[V/I/M/F/W/C], and/or S74[A/I/V/L/M/F/W/C], and, optionally, E22[V/I/L/F/W/C], G75[A/I/V/L/M/F/W/C] and/or E99[V/I/L/F/W/C]) are made and evaluated for immunostimulatory activity as described herein. Although the model does not predict direct participation of M69 and A70 in these interactions, M69 and A70 are located close to the LT-IIb-Bs/TLR2 interface and, moreover, are key residues of the hydrophobic surface of LT-IIb-B₅. Indeed, the M69E and A70D mutations abrogate the LT-IIb-B₅-TLR2/l interaction, perhaps by disrupting the hydrophobic nature of the interface. For these reasons, modifications to enhance the hydrophobic character of residues M69 and A70 might result in molecules with increased capacity to interact with and activate TLR2.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A purified B subunit polypeptide from a type II heat-labile enterotoxin, said polypeptide comprising an amino acid modification at least one residue at a position selected from the group consisting of position 69, 70, 73, and 74 numbered relative to residues 24-123 of SEQ ID NO:1, wherein the at least one amino acid modification comprises replacement of: a Met at position 69 with a residue having greater hydrophobicity than Met, an Ala at position 70 with a residue having greater hydrophobicity than Ala, a Leu at position 73 with a residue having greater hydrophobicity than Leu, a Ser at position 74 with a residue having greater hydrophobicity than Ser.

2. The polypeptide of claim 1 , wherein the Met at position 69 is replaced with an amino acid residue selected from the group consisting of VaI, He, Leu, Phe, Trp, and Cy s.

3. The polypeptide of claim 1 , wherein the Ala at position 70 is replaced with an amino acid residue selected from the group consisting of He, Leu, Phe, Trp, and Cy s.

4. The polypeptide of claim 1, wherein the Leu at position 73 is replaced with an amino acid residue selected from the group consisting of VaI, He, Met, Phe, Trp, and Cy s.

5. The polypeptide of claim 1, wherein the Ser at position 74 is replaced with an amino acid residue selected from the group consisting of Ala, He, VaI, Leu, Met, Phe, Trp, and Cy s.

6. The polypeptide of claim 1 further comprising an amino acid modification at position 22 numbered relative to residues 24-123 of SEQ ID NO: 1.

7. The polypeptide of claim 6, wherein a GIu at position 22 is replaced with an amino acid residue selected from the group consisting of VaI, He, Leu, Phe, Trp, and Cy s.

8. The polypeptide of claim 1 further comprising an amino acid modification at position 75 numbered relative to residues 24-123 of SEQ ID NO: 1.

9. The polypeptide of claim 8, wherein a GIy at position 75 is replaced with an amino acid residue selected from the group consisting of Ala, He, VaI, Leu, Met, Phe, Trp, and Cy s.

10. The polypeptide of claim 1 further comprising an amino acid modification at position 99 numbered relative to residues 24-123 of SEQ ID NO: 1.

11. The polypeptide of claim 10, wherein a GIu at position 99 is replaced with an amino acid residue selected from the group consisting of VaI, He, Leu, Phe, Trp, and Cy s.

12. The polypeptide of claim 1, wherein said polypeptide has the amino acid sequence shown in SEQ ID NO:2.

13. The polypeptide of claim 1 , wherein said polypeptide has the amino acid sequence shown in SEQ ID NO:3.

14. An isolated nucleic acid encoding the polypeptide of any of claims 1- 13.

15. A pentamer of subunit B polypeptides from a type II heat-labile enterotoxin, wherein at least one of said polypeptides in said pentamer is the polypeptide of any of claims 1-13.

16. The pentamer of claim 15 , wherein said pentamer exhibits immunostimulatory activity.

17. The pentamer of claim 15, wherein said pentamer acts as an adjuvant.

18. A pharmaceutical composition comprising the polypeptide of any of claims 1-13 or the pentamer of any of claims 15-17 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the polypeptide of any of claims 1-13 or the pentamer of any of claims 15-17 and an antigen.

20. The pharmaceutical composition of claim 19, wherein said antigen is selected from the group consisting of AgI/II adhesion protein of Streptococcus mutans; saliva-binding region of AgI/II adhesion protein of Streptococcus mutans; PspA and PspC virulence proteins of Streptococcus pneumoniae; fimbriae of Porphyromonas gingivalis; lectin antigen of Entamoeba histolytica; P6 surface protein of nontypeable Haemophilus influenza; HBsAg of Hepatitis B virus; and TbpA and TbpB transferrin-binding proteins of ^'Neisseria gonorrhoeae.

21. A method of stimulating a mammal ' s immune response to an antigen comprising the steps of administering to the mammal: the polypeptide of any of claims 1-13 and said antigen, the pentamer of any of claims 15-17 and said antigen, the pharmaceutical composition of claim 18 and said antigen, or the pharmaceutical composition of claims 19 or 20.

22. The method of claim 21 , wherein said administration is to a mucosal surface.

23. The method of claim 22, wherein said mucosal surface is selected from the group consisting of intranasal, oral, parenteral, rectal, or vaginal.

24. The method of claim 21 , wherein the mammal is a human.

25. The method of claim 21 , wherein the polypeptide, the pentamer or the pharmaceutical composition and the antigen are administered simultaneously.

26. The method of claim 21 , wherein the polypeptide, the pentamer or the pharmaceutical composition and the antigen are administered sequentially.

27. The method of claim 21 , wherein the polypeptide, the pentamer or the pharmaceutical composition and the antigen are administered together in the same composition.

28. The method of claim 21 , wherein the polypeptide, the pentamer or the pharmaceutical composition and the antigen are administered by a route selected from the group consisting of intranasal, oral, gastrointestinal, rectal, vaginal, and genitourinary tract.

29. The method of claim 21 , wherein secretion of one or more cytokines by dendritic cells or macrophages is indicative of said stimulated immune response in said mammal.

30. The method of claim 29, wherein said one or more cytokines are selected from the group consisting of IL-12, IL-6, IL-Ib, IL-IO, and TNF-α.

31. The method of claim 21 , wherein an activation of naϊve T-cells is indicative of said stimulated immune response in said mammal.

32. The method of claim 31 , wherein said activation of naϊve T-cells is indicated by the presence of one or more T-cell activation markers selected from the group consisting of CD44 and CD69.

33. The method of claim 21 , wherein said activation of naϊve T-cells is indicated by a decrease in fluorescence intensity of carboxyfluorescein succinimidyl ester (CFSE)-stained cytolytic T-cells.

34. An article of manufacture comprising packaging material, one or more of the polypeptides of any of claims 1-13, the pentamer of any of claims 15-17 or the pharmaceutical composition of any of claims 18-20, and written instructions for using one or more of the polypeptides, the pentamers or the pharmaceutical compositions to stimulate the immune response of a mammal.