WO2006050420A2

WO2006050420A2 - Chimeric immunogens that comprise ovalbumin

Info

Publication number: WO2006050420A2
Application number: PCT/US2005/039620
Authority: WO
Inventors: Randall Mrsny
Original assignee: Trinity Biosystems, Inc.
Priority date: 2004-11-01
Filing date: 2005-11-01
Publication date: 2006-05-11
Also published as: WO2006050420A3

Abstract

The present invention relates to chimeric immunogens that comprise a receptor binding domain, a translocation domain, and ovalbumin, or a portion thereof. The invention also relates to methods of using and making the chimeric immunogens of the invention, polynucleotides encoding the chimeric immunogens of the invention, expression vectors comprising the polynucleotides of the invention, and cells comprising the expression vectors of the invention.

Description

Chimeric Immunogens that Comprise Ovalbumin

1. FIELD OF THE INVENTION

[0001] The present invention relates, in part, to methods and compositions for assessing the immune response induced by a chimeric immunogen that comprises ovalbumin, or a portion thereof. The methods and compositions rely, in part, on administering a chimeric immunogen comprising ovalbumin, or a portion thereof, to a subject to be immunized.

2. BACKGROUND

[0002] Immunization against bacterial or viral infection has greatly contributed to relief from infectious disease. Generally, immunization relies on administering an inactivated or attenuated pathogen to the subject to be immunized. For example, hepatitis B vaccines can be made by inactivating viral particles with formaldehyde, while some polio vaccines consist of attenuated polio strains that cannot mount a full-scale infection. In either case, the subject's immune system is stimulated to mount a protective immune response by interacting with the inactivated or attenuated pathogen. See, e.g., Kuby, 1997, Immunology W.H. Freeman and Company, New York.

[0003] This approach has proved successful for immunizing against a number of pathogens. Indeed, many afflictions that plagued mankind for recorded history have been essentially eliminated by immunization with attenuated or inactivated pathogens. See id. Nonetheless, this approach is not effective to immunize against infection by many pathogens that continue to pose significant public health problems. In particular, no vaccine presently exists that has been approved for immunization against numerous bacterial and viral infections, including, for example, Pseudomonas, Chlamydia, HIV, HCV, etc. The absence of such a vaccine presents significant public health problems.

[0004] One approach for exploring immune responses and the immune system to assist in design and construction of such vaccines has been to characterize immune responses induced against well characterized molecules such as, for example, ovalbumin, keyhole limpet hemocyanin, hemeagglutinin, and the like. Ovalbumin, which is the main proteinaceous component of egg white, has been frequently used as a soluble protein in immunological studies. See, e.g., Rosenwasser & Gelfand, 1999, Am. J. Respir. Cell MoI. Biol. 21 :4-6. One particularly useful feature of ovalbumin as a test antigen is its ability to induce cell-mediated responses against amino acids 257-263, which has the amino acid sequence SIINFEKL. See, e.g., Karman et al, 2004, J Immunol. 173(4):2353-61.

[0005] Further, chimeric proteins constructed from Pseudomonas exotoxin A ("PE") derivatives have been tested for their ability to induce a protective immune response. See, e.g., Hertle et al, 2001, Infect. Immun. 69:6962-6969 and International Patent Publication Nos. WO 99/02712 and WO 99/02713. However, none of these attempts has to date resulted in a vaccine that has been approved as effective to immunize against infection by any pathogen. Further characterization of immune responses induced by such chimeric immunogens is needed to explore the mechanism and specificity of immune responses by such chimeric immunogens. Such characterization would be useful, for example, in guiding design and construction of new PE derivatives for use in vaccines against pathogens for which no vaccines are currently available. These and other unmet needs are satisfied by the present invention.

3. SUMMARY OF THE INVENTION

[0006] The chimeric immunogens of the invention comprise a heterologous antigen and can elicit humoral, cell-mediated and secretory immune responses against the heterologous antigen. Such chimeras are useful, for example, in characterizing the immune response induced by the chimeric immunogen against the heterologous antigen and can inform efforts to construct chimeric immunogens that can be used to immunize against infection by pathogens.

[0007] Accordingly, in certain aspects, the invention provides a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. In certain embodiments, the chimeric immunogen, when administered to a subject, induces an immune response in the subject that is specific for one or more ovalbumin epitope(s). The immune response can be a humoral, cell-mediated, or secretory immune response, or any combination thereof.

[0008] In another aspect, the invention provides a method for inducing an immune response in a subject that comprises administering to the subject an effective amount of a chimeric immunogen comprising a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. Administration of the chimeric immunogen can induce an immune response in the subject that is specific for one or more ovalbumin epitope(s).

[0009] In yet another aspect, the invention provides a method for generating in a subject antibodies specific for one or more ovalbumin epitope(s). The method comprises administering to said subject an effective amount of a chimeric immunogen comprising a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof.

[0010] In still another aspect, the invention provides a polynucleotide that encodes a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a a heterologous antigen that is ovalbumin, or a portion thereof.

[0011] In yet another aspect, the invention provides expression vectors that comprise a polynucleotide of the invention.

[0012] In still another aspect, the invention provides cells comprising an expression vector of the invention.

[0013] In yet another aspect, the invention provides a composition comprising a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. In certain embodiments, the composition further comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 presents an exemplary amino acid sequence of P 'seudomonas aeruginosa exotoxin A, identified as SEQ ID NO.: 1.

[0015] Figure 2 presents an exemplary amino acid sequence of chicken ovalbumin, identified as SEQ ID NO.. -2.

5. DETAILED DESCRIPTION OF THE INVENTION 5.1. DEFINITIONS

[0016] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0017] A "ligand" is a compound that specifically binds to a target molecule. Exemplary ligands include, but are not limited to, an antibody, a cytokine, a substrate, a signaling molecule, and the like.

[0018] A "receptor" is compound that specifically binds to a ligand.

[0019] A ligand or a receptor (e.g., an antibody) "specifically binds to" or "is specifically immunoreactive with" another molecule when the ligand or receptor functions in a binding reaction that indicates the presence of the molecule in a sample of heterogeneous compounds. Thus, under designated assay (e.g., immunoassay) conditions, the ligand or receptor binds preferentially to a particular compound and does not bind in a significant amount to other compounds present in the sample. For example, a polynucleotide specifically binds under hybridization conditions to another polynucleotide comprising a complementary sequence and an antibody specifically binds under immunoassay conditions to an antigen bearing an epitope used to induce the antibody.

[0020] "Immunoassay" refers to a method of detecting an analyte in a sample involving contacting the sample with an antibody that specifically binds to the analyte and detecting binding between the antibody and the analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. In one example, an antibody that binds a particular antigen with an affinity (K_m) of about 10 μM specifically binds the antigen.

[0021] "Vaccine" refers to an agent or composition containing an agent effective to confer an at least partially prophylactic or therapeutic degree of immunity on an organism while causing only very low levels of morbidity or mortality. Methods of making vaccines are, of course, useful in the study of the immune system and in preventing and treating animal or human disease. [0022] An "immune response" refers to one or more biological activities mediated by cells of the immune system in a subject. Such biological activities include, but are not limited to, production of antibodies; activation and proliferation of immune cells, such as, e.g., B cells, T cells, macrophages, leukocytes, lymphocytes, etc. ; release of messenger molecules, such as cytokines, chemokines, interleukins, tumor necrosis factors, growth factors, etc. ; and the like. An immune response is typically mounted when a cell of the immune system encounters non- self antigen that is recognized by a receptor present on the surface of the immune cell. The immune response preferably protects the subject to some degree against infection by a pathogen that bears the antigen against which the immune response is mounted.

[0023] An immune response may be "elicited," "induced," or "induced against" a particular antigen. Each of these terms is intended to be synonymous as used herein and refers to the ability of the chimeric immunogen to generate an immune response upon administration to a subject.

[0024] An "immunogen" is a molecule or combination of molecules that can induce an immune response in a subject when the immunogen is administered to the subject.

[0025] "Immunizing" refers to administering an immunogen to a subject.

[0026] An "immunogenic amount" of a compound is an amount of the compound effective to elicit an immune response in a subject.

[0027] "Linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a. nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.

[0028] "Ovalbumin" refers to a polypeptide that is found in the whites of eggs from, for example, chickens, turkeys, ducks, pheasants, ostriches, and the like. A representative sequence of ovalbumin is presented as Figure 2, however, any polypeptide with at least 70% identity or 80% homology to the sequence presented in Figure 2 can also be considered to be ovalbumin.

[0029] "Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammal. A pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of an agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers, and excipients, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and formulations are described in Remington's Pharmaceutical Sciences, 19th Ed. 1995, Mack Publishing Co., Easton. A "pharmaceutically acceptable salt" is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0030] Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent. Typical modes of administration include enteral {e.g., oral, intranasal, rectal, or vaginal) or parenteral (e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0031] "Small organic molecule" refers to organic molecules of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes organic biopolymers (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, up to about 2000 Da, or up to about 1000 Da.

[0032] A "subject" of diagnosis, treatment, or administration is a human or non-human animal, including a mammal, such as a rodent (e.g., a mouse or rat), a lagomorph (e.g., a rabbit), or a primate. A subject of diagnosis, treatment, or administration is preferably a primate, and more preferably a human.

[0033] "Treatment" refers to prophylactic treatment or therapeutic treatment. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology for the purpose of diminishing, slowing the progression, eliminating, or halting those signs.

[0034] "Pseudomonas exotoxin A" or "PE" is secreted by Pseudomonas aeruginosa as a 67 kD protein composed of three prominent globular domains (Ia, II, and III) and one small subdomain (Ib) that connects domains II and III. See A.S. Allured et al, 1986, Proc. Natl. Acad. Sci. 83:1320-1324, and Figure 1, which presents the amino acid sequence of native PE. Without intending to be bound to any particular theory or mechanism of action, domain Ia of PE is believed to mediate cell binding because domain Ia specifically binds to the low density lipoprotein receptor-related protein ("LRP"), also known as the α2-macroglobulin receptor ("α2-MR") and CD-91. See M.Z. Kounnas et al, 1992, J Biol. Chem. 267:12420-23. Domain Ia spans amino acids 1-252. Domain II of PE is believed to mediate translocation to the interior of a cell following binding of domain Ia to the α2-MR. Domain II spans amino acids 253-364. Domain Ib has no known function and spans amino acids 365-399. Domain III mediates cytotoxicity of PE and includes an endoplasmic reticulum retention sequence. PE cytotoxicity is believed to result from ADP ribosylation of elongation factor 2, which inactivates protein synthesis. Domain III spans amino acids 400-613 of PE. Deleting amino acid E553 ("ΔE553") from domain III eliminates EF2 ADP ribosylation activity and detoxifies PE. PE having the mutation ΔE553 is referred to herein as "PEΔE553." Genetically modified forms of PE are described in, e.g., United States patent nos. 5,602,095; 5,512,658 and 5,458,878. Pseudomonas exotoxin, as used herein, also includes genetically modified, allelic, and chemically inactivated forms of PE within this definition. See, e.g., Vasil et al, 1986, Infect. Immunol. 52:538-48. Further, reference to the various domains of PE is made herein to the reference PE sequence presented as Figure 1. However, one or more domain from modified PE, e.g., genetically or chemically modified PE, or a portion of such domains, can also be used in the chimeric immunogens of the invention so long as the domains retain functional activity. One of skill in the art can readily identify such domains of such modified PE based on, for example, homology to the PE sequence exemplified in Figure 1 and test for functional activity using, for example, the assays described below.

[0035] "Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA") as well as nucleic acid analogs. Nucleic acid analogs include those which include non-naturally occurring bases, nucleotides that engage in linkages with other nucleotides other than the naturally occurring phosphodiester bond or which include bases attached through linkages other than phosphodiester bonds. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" typically refers to large polynucleotides. The term "oligonucleotide" typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces π

[0036] Conventional notation is used herein to describe polynucleotide sequences: the left- hand end of a single-stranded polynucleotide sequence is the 5 '-end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5 '-direction.

[0037] The direction of 5' to 3' addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the "coding strand"; sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5' to the 5 '-end of the RNA transcript are referred to as "upstream sequences"; sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as "downstream sequences."

[0038] "Complementary" refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides. Thus, the two molecules can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. Thus, the polynucleotide whose sequence 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5 '-GTATA-3'.

[0039] The term "% sequence identity" is used interchangeably herein with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm, and means that a given sequence is at least 80% identical to another length of another sequence. Exemplary levels of sequence identity include, but are not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequence identity to a given sequence.

[0040] The term "% sequence homology" is used interchangeably herein with the term "% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence. Exemplary levels of sequence homology include, but are not limited to, 60, 70, 80, 85, 90, 95, 98% or more sequence homology to a given sequence.

[0041] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et al, 1990, J. MoI. Biol. 215:403-10 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et al, 1997, Nucleic Acids Res., 25:3389-3402. Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases. The BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See id.

[0042] A preferred alignment of selected sequences in order to determine "% identity" between two or more sequences, is performed using for example, the CLUSTAL-W program in MacVector version 6.5, operated with default parameters, including an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM 30 similarity matrix.

[0043] "Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Asn (N), GIn (Q) Ser (S) and Thr (T).

[0044] "Nonpolar Amino Acid" refers to a hydrophobic amino acid having a side chain that is uncharged at physiological pH and which has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded nonpolar amino acids include Ala (A), GIy (G), He (I), Leu (L), Met (M) and VaI (V) .

[0045] "Hydrophilic Amino Acid" refers to an amino acid exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al. , 1984, J. MoI. Biol. 179:125-142. Genetically encoded hydrophilic amino acids include Arg (R), Asn (N), Asp (D), GIu (E), GIn (Q), His (H), Lys (K), Ser (S) and Thr (T).

[0046] "Hydrophobic Amino Acid" refers to an amino acid exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al, 1984, J. MoI. Biol. 179:125-142. Genetically encoded hydrophobic amino acids include Ala (A), GIy (G), He (I), Leu (L), Met (M), Phe (F), Pro (P), Trp (W), Tyr (Y) and VaI (V).

[0047] "'Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp (D) and GIu (E).

[0048] "Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with a hydrogen ion. Genetically encoded basic amino acids include Arg (R), His (H) and Lys (K).

[0049] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and RNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0050] "Amplification" refers to any means by which a polynucleotide sequence is copied and thus expanded into a larger number of polynucleotide molecules, e.g., by reverse transcription, polymerase chain reaction, ligase chain reaction, and the like.

[0051] "Primer" refers to a polynucleotide that is capable of specifically hybridizing to a designated polynucleotide template and providing a point of initiation for synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions in which synthesis is induced, i.e., in the presence of nucleotides, a complementary polynucleotide template, and an agent for polymerization such as DNA polymerase. A primer is typically single-stranded, but may be double-stranded. Primers are typically deoxyribonucleic acids, but a wide variety of synthetic and naturally occurring primers are useful for many applications. A primer is complementary to the template to which it is designed to hybridize to serve as a site for the initiation of synthesis, but need not reflect the exact sequence of the template. In such a case, specific hybridization of the primer to the template depends on the stringency of the hybridization conditions. Primers can be labeled with, e.g., chromo genie, radioactive, or fluorescent moieties and used as detectable moieties.

[0052] "Probe," when used in reference to a polynucleotide, refers to a polynucleotide that is capable of specifically hybridizing to a designated sequence of another polynucleotide. A probe specifically hybridizes to a target complementary polynucleotide, but need not reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target depends on the stringency of the hybridization conditions. Probes can be labeled with, e.g., chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In instances where a probe provides a point of initiation for synthesis of a complementary polynucleotide, a probe can also be a primer. [0053] "Hybridizing specifically to" or "specific hybridization" or "selectively hybridize to", refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.

[0054] The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences. "Stringent hybridization" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids can be found in Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, NY; Sambrook et al, 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3^rd ed., NY; and Ausubel et al. , eds. , Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0055] Generally, highly stringent hybridization and wash conditions are selected to be about 5° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.

[0056] One example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than about 100 complementary residues on a filter in a Southern or northern blot is 50% formalin with 1 mg of heparin at 42° C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72° C for about 15 minutes. An example of stringent wash conditions is a 0.2X SSC wash at 65° C for 15 minutes. See Sambrook et al. for a description of SSC buffer. A high stringency wash can be preceded by a low stringency wash to remove background probe signal. An exemplary medium stringency wash for a duplex of, e.g., more than about 100 nucleotides, is Ix SSC at 45° C for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than about 100 nucleotides, is 4-6x SSC at 40° C for 15 minutes, hi general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization.

[0057] "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Conventional notation is used herein to portray polypeptide sequences; the beginning of a polypeptide sequence is the amino-terminus, while the end of a polypeptide sequence is the carboxyl-terminus.

[0058] The term "protein" typically refers to large polypeptides, for example, polypeptides comprising more than about 50 amino acids. The term "protein" can also refer to dimers, trimers, and multimers that comprise more than one polypeptide.

[0059] The term "peptide" typically refers to short polypeptides, for example, polypeptides comprising about 50 or less amino acid's.

[0060] "Conservative substitution" refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I)₃ Leucine (L), Methionine (M), and Valine (V) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W).

5.2. Chimeric Immunogens

[0061] Generally, the chimeric immunogens of the present invention are polypeptides that comprise structural domains corresponding to domains Ia and II of PE. The chimeric immunogens can optionally comprise structural domains corresponding to the other domains of PE, domains Ib and III. These structural domains perform certain functions, including, but not limited to, cell recognition, translocation and endoplasmic reticulum retention, that correspond to the functions of the domains of PE. By including or omitting the optional domains of PE, the character of the induced immune response can be modulated, as described below.

[0062] In addition to the portions of the molecule that correspond to PE functional domains, the chimeric immunogens of this invention further comprise a heterologous antigen that is ovalbumin, or a portion thereof. The heterologous antigen can be introduced into or replace some or all of the Ib domain of PE, or the heterologous antigen can be introduced into or replace any other portion of the molecule that does not disrupt a cell-binding or translocation activity. An immune response specific for the heterologous antigen is elicited upon administration of the chimeric immunogen to a subject.

[0063] Accordingly, the chimeric immunogens of the invention generally comprise the following structural elements, each element imparting particular functions to the chimeric immunogen: (1) a "receptor binding domain" that functions as a ligand for a cell surface receptor and that mediates binding of the protein to a cell; (2) a "translocation domain" that mediates translocation from the exterior of the cell to the interior of the cell; (3) the heterologous antigen that is ovalbumin, or a portion thereof; and, optionally, (4) an "endoplasmic reticulum ("ER") retention domain" that translocates the chimeric immunogen from the endosome to the endoplasmic reticulum, from which it enters the cytosol. The chimeric immunogen can still induce an immune response in the absence of the ER retention domain, though this absence changes the nature of the induced immune response, as described below.

[0064] The domains of the chimeric immunogens other than the heterologous antigen can be present in the order set forth above, i.e., domain Ia is closest to the N-terminus, then the translocation domain, then the ER retention domain. In fact, this arrangement is preferred. However, the domains of the chimeric immunogen can be in any order as long as the domains retain their functional activities. Several representative assays to test such functional activities are set forth below.

[0065] Such chimeric immunogens offer several advantages over conventional immunogens. To begin with, certain embodiments of the chimeric immunogens can be constructed and expressed in recombinant systems. These systems eliminate any requirement to crosslink the heterologous antigen to a carrier protein. Recombinant technology also allows one to make a chimeric immunogen having an insertion site designed for introduction of any desired heterologous antigen. Such insertion sites allow the skilled artisan to quickly and easily produce chimeric immunogens that comprise either known variants of a heterologous antigen or emerging variants of evolving heterologous antigens.

[0066] Further, the chimeric immunogens can be engineered to alter the function of their domains in order to tailor the activity of the immunogen to its intended use. For example, by selecting the appropriate receptor binding domain, the skilled artisan can target the chimeric immunogen to bind to a desired cell or cell line.

[0067] In addition, because certain embodiments of the chimeric immunogens include a constrained cysteine-cysteine loop, heterologous antigens that are so constrained in nature can be presented in native or near-native conformation. By doing so, the induced immune response is specific for antigen in its native conformation, and can more effectively protect the subject from infection by the pathogen. For example, a turn-turn-helix motif can be observed in peptides constrained by a disulfide bond, but not in linear peptides. See Ogata et al, 1990, Biol. Chem. 265:20678-85.

[0068] Moreover, the chimeric immunogens can be used to elicit a humoral, a cell-mediated or a secretory immune response. Depending on the pathway by which the chimeric immunogen is processed in an antigen-presenting cell, the chimeric immunogen can induce an immune response mediated by either class I or class II MHC. See Becerrra et al, 2003, Surgery 133:404-410 and Lippolis et al, 2000, Cell. Immunol. 203:75-83. Further, if the PE chimeras are administered to a mucosal surface of the subject, a secretory immune response involving IgA can be induced. See, e.g., Mrsny et al, 1999, Vaccine 17:1425-1433 and Mrsny et al, 2002, Drug Discovery Today 7:247-258.

[0069] The chimeric immunogens of the invention can also be used to elicit a protective immune response without using attenuated or inactivated pathogens. The inactivation or attenuation of such pathogens can sometimes be incomplete, or the pathogen can revert to be fully infectious, leading to infection by the pathogen upon administration of the vaccine. For example, administration of attenuated polio vaccine actually results in paralytic polio in about 1 in 4 million subjects receiving the vaccine. See Kuby, 1997, Immunology Ch. 18, W.H. Freeman and Company, New York. [0070] Thus, in certain aspects, the invention provides a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. In certain embodiments, the chimeric immunogen, when administered to a subject, generates an immune response in the subject that is specific for one or more ovalbumin epitope(s).

[0071] In certain embodiments, the chimeric immunogen further comprises an endoplasmic reticulum retention domain. In certain embodiments, the heterologous antigen that is ovalbumin, or a portion thereof, is located between the translocation domain and the endoplasmic reticulum retention domain. In certain embodiments, the endoplasmic reticulum retention domain is an enzymatically inactive domain III of Ps eudomonas exotoxin A. In certain embodiments, the enzymatically inactive domain III of Ps eudomonas exotoxin A is inactivated by deleting a glutamate at position 553.

[0072] In certain embodiments, the endoplasmic reticulum retention domain comprises an ER retention signal that has an amino acid sequence selected from the group of RDEL (SEQ ID NO.:3) or KDEL (SEQ ID NO.:4). In certain embodiments, the ER retention signal is sufficiently near the C-terminus of said endoplasmic reticulum retention domain to result in retention of the chimeric immunogen in the endoplasmic reticulum.

[0073] In certain embodiments, the chimeric immunogen comprises a translocation domain that is selected from the group consisting translocation domains from Pseudomonas exotoxin A, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin. In further embodiments, the translocation domain is domain II of Pseudomonas exotoxin A. In yet further embodiments, the translocation domain comprises amino acids 280 to 364 of domain II of Pseudomonas exotoxin A.

[0074] In certain embodiments, the chimeric immunogen comprises more than one heterologous antigen that is ovalbumin, or a portion thereof.

[0075] In certain embodiments, the chimeric immunogen comprises a receptor binding domain that is selected from the group consisting of domain Ia of Pseudomonas exotoxin A; a receptor binding domains from cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin; a monoclonal antibody, a polyclonal antibody, or a single-chain antibody; TGFα, TGFβ, EGF, PDGF, IGF, or FGF; IL-I, IL-2, IL-3, or IL-6; and MIP-Ia, MIP-Ib, MCAF, or IL- 8. In further embodiments, the receptor binding domain is domain Ia of Pseudomonas exotoxin A. In yet further embodiments, the domain Ia of P 'seudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:5.

[0076] In certain embodiments, the receptor binding domain binds to α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycopolypeptide receptor, CD3, CD4, CD8, chemokine receptor, CD25, CDl IB, CDl 1C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, or VEGF receptor. In further embodiments, the receptor binding domain binds to α2-macroglobulin receptor.

5.2.1. Receptor Binding Domain

[0077] The chimeric immunogens of the invention generally comprise a receptor binding domain. The receptor binding domain can be any receptor binding domain that binds to a cell surface receptor without limitation. Such receptor binding domains are well-known to those of skill in the art. Preferably, the receptor binding domain binds specifically to the cell surface receptor. The receptor binding domain should bind to the cell surface receptor with sufficient affinity to hold the chimeric immunogen in proximity to the cell surface to allow endocytosis of the chimeric immunogen. Representative assays that can routinely be used by the skilled artisan to assess binding of the receptor binding domain to a cell surface receptor are described below.

[0078] In certain embodiments, the receptor binding domain can comprise a polypeptide, a peptide, a protein, a lipid, a carbohydrate, or a small organic molecule, or a combination thereof. Examples of each of these molecules that bind to cell surface receptors are well known to those of skill in the art. Suitable peptides, polypeptides, or proteins include, but are not limited to, bacterial toxin receptor binding domains, such as the receptor binding domains from PE, cholera toxin, diptheria toxin, shiga toxin, shiga-like toxin, etc. ; antibodies, including monoclonal, polyclonal, and single-chain antibodies, or derivatives thereof, growth factors, such as TGFα, TGFβ, EGF, PDGF, IGF, FGF, etc.; cytokines, such as IL-I, IL-2, IL-3, IL-6, etc; chemokines, such as MIP-Ia, MIP-Ib, MCAF, IL-8, etc.; and other ligands, such as CD4, cell adhesion molecules from the immunoglobulin superfamily, integrins, ligands specific for the IgA receptor, etc. See, e.g., Pastan et al, 1992, Anna. Rev. Biochem. 61:331-54; and U.S. Patent Nos. 5,668,255, 5,696,237, 5,863,745, 5,965,406, 6,022,950, 6,051,405, 6,251,392, 6,440,419, and 6,488,926. The skilled artisan can select the appropriate receptor binding domain based upon the expression pattern of the receptor to which the receptor binding domain binds.

[0079] Lipids suitable for receptor binding domains include, but are not limited to, lipids that themselves bind cell surface receptors, such as sphingosine-1 -phosphate, lysophosphatidic acid, sphingosylphosphorylcholine, retinoic acid, etc. ; lipoproteins such as apolipoprotein E, apolipoprotein A, etc. , and glycolipids such as lipopolysaccharide, etc. ; glycosphingolipids such as globotriaosylceramide and galabiosylceramide; and the like. Carbohydrates suitable for receptor binding domains include, but are not limited to, monosaccharides, disaccharides, and polysaccharides that comprise simple sugars such as glucose, fructose, galactose, etc. ; and glycoproteins such as mucins, selectins, and the like. Suitable small organic molecules for receptor binding domains include, but are not limited to, vitamins, such as vitamin A, Bi, B₂, B₃, B₆, B₉, Bj₂, C, D, E, and K, amino acids, and other small molecules that are recognized and/or taken up by receptors present on the surface of epithelial cells.

[0080] In certain embodiments, the receptor binding domain can bind to a receptor found on an epithelial cell. In further embodiments, the receptor binding domain can bind to a receptor found on the apical membrane of an epithelial cell. In still further embodiments, the receptor binding domain can bind to a receptor found on the apical membrane of a mucosal epithelial cell. The receptor binding domain can bind to any receptor known to be present on the apical membrane of an epithelial cell by one of skill in the art without limitation. For example, the receptor binding domain can bind to α2-MR. An example of a receptor binding domain that can bind to α2-MR is domain Ia of PE. Accordingly, in certain embodiments, the receptor binding domain is domain Ia of PE. In other embodiments, the receptor binding domain is a portion of domain Ia of PE that can bind to α2-MR.

[0081] In certain embodiments, the receptor binding domain can bind to a receptor present on an antigen presenting cell, such as, for example, a dendritic cell or a macrophage. The receptor binding domain can bind to any receptor present on an antigen presenting cell without limitation. For example, the receptor binding domain can bind to any receptor identified as present on a dendritic or other antigen presenting cell identified in Figdor, 2003, Pathol. Biol. (Paris). 51(2):61-3; Coombεs et al., 2001, Immunol Lett. 3;78(2):103-l l; Shortman K et α/., 1997, Ciba Found Symp. 204:130-8; discussion 138-41; Katz, 1998, Curr Opin Immunol. l(2):213-9; and Goldsby et al, 2003, Immunology, 5th Edition W. H. Freeman & Company, New York, NY. In particular, the receptor binding domain can bind to α2-MR, which is also expressed on the surface of antigen presenting cells. Thus, in certain embodiments, the receptor binding domain can bind to a receptor that is present on both an epithelial cell and on an antigen presenting cell.

[0082] In certain embodiments, the receptor binding domains can bind to a cell surface receptor that is selected from the group consisting of α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycopolypeptide receptor, CD3, CD4, CD8, chemokine receptor, CD25, CDl IB, CDl 1C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, and VEGF receptor.

[0083] In certain embodiments, the chimeric irnrnunogens of the invention comprise more than one domain that can function as a receptor binding domain. For example, the chimeric immunogen could comprise PE domain Ia in addition to another receptor binding domain.

[0084] The receptor binding domain can be attached to the remainder of the chimeric immunogen by any method or means known by one of skill in the art to be useful for attaching such molecules, without limitation. In certain embodiments, the receptor binding domain is expressed together with the remainder of the chimeric immunogen as a fusion protein. Such embodiments are particularly useful when the receptor binding domain and the remainder of the immunogen are formed from peptides or polypeptides.

[0085] In other embodiments, the receptor binding domain is connected with the remainder of the chimeric immunogen with a linker. In yet other embodiments, the receptor binding domain is connected with the remainder of the chimeric immunogen without a linker. Either of these embodiments are useful when the receptor binding domain comprises a peptide, polypeptide, protein, lipid, carbohydrate, nucleic acid, or small organic molecule.

[0086] In certain embodiments, the linker can form a covalent bond between the receptor binding domain and the remainder of the chimeric immunogen. In other embodiments, the linker can link the receptor binding domain to the remainder of the chimeric immunogen with one or more non-covalent interactions of sufficient affinity. One of skill in the art can readily recognize linkers that interact with each other with sufficient affinity to be useful in the chimeric immunogens of the invention. For example, biotin can be attached to the receptor binding domain, and streptavidin can be attached to the remainder of the molecule. In certain embodiments, the linker can directly link the receptor binding domain to the remainder of the molecule. In other embodiments, the linker itself comprises two or more molecules that associate in order to link the receptor binding domain to the remainder of the molecule. Exemplary linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, substituted carbon linkers, unsaturated carbon linkers, aromatic carbon linkers, peptide linkers, etc.

[0087] In embodiments where a linker is used to connect the receptor binding domain to the remainder of the chimeric immunogen, the linkers can be attached to the receptor binding domain and/or the remainder of the chimeric immunogen by any means or method known by one of skill in the art without limitation. For example, the linker can be attached to the receptor binding domain and/or the remainder of the chimeric immunogen with an ether, ester, thioether, thioester, amide, imide, disulfide or other suitable moiety. The skilled artisan can select the appropriate linker and means for attaching the linker based on the physical and chemical properties of the chosen receptor binding domain and the linker. The linker can be attached to any suitable functional group on the receptor binding domain or the remainder of the molecule. For example, the linker can be attached to sulfhydryl (-S), carboxylic acid (COOH) or free amine (-NH2) groups, which are available for reaction with a suitable functional group on a linker. These groups can also be used to connect the receptor binding domain directly connected with the remainder of the molecule in the absence of a linker.

[0088] Further, the receptor binding domain and/or the remainder of the chimeric immunogen can be derivatized in order to facilitate attachment of a linker to these moieties. For example, such derivatization can be accomplished by attaching suitable derivative such as those available from Pierce Chemical Company, Rockford, Illinois. Alternatively, derivatization may involve chemical treatment of the receptor binding domain and/or the remainder of the molecule. For example, glycol cleavage of the sugar moiety of a carbohydrate or glycoprotein receptor binding domain with periodate generates free aldehyde groups. These free aldehyde groups may be reacted with free amine or hydrazine groups on the remainder of the molecule in order to connect these portions of the molecule. See U.S. Patent No.4,671, 958. Further, the skilled artisan can generate free sulfhydryl groups on proteins to provide a reactive moiety for making a disulfide, thioether, theioester, etc. linkage. See U.S. Pat. No. 4,659,839.

[0089] Any of these methods for attaching a linker to a receptor binding domain and/or the remainder of a chimeric immunogen can also be used to connect a receptor binding domain with the remainder of the chimeric immunogen in the absence of a linker. In such embodiments, the receptor binding domain is coupled with the remainder of the immunogen using a method suitable for the particular receptor binding domain. Thus, any method suitable for connecting a protein, peptide, polypeptide, nucleic acid, carbohydrate, lipid, or small organic molecule to the remainder of the chimeric immunogen known to one of skill in the art, without limitation, can be used to connect the receptor binding domain to the remainder of the immunogen. In addition to the methods for attaching a linker to a receptor binding domain or the remainder of an immunogen, as described above, the receptor binding domain can be connected with the remainder of the immunogen as described in U.S. Patent Nos. 6,673,905; 6,585,973; 6,596,475; 5,856,090; 5,663,312; 5,391,723; 6,171,614; 5,366,958; and 5,614,503.

[0090] In certain embodiments, the receptor binding domain can be a monoclonal antibody or antigen-binding portion of an antibody . In some of these embodiments, the chimeric immunogen is expressed as a fusion protein that comprises an immunoglobulin heavy chain from an immunoglobulin specific for a receptor on a cell to which the chimeric immunogen is intended to bind, or antigen-binding portion thereof. The light chain of the immunoglobulin, or antigen-binding portion thereof, then can be co-expressed with the chimeric immunogen, thereby forming an antigen-binding light chain-heavy chain dimer. In other embodiments, the antibody, or antigen-binding portion thereof, can be expressed and assembled separately from the remainder of the chimeric immunogen and chemically linked thereto.

5.2.2. Translocation Domain

[0091] The chimeric immunogens of the invention also comprise a translocation domain. The translocation domain can be any translocation domain known by one of skill in the art to effect translocation of chimeric proteins that have bound to a cell surface receptor from outside the cell to inside the cell, e.g., the outside of an epithelial cell, such as, for example, a polarized epithelial cell. In certain embodiments, the translocation domain is a translocation domain from PE, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, or shiga-like toxin. See, for example, U.S. Patent Nos. 5,965,406, and 6,022,950. In preferred embodiments, the translocation domain is domain II of PE. In certain embodiments, the translocation domain of domain II of PE has an amino acid sequence that is SEQ ID NO:6. [0092] The translocation domain need not, though it may, comprise the entire amino acid sequence of domain II of native PE, which spans residues 253-364 of PE. For example, the translocation domain can comprise a portion of PE that spans residues 280-344 of domain II of PE. The amino acids at positions 339 and 343 appear to be necessary for translocation. See Siegall et at, 1991, Biochemistry 30:7154-59. Further, conservative or nonconservative substitutions can be made to the amino acid sequence of the translocation domain, as long as translocation activity is not substantially eliminated. A representative assay that can routinely be used by one of skill in the art to determine whether a translocation domain has translocation activity is described below.

[0093] Without intending to be limited to any particular theory or mechanism of action, the translocation domain is believed to perform at least two important functions in the chimeric immunogens of the invention. First, the translocation domain permits the trafficking of the chimeric immunogen through a polarized epithelial cell into the bloodstream after the immunogen binds to a receptor present on the apical surface of the polarized epithelial cell. This trafficking results in the release of the chimeric immunogen from the basal-lateral membrane of the polarized epithelial cell. Second, the translocation domain facilitates endocytosis of the chimeric immunogen into an antigen presenting cell after the immunogen binds to a receptor present on the surface of the antigen presenting cell.

5.2.3. Heterologous Antigen

[0094] The chimeric immunogens of the invention also comprise a heterologous antigen. The antigen is "heterologous" because it is heterologous to a portion of the remainder of the immunogen; i.e., not ordinarily found in a molecule from which one of the other domains of the chimeric immunogen is derived. The heterologous antigen can be any molecule, macromolecule, combination of molecules, etc. against which an immune response is desired. Thus, the heterologous antigen can be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which the skilled artisan wishes to induce an immune response. Preferably, the heterologous antigen is an antigen that is present on a pathogen. More preferably, the heterologous antigen is an antigen that, when administered to a subject as part of a chimeric immunogen, results in an immune response against the heterologous antigen that protects the subject from infection by a pathogen from which the heterologous antigen is derived. [0095] The heterologous antigen can be attached to the remainder of the chimeric immunogen by any method known by one of skill in the art without limitation. In certain, embodiments, the heterologous antigen is expressed together with the remainder of the chimeric immunogen as a fusion protein. In such embodiments, the heterologous antigen can be inserted into or replace any portion of the chimeric immunogen, so long as the receptor binding domain, the translocation domain, and the optional ER retention signal domain retain their activities, and the immune response induced against the heterologous antigen retains specificity. Methods for assessing the specificity of the immune response against the heterologous antigen are extensively described below. The heterologous antigen is preferably inserted into or replaces all or a portion of the Ib loop of PE, into the ER retention domain, or attached to or near the C-terminal end of the translocation domain.

[0096] In native PE, the Ib loop (domain Ib) spans amino acids 365 to 399, and is structurally characterized by a disulfide bond between two cysteines at positions 372 and 379. This portion of PE is not essential for any known activity of PE, including cell binding, translocation, ER retention or ADP ribosylation activity. Accordingly, domain Ib can be deleted entirely, or modified to contain a heterologous antigen.

[0097] Thus, in certain embodiments, the heterologous antigen can be inserted into domain Ib. If desirable, the heterologous antigen can be inserted into domain Ib wherein the cysteines at positions 372 and 379 are not crosslinked. This can be accomplished by reducing the disulfide linkage between the cysteines, by deleting one or both of the cysteines entirely from the Ib domain, by mutating one or both of the cysteines to other residues, such as, for example, serine, or by other similar techniques. Alternatively, the heterologous antigen can be inserted into the Ib loop between the cysteines at positions 372 and 379. In such embodiments, the disulfide linkage between the cysteines can be used to constrain the heterologous antigen domain.

[0098] This arrangement offers several advantages. The chimeric immunogens can be used in this manner to present heterologous antigens that naturally comprise a cysteine-cysteine disulfide bond in native or near-native conformation. Further, without intending to be bound to any particular theory or mechanism of action, it is believed that charged amino acid residues in the native Ib domain result in a hydrophilic structure that protrudes from the molecule and into the solvent. Thus, inserting the heterologous antigen into the Ib loop gives immune system components unfettered access to the antigen, resulting in more effective antigen presentation. Such access is particularly useful the heterologous antigen is a B cell antigen for inducing a humoral immune responses. Further, changes, including mutations or insertions, to domain Ib do not appear to affect activity of the other PE domains. Accordingly, although native Ib domain has only six amino acids between the cysteine residues, much longer sequences can be inserted into the loop without disrupting the other functions of the chimeric immunogen.

[0099] In other embodiments, the heterologous antigen can be inserted into the optional ER retention domain of the chimeric immunogen. Without intending to be bound to any particular theory or mechanism of action, it is believed that the nature of the immune response against the heterologous antigen varies depending on the degree of separation between the antigen and the ER retention signal. In particular, the degree to which the heterologous antigen is processed by the Class I or II MHC pathways can vary depending on this degree of separation. By placing the heterologous antigen close to the ER retention signal, e.g., inserting the heterologous antigen into the ER retention domain of the chimeric immunogen near the ER retention signal, more of the heterologous antigen can be directed into the Class I MHC processing pathway, thereby inducing a cellular immune response. Conversely, when the heterologous antigen is further from the ER retention signal, more of the antigen is directed into the Class II MHC processing pathway, thereby facilitating induction of a humoral immune response. If the immune response is intended to be primarily humoral, with essentially no Class I MHC cell mediated response, the ER retention domain can be deleted entirely, and the heterologous antigen attached to the immunogen in another location, such as, for example, to the C terminus of the translocation domain. Thus, by controlling the spatial relationship between the heterologous antigen and the ER retention signal, the skilled artisan can modulate the immune response that is induced against the heterologous antigen.

[0100] In embodiments where the heterologous antigen is expressed together with another portion of the chimeric immunogen as a fusion protein, the heterologous antigen can be can be inserted into the chimeric immunogen by any method known to one of skill in the art without limitation. For example, amino acids corresponding to the heterologous antigen can be directly into the chimeric immunogen, with or without deletion of native amino acid sequences. In certain embodiments, all or part of the Ib domain of PE can be deleted and replaced with the heterologous antigen. In certain embodiments, the cysteine residues of the Ib loop are deleted so that the heterologous antigen remains unconstrained. In other embodiments, the cysteine residues of the Ib loop are linked with a disulfide bond and constrain the heterologous antigen.

[0101] In embodiments where the heterologous antigen is not expressed together with the remainder of the chimeric immunogen as a fusion protein, the heterologous antigen can be connected with the remainder of the chimeric immunogen by any suitable method known by one of skill in the art, without limitation. More specifically, the exemplary methods described above for connecting a receptor binding domain to the remainder of the molecule are equally applicable for connecting the heterologous antigen to the remainder of the molecule.

[0102] In certain embodiments, the heterologous antigen is a peptide, polypeptide, or protein. The heterologous antigen can be any peptide, polypeptide, or protein against which an immune response is desired to be induced. In certain embodiments, the heterologous antigen is a peptide that comprises about 5, about 8, about 10, about 12, about 15, about 17, about 20, about 25, about 30, about 40, about 50, or about 60, about 70, about 80, about 90, about 100, about 200, about 400, about 600, about 800, or about 1000 amino acids. In certain embodiments, the heterologous antigen is ovalbumin, or a portion thereof. In further embodiments, the heterologous antigen is a peptide derived from ovalbumin. In a preferred embodiment, the heterologous antigen is a ovalbumin and has an amino acid sequence that is SEQ ID NO.:2.

[0103] In certain embodiments, the heterologous antigen is a carbohydrate. The heterologous antigen can be any carbohydrate against which an immune response is desired to be induced. In certain embodiments, the heterologous antigen is a carbohydrate that comprises about 1, about 2, about 3, about 4, about 5, about 8, about 10, about 12, about 15, about 17, about 20, about 25, about 30, about 40, about 50, or about 60, about 70, about 80, about 90, or about 100 sugar monomers.

[0104] In other embodiments, the heterologous antigen can be a glycoprotein, or a portion thereof. The heterologous antigen can be any glycoprotein, or portion of a glycoprotein, against which an immune response is desired to be induced. In certain embodiments, the heterologous antigen is a glycoprotein or glycoprotein portion that comprises about 5, about 8, about 10, about 12, about 15, about 17, about 20, about 25, about 30, about 40, about 50, or about 60, about 70, about 80, about 90, about 100, about 200, about 400, about 600, about 800, or about 1000 amino acids.

[0105] In addition to the protein component, the glycoprotein or glycoprotein portion also comprises a carbohydrate moiety. The carbohydrate moiety of the glycoprotein or glycoprotein portion comprises about 1, about 2, about 3, about 4, about 5, about 8, about 10, about 12, about 15, about 17, about 20, about 25, about 30, about 40, about 50, or about 60, about 70, about 80, about 90, or about 100 sugar monomers.

[0106] In general, the skilled artisan may select the heterologous antigen at her discretion, guided by the following discussion. One important factor in selecting the heterologous antigen is the type of immune response that is to be induced. For example, when a humoral immune response is desired, the heterologous antigen should be selected to be recognizable by a B-cell receptor and to be antigenically similar to a region of the source molecule that is available for antibody binding.

[0107] Important factors to consider when selecting a B-cell antigen include, but are not limited to, the size and conformation of the antigenic determinant to be recognized, both in the context of the chimeric immunogen and in the native molecule from which the heterologous antigen is derived; the hydrophobicity or hydrophilicity of the heterologous antigen; the topographical accessibility of the antigen in the native molecule from which the heterologous antigen is derived; and the flexibility or mobility of the portion of the native molecule from which the heterologous antigen is derived. See, e.g., Kuby, 1997, Immunology Chapter 4, W.H. Freeman and Company, New York. Based on these criteria, the skilled artisan can, when appropriate, select a portion of a large molecule, such as a protein, to be the heterologous antigen. If the source of the heterologous antigen cannot be effectively represented by selecting a portion of it, then the skilled artisan can select the entire molecule to be the heterologous antigen. Such embodiments are particularly useful in the cases of B- cell antigens that are formed by non-sequential amino acids, i.e., antigens formed by amino acids that are not adjacent in the primary structure of the source protein.

[0108] Similarly, if the skilled artisan wishes to deliver a heterologous antigen to activate T cells, several factors must be considered in the selection of the heterologous antigen. Principle among such factors is whether helper T cells or cytotoxic T cells are to be stimulated. As described below, helper T cells recognize antigen presented by Class II MHC molecules, while cytotoxic T cells recognize antigen present by Class I MHC. Accordingly, in order to selectively activate these populations, the skilled artisan should select the heterologous antigen to be presentable by the appropriate type of MHC. For example, the skilled artisan can select the heterologous antigen to be a peptide that is presented by Class I MHC when a response mediated by cytotoxic T cells is desired. Similarly, the skilled artisan can select the heterologous antigen to be a peptide that is presented by Class II MHC when a response mediated by helper T cells is desired.

[0109] Further, both Class I and Class II MHC exhibit significant allelic variation in studied populations. Much is known about Class I and II MHC alleles and the effects of allelic variation on antigens that can be presented by the different alleles. For example, rules for interactions between Class I MHC haplotype and antigens that can be effectively presented by these molecules are reviewed in Stevanovic, 2002, Transpl Immunol 10:133-136. Further guidance on selection of appropriate peptide antigens for Class I and II MHC molecules may be found in US Patent Nos. 5,824,315 and 5,747,269, and in Germain et al , 1993, Annu. Rev. Immunol. 11 :403-450; Sinigaglia et al, 1994, Curr. Opin. Immunol. 6:52-56; Margalit et al, 2003, Novartis FoundSymp. 254:77-101, 216-22, and250-252; Takahashi, 2003, Comp Immunol Microbiol Infect Dis. 26:309-328; Yang, 2003, Microbes Infect. 5:39-47; and Browning et al, 1996, HLA and MHC: Genes, Molecules and Function (Davenport and Hill, eds.) A BIOS Scientific Publishers, Oxford. An empirical system for identifying peptide antigens for presentation on Class II MHC, and that can be adapted for identifying peptide antigens for presentation on Class I MHC, is presented in US Patent No. 6,500,641.

[0110] Further, the chimeric immunogen can comprise one or more antigens in addition to ovalbumin, or a portion thereof, which can be a molecule that potentiates an immune response. Any antigen that can act as immune stimulant known by one of skill in the art without limitation can be used as an antigen in such embodiments. For example, the heterologous antigen can be a nucleic acid with an unmethylated CpG motif, with a methylated CpG motif, or without any CpG motifs, as described in U.S. Patent Nos. 6,653,292 and 6,239,116 and Published U.S. Application 20040152649, lipopolysaccharide (LPS) or an LPS derivative such as mono- or diphosphoryl lipid A, or any of the LPS derivatives or other adjuvants described in U.S. Patent Nos. 6,716,623, 6,720,146, and 6,759,241. 5.2.4. Endoplasmic Reticulum Retention Domain

[0111] The chimeric immunogens of the invention can optionally comprise an endoplasmic reticulum retention domain. This domain comprises an endoplasmic reticulum signal sequence, which functions in translocating the chimeric immunogen from the endosome to the endoplasmic reticulum, and from thence into the cytosol. Native PE comprises an ER retention domain in domain III. The ER retention domain comprises an ER retention signal sequence at its carboxy terminus. In native PE, this ER retention signal is REDLK (SEQ ID NO.:7). The terminal lysine can be eliminated (i.e., REDL (SEQ ID NO.:2)) without an appreciable decrease in activity. However, any ER retention signal sequence known to one of skill in the art without limitation can be used in the chimeric immunogens of the invention. Other suitable ER retention signal sequences include, but are not limited to, KDEL (SEQ ID NO.:3), or dimers or multimers of these sequences. See Ogata et ah, 1990, J Biol. Chem. 265:20678-85; U.S. Patent 5,458,878; and Pastan et al, 1992, Amu. Rev. Biochem. 61:331- 54.

[0112] In certain embodiments, the chimeric immunogen comprises domain III of native PE, or a portion thereof. Preferably, the chimeric immunogen comprises domain III of ΔE553 PE. In certain embodiments, domain III, including the ER retention signal, can be entirely eliminated from the chimeric immunogen. In other embodiments, the chimeric immunogen comprises an ER retention signal sequence and comprises a portion or none of the remainder of PE domain III. In certain embodiments, the portion of PE domain III other than the ER retention signal can be replaced by another amino acid sequence. This amino acid sequence can itself be non immunogenic, slightly immunogenic, or highly immunogenic. A highly immunogenic ER retention domain is preferable for use in eliciting a humoral immune response. For example, PE domain III is itself highly immunogenic and can be used in chimeric immunogens where a robust humoral immune response is desired. Chimeras in which the ER retention domain is only slightly immunogenic will be more useful when an Class I MHC-dependent cell-mediated immune response is desired.

[0113] ER retention domain activity can routinely be assessed by those of skill in the art by testing for translocation of the protein into the target cell cytosol using the assays described below.

[0114] In native PE, the ER retention sequence is located at the C-terminus of domain III. Native PE domain III has at least two observable activities. Domain III mediates ADP- ribosylation and therefore toxicity. Further, the ER retention signal present at the C-terminus directs endocytosed toxin into the endoplasmic reticulum and from thence, into the cytosol. Eliminating the ER retention sequence from the chimeric immunogens does not alter the activity of Pseudomonas exotoxin as a superantigen, but does prevent it from eliciting an MHC Class I-dependent cell-mediated immune response.

[0115] The PE domain that mediates ADP -ribosylation is located between about amino acids 400 and 600 of PE. This toxic activity of native PE is preferably eliminated in the chimeric immunogens of the invention. By doing so, the chimeric immunogen can be used as a vehicle for delivering heterologous antigens to be processed by the cell and presented on the cell surface with MHC Class I or Class II molecules, as desired, rather than as a toxin. ADP ribosylation activity can be eliminated by, for example, deleting amino acid E553. See, e.g., Lukac et al, 1988, Infect, andlmmun. 56:3095-3098. Alternatively, the amino acid sequence of domain III, or portions of it, can be deleted from the protein. Of course, an ER retention sequence should be included at the C-terminus if a Class I MHC -mediated immune response is to be induced.

[0116] In certain embodiments, the ER retention domain is substantially identical to the native amino acid sequences of PE domain III, or a fragment thereof. In certain embodiments, the ER retention domain is domain III of PE. In other embodiments, the ER retention domain is domain III of ΔE553 PE. In still other embodiments, the ER retention domain comprises an amino acid sequence that is selected from the group consisting of RDELK, RDEL, and KDEL.

5.3. Methods for Inducing an Immune Response

[0117] In another aspect, the invention provides methods of inducing an immune response against a heterologous antigen. The methods allow one of skill in the art to induce a cellular, humoral, or secretory immune response. These methods generally rely on administration of a chimeric immunogen of the invention to a subject in whom the immune response is to be induced. As described above, the chimeric immunogens can be used to induce an immune response that is specific for a heterologous antigen.

[0118] Accordingly, the invention provides methods for inducing an immune response against a heterologous antigen. In certain embodiments, the methods comprise administering to a subject in whom the immune response is to be induced a chimeric immunogen bearing the heterologous antigen. The chimeric immunogen can be administered as a composition, as described below.

[0119] In certain aspects, the invention provides a method for inducing an immune response in a subject that comprising administering to the subject an effective amount of a chimeric immunogen comprising a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. In certain embodiments, administration of the chimeric immunogen induces a humoral immune response. In certain embodiments, administration of the chimeric immunogen induces a cell-mediated immune response. In certain embodiments, administration of the chimeric immunogen induces a secretory immune response. In certain embodiments, administration of the chimeric immunogen induces a humoral and a cell-mediated immune response. In certain embodiments, administration of the chimeric immunogen induces a humoral and secretory immune response. In certain embodiments, administration of the chimeric immunogen induces a cell-mediated and secretory immune response. In certain embodiments, administration of the chimeric immunogen induces a humoral, cell-mediated and secretory immune response.

[0120] In certain embodiments, the subject is a mouse, rat, rabbit, chicken, goat, sheep, cow, or horse. In certain embodiments, the chimeric immunogen is administered to said subject nasally or orally.

[0121] In certain embodiments, the chimeric immunogen is administered in the form of a pharmaceutical composition that comprises the chimeric immunogen and a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In certain embodiments, the pharmaceutical composition is formulated for nasal or oral administration.

[0122] In other embodiments, the invention provides a method for generating in a subject antibodies specific for one or more ovalbumin epitope(s). The method comprises administering to the subject an effective amount of a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. Administration of such chimeric immunogens generates antibodies specific for one or more ovalbumin epitope(s).

[0123] In certain embodiments, the subject is a mammal. In further embodiments, the subject is a rodent, lagomorph or primate. In a preferred embodiments, the subject is a mouse. 5.3.1. Humoral Immune Responses

[0124] In certain embodiments, the invention provides a method for inducing a humoral immune response against the heterologous antigen in a subject. The methods generally comprise administering to a subject a chimeric immunogen that is configured to produce a humoral immune response. Such immune responses generally involve the production of antibodies specific for at least one epitope of the heterologous antigen. Certain embodiments of the chimeric immunogens have properties that allow the skilled artisan to induce a humoral immune response against the heterologous antigen. For example, when the heterologous antigen is inserted into PE domain Ib, the flanking cysteines cause the heterologous antigen to be extended from the remainder of the immunogen and facilitate recognition of the antigen by a B cell through an interaction with a B -cell receptor. Interaction between the heterologous antigen and the B cell receptor stimulates clonal expansion of the B cell bearing the receptor, eventually resulting in a population of plasma cells that secrete antibodies specific for the antigen.

[0125] In most circumstances, B cell recognition of antigen is necessary, but not sufficient, to induce a robust humoral immune response. The humoral response is greatly potentiated by CD4⁺ (helper) T cell signaling to B cells primed by antigen recognition. Helper T cells are activated to provide such signals to B cells by recognition of antigen processed through the Class II MHC pathway. The antigen recognized by the T cell can, but need not, be the same antigen recognized by the B cell. The chimeric immunogens of the invention can be targeted to such antigen presenting cells for processing in the Class II MHC pathway in order to stimulate helper T cells to activate B cells. By doing so, the chimeric immunogens can be used to stimulate a robust humoral immune response that is specific for the heterologous antigen.

[0126] Further, the chimeric immunogens are attractive vehicles for inducing a humoral immune response against heterologous antigens that are constrained within their native environment. By inserting the heterologous antigen into the Ib loop of PE antigens, the antigen can be presented to immune cells in near-native conformation. The resulting antibodies generally recognize the native antigen better than those raised against unconstrained versions of the heterologous antigen. The Ib loop can also be used to present B cell antigens that are not constrained in their native environment. In such embodiments, the antigen inserted into the Ib loop should be flanked by a sufficient number of amino acids that give conformational flexibility, such as, e.g., glycine, serine, etc., to allow the antigen to fold into its native form and avoid constraint by the disulfide linkage between the cysteines of the Ib loop.

[0127] In addition, the method of administration of the chimeric immunogen can also affect the type of immune response that is induced with a chimeric immunogen of the invention. In particular, parenteral administration, rather than administration to a mucous membrane of the subject, is believed to favor a humoral rather than secretory immune response. Accordingly, in certain embodiments, the methods of inducing a humoral immune response comprise administering a chimeric immunogen of the invention to a subject parenterally. In certain embodiments, the administration is intramuscular. In other embodiments, the administration is subcutaneous. In still other embodiments, the administration is intravenous or intra¬ arterial.

[0128] The humoral immune response induced by the chimeric immunogens can be assessed using any method known by one of skill in the art without limitation. For example, an animal's immune response against the heterologous antigen can be monitored by taking test bleeds and determining the titer of antibody reactivity to the heterologous antigen. When appropriately high titers of antibody to the heterologous antigen are obtained, blood can be collected from the animal and antisera prepared. The antisera can be further enriched for antibodies reactive to the heterologous antigen, when desired. See, e.g., Coligan, 1991, Current Protocols in Immunology, Greene Publishing Associates and Wiley Interscience, NY; and Harlow and Lane, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY.

[0129] Antibodies produced in response to administration of the chimeric immunogens can then be used for any purpose known by one of skill in the art, without limitation. The antibodies are believed to be equivalent to antibodies induced using conventional techniques, such as coupling peptides to an immunogen. For example, the antibodies can be used to make monoclonal antibodies, humanized antibodies, chimeric antibodies or antibody fragments. Techniques for producing such antibody derivatives may be found in, for example, Stites et al. eds., 1997, Medical Immunology (9th ed.)₅ McGraw-Hill/ Appleton & Lange, CA; Harlow and Lane, 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY; Goding, 1986, Monoclonal Antibodies: Principles and Practice (2d ed.), Academic Press, NY; Kohler and Milstein, 1975, Nature 256: 495-497; and U.S. Patent No. 5,585,089.

5.3.2. Cell-Mediated Immune Responses

[0130] In other embodiments, the invention provides methods for eliciting a cell-mediated immune response against cells expressing the heterologous antigen. The methods generally comprise administering to a subject a chimeric immunogen that comprises the heterologous antigen that is configured to produce a cell-mediated immune response. Such immune responses generally involve the activation of cytotoxic T lymphocytes that can recognize and kill cells that display the antigen on their surfaces. However, certain aspects of humoral immune responses give rise to cell-mediated effects as well, as described below. Certain embodiments of the chimeric immunogens have properties that allow the skilled artisan to induce a cell-mediated immune response against the heterologous antigens.

[0131] In particular, heterologous antigens that are inserted into a chimeric immunogen near a ER retention signal tend to induce a cell-mediated immune response. Without intending to be bound to any particular theory or mechanism of action, it is believed that the ER retention signal causes the chimeric immunogen to be trafficked from an endosome to the ER, and from thence into the cytosol. Once in the cytosol, peptides from the immunogen, including the heterologous antigen, enter the Class I MHC processing pathway. The peptides associate with Class I MHC and are presented on the surface of the cell into which the immunogen has been introduced. CD 8 (cytotoxic) T lymphocytes then recognize the heterologous antigen in association with Class I MHC and thereby become activated and primed to kill cells that similarly have the heterologous antigen associated with Class I MHC on their surfaces.

[0132] Part of the processing that occurs during presentation on Class I MHC is believed to result in degradation of the chimeric immunogen into peptides that can associate with the MHC molecule. This proteolysis is believed to begin in the endosome and to continue in the cytosol. If, in the course of this process, the heterologous antigen is separated from the ER retention signal before the heterologous antigen is trafficked to the cytosol, it is believed that the heterologous antigen cannot associate with Class I MHC. In such circumstances, the heterologous antigen can remain in the endosome, and can be directed to the Class II MHC processing pathway. Accordingly, it is believed that the distance, e.g., the number of amino acids, between the heterologous antigen and the ER retention signal can affect the degree to which the antigen is presented in association with Class I or Class II MHC. [0133] Features of peptides that associate with the various allelic forms of Class I MHC have been well characterized. For example, peptides bound by HLA-Al generally comprise a first conserved residue of T, S or M, a second conserved residue of D or E, and a third conserved residue of Y, wherein the first and second residues are adjacent, and both are separated from the third residue by six or seven amino acids. Peptides that bind to other alleles of Class I MHC have also been characterized. Using this knowledge, the skilled artisan can select heterologous antigens that can associate with a Class I MHC allele that is expressed in the subject. By administering chimeric immunogens comprising such antigens near the ER retention signal, a cell-mediated immune response can be induced.

[0134] Cell-mediated immune responses can also arise as a consequence of humoral immune responses. Antibodies produced in the course of the humoral immune response bind to their cognate antigen; if this antigen is present on the surface of a cell, the antibody binds to the cell surface. Cells bound by antibodies in this manner are subject to antibody-dependent cell- mediated cytotoxicity, in which immune cells that bear Fc receptors attack the marked cells. For example, natural killer cells and macrophages have Fc receptors and can participate in this phenomenon.

5.3.3. Secretory Immune Response

[0135] In other embodiments, the invention provides methods for eliciting a secretory immune response against the heterologous antigen. The methods generally comprise administering to a mucous membrane of the subject a chimeric immunogen that comprises the heterologous antigen that is configured to bind to a receptor present on the mucous membrane. The mucous membrane can be any mucous membrane known by one of skill in the art to be present in the subject, without limitation. For example, the mucous membrane can be present in the eye, nose, mouth, trachea, lungs, esophagus, stomach, small intestine, large intestine, rectum, anus, sweat glands, vulva, vagina, or penis of the subject. Certain embodiments of the chimeric immunogens have properties that allow the skilled artisan to induce a secretory immune response against the heterologous antigens.

[0136] In particular, chimeric immunogens that comprise receptor binding domains that can bind to a receptor present on the apical membrane of an epithelial cell can be used to induce a secretory immune response. Such receptor binding domains are extensively described above. Without intending to be bound by any particular theory or mechanism of action, it is believed that the original encounter with the antigen at the mucosal surface directs the immune system to produce a secretory rather than humoral immune response.

[0137] Secretory immune responses are desirable for protecting against any pathogen that enters the body through a mucous membrane. Mucous membranes are primary entryways for many infectious pathogens, including, for example, HIV, herpes, vaccinia, cytomegalovirus, yersinia, vibrio, and Pseudomonas spp. Mucous membranes can be found in the mouth, nose, throat, lung, vagina, rectum and colon. As one defense against entry by these pathogens, the body secretes secretory IgA from mucosal epithelial membranes that can bind the pathogens and prevent or deter pathogenesis. Furthermore, antigens presented at one mucosal surface can trigger responses at other mucosal surfaces due to trafficking of antibody-secreting cells between the mucous membranes. The structure of secretory IgA appears to be crucial for its sustained residence and effective function at the luminal surface of a mucous membrane. "Secretory IgA" or "slgA" generally refers to a polymeric molecule comprising two IgA immunoglobulins joined by a J chain and further bound to a secretory component. While mucosal administration of antigens can generate an IgG response, parenteral administration of immunogens rarely produces strong slgA responses.

[0138] The chimeric immunogens can be administered to the mucous membrane of the subject by any suitable method or in any suitable formulation known to one of skill in the art without limitation. For example, the chimeric immunogens can be administered in the form of liquids or solids, e.g., sprays, ointments, suppositories or erodible polymers impregnated with the immunogen. Administration can involve applying the immunogen to a one or more different mucosal surfaces. Further, in certain embodiments, the chimeric immunogen can be administered in a single dose. In other embodiments, the chimeric immunogen can be administered in a series of two or more administrations. In certain embodiments, the second or subsequent administration of the chimeric immunogen is administered parenterally, e.g., subcutaneously or intramuscularly.

[0139] The slgA response is strongest on mucosal surfaces exposed to the immunogen. Therefore, in certain embodiment, the immunogen is applied to a mucosal surface that is likely to be a site of exposure to the pathogen. Accordingly, chimeric immunogens against pathogens encountered on vaginal, anal, or oral mucous membranes are preferably administered to vaginal, anal or oral mucosal surfaces, respectively. However, nasal administration of the chimeric immunogens can also induce robust secretory immune responses from other mucous membranes. See, for example, Boyaka et ah, 2003, Cur. Pharm. Des. 9:1965-1972.

[0140] Mucosal administration of the chimeric immunogens of this invention result in strong memory responses, both for IgA and IgG. These memory responses can advantageously be boosted by re-administering the chimeric immunogen after a period of time. Such booster administrations can be administered either mucosally or parenterally. The memory response can be elicited by administering a booster dose more than a year after the initial dose. For example, a booster dose can be administered about 12, about 16, about 20 or about 24 months after the initial dose.

5.4. Polynucleotides Encoding Chimeric Immunogens

[0141] In another aspect, the invention provides polynucleotides comprising a nucleotide sequence encoding a chimeric immunogen of the invention. These polynucleotides are useful, for example, for making the chimeric immunogens. In yet another aspect, the invention provides an expression system that comprises a recombinant polynucleotide sequence encoding a receptor binding domain, a translocation domain, an optional ER retention domain, and an insertion site for a polynucleotide sequence encoding a heterologous antigen. The insertion site can be anywhere in the polynucleotide sequence so long as the insertion does not disrupt the receptor binding domain, the translocation domain, or the optional ER retention domain. Preferably, the insertion site is between the translocation domain and the ER retention domain. In other equally preferred embodiments, the insertion site is in the ER retention domain.

[0142] In certain embodiments, the recombinant polynucleotides are based on polynucleotides encoding PE, or portions or derivatives thereof. In other embodiments, the recombinant polynucleotides are based on polynucleotides that hybridize to a polynucleotide that encodes PE under stringent hybridization conditions. A nucleotide sequence encoding PE is presented as SEQ ID NO.:8. This sequence can be used to prepare PCR primers for isolating a nucleic acid that encodes any portion of this sequence that is desired. For example, PCR can be used to isolate a nucleic acid that encodes one or more of the functional domains of PE. A nucleic acid so isolated can then be joined to nucleic acids encoding other functional domains of the chimeric immunogens using standard recombinant techniques. [0143] Other in vitro methods that can be used to prepare a polynucleotide encoding PE, PE domains, or any other functional domain useful in the chimeric immunogens of the invention include, but are not limited to, reverse transcription, the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self- sustained sequence replication system (3SR) and the QP replicase amplification system (QB). Any such technique known by one of skill in the art to be useful in construction of recombinant nucleic acids can be used. For example, a polynucleotide encoding the protein or a portion thereof can be isolated by polymerase chain reaction of cDNA using primers based on the DNA sequence of PE or another polynucleotide encoding a receptor binding domain.

[0144] Guidance for using these cloning and in vitro amplification methodologies are described in, for example, U.S. Patent No. 4,683,195; Mullis et al, 1987, Cold Spring Harbor Symp. Quant. Biol.51 :263; and Erlich, ed, 1989, PCi? Technology, Stockton Press, NY. Polynucleotides encoding a chimeric immunogen or a portion thereof also can be isolated by screening genomic or cDNA libraries with probes selected from the sequences of the desired polynucleotide under stringent, moderately stringent, or highly stringent hybridization conditions.

[0145] Construction of nucleic acids encoding the chimeric immunogens of the invention can be facilitated by introducing an insertion site for a nucleic acid encoding the heterologous antigen into the construct. In certain embodiments, an insertion site for the heterologous antigen can be introduced between the nucleotides encoding the cysteine residues of domain Ib. In other embodiments, the insertion site can be introduced anywhere in the nucleic acid encoding the immunogen so long as the insertion does not disrupt the functional domains encoded thereby. In certain embodiments, the insertion site can be in the ER retention domain. In certain embodiments, the insertion site is introduced into the nucleic acid encoding the chimeric immunogen. In other embodiments, the nucleic acid comprising the insertion site can replace a portion of the nucleic acid encoding the immunogen, as long s the replacement does not disrupt the receptor binding domain or the translocation domain.

[0146] In more specific embodiments, the insertion site comprises that includes a cloning site cleaved by a restriction enzyme. In certain embodiments, the cloning site can be recognized and cleaved by a single restriction enzyme, for example, by Pstl. In such examples, a polynucleotide encoding heterologous antigen that is flanked by Pstl sequences can be inserted into the vector. In other embodiments, the insertion site comprises a polylinker that comprises about one, about two, about three, about four, about five, about ten, about twenty or more cloning sites, each of which can be cleaved by one or more restriction enzymes.

[0147] Further, the polynucleotides can also encode a secretory sequence at the amino terminus of the encoded chimeric immunogen. Such constructs are useful for producing the chimeric immunogens in mammalian cells as they simplify isolation of the immunogen.

[0148] Furthermore, the polynucleotides of the invention also encompass derivative versions of polynucleotides encoding a chimeric immunogen. Such derivatives can be made by any method known by one of skill in the art without limitation. For example, derivatives can be made by site-specific mutagenesis, including substitution, insertion, or deletion of one, two, three, five, ten or more nucleotides, of polynucleotides encoding the chimeric immunogen. Alternatively, derivatives can be made by random mutagenesis. One method for randomly mutagenizing a nucleic acid comprises amplifying the nucleic acid in a PCR reaction in the presence of 0.1 mM MnCl₂ and unbalanced nucleotide concentrations. These conditions increase the misincorporation rate of the polymerase used in the PCR reaction and result in random mutagenesis of the amplified nucleic acid.

[0149] Several site-specific mutations and deletions in chimeric molecules derived from PE have been made and characterized. For example, deletion of nucleotides encoding amino acids 1-252 of PE yields a construct referred to as "PE40." Deleting nucleotides encoding amino acids 1-279 of PE yields a construct referred to as "PE37." See U.S. Patent No. 5,602,095. In both of these constructs, the receptor binding domain of PE, i.e., domain Ia, has been deleted. Nucleic acids encoding a receptor binding domain can be ligated to these constructs to produce chimeric immunogens that are targeted to the cell surface receptor recognized by the receptor binding domain. Of course, these constructs are particularly useful for expressing chimeric immunogens that have a receptor binding domain that is not domain Ia of PE. The constructs can optionally encode an amino-terminal methionine to assist in expression of the construct. In certain embodiments, the receptor binding domain can be ligated to the 5' end of the polynucleotide encoding the translocation domain and optional ER retention domain. In other embodiments, the polynucleotide can be inserted into the constructs in the nucleotide sequence encoding the ER retention domain. [0150] Other nucleic acids encoding mutant forms of PE that can be used as a source of nucleic acids for constructing the chimeric immunogens of the invention include, but are not limited to, PEΔ553 and those described in U.S. Patent Nos. 5,602,095; 5,512,658 and 5,458,878, and in Vasil et al, 1986, Infect. Immunol. 52:538-48.

[0151] Accordingly, in certain aspects, the invention provides a polynucleotide that encodes a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalbumin, or a portion thereof. In certain embodiments, the chimeric immunogen, when administered to a subject, induces an immune response in the subject that is specific for one or more ovalbumin epitope(s).

[0152] In certain embodiments, polynucleotide encodes a chimeric immunogen further comprising an endoplasmic reticulum retention domain. In further embodiments, the ovalbumin heterologous antigen is located between the translocation domain and the endoplasmic reticulum retention domain. In certain embodiments, the endoplasmic reticulum retention domain is an enzymatically-inactive domain III of Pseudomonas exotoxin A. In certain embodiments, the enzymatically inactive domain III of Pseudomonas exotoxin A is inactivated by deleting a glutamate at position 553. In certain embodiments, the endoplasmic reticulum retention domain comprises an amino acid sequence that is selected from the group of RDEL (SEQ ID NO.:2) or KDEL (SEQ ID NO.:3) that is sufficiently near the C-terminus of said endoplasmic reticulum retention domain to result in retention of said chimeric immunogen in the endoplasmic reticulum.

[0153] In certain embodiments, the polynucleotide encodes a translocation domain that is selected from the group consisting translocation domains from Pseudomonas exotoxin A, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin. In certain embodiments, the translocation domain is domain II of Pseudomonas exotoxin A. In further embodiments, the translocation domain comprises amino acids 280 to 364 of domain II of Pseudomonas exotoxin A.

[0154] In certain embodiments, the polynucleotide encodes a receptor binding domain that is selected from the group consisting of domain Ia of Pseudomonas exotoxin A; a receptor binding domains from cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin; a monoclonal antibody, a polyclonal antibody, or a single-chain antibody; TGFα, TGFβ, EGF, PDGF, IGF, or FGF; IL-I, IL-2, IL-3, or IL-6; and MIP-Ia, MIP-Ib, MCAF, or IL-8. In certain embodiments, the receptor binding domain is domain Ia of Pseudomonas exotoxin A. In further embodiments, the domain Ia of Pseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:5.

[0155] In certain embodiments, the receptor binding domain binds to α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycopolypeptide receptor, CD3, CD4, CD8, chemokine receptor, CD25, CDI lB, CDl 1C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, or VEGF receptor. In certain embodiments, the receptor binding domain binds to α2-macroglobulin receptor.

5.5. Expression Vectors

[0156] In still another aspect, the invention provides expression vectors for expressing the chimeric immunogens of the invention. Generally, expression vectors are recombinant polynucleotide molecules comprising expression control sequences operatively linked to a nucleotide sequence encoding a polypeptide. Expression vectors can readily be adapted for function in prokaryotes or eukaryotes by inclusion of appropriate promoters, replication sequences, selectable markers, etc. to result in stable transcription and translation of mRNA. Techniques for construction of expression vectors and expression of genes in cells comprising the expression vectors are well known in the art. See, e.g., Sambrook et al, 2001, Molecular Cloning — A Laboratory Manual, 3^r edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al, eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0157] Useful promoters for use in expression vectors include, but are not limited to, a metallothionein promoter, a constitutive adenovirus major late promoter, a dexamethasone- inducible MMTV promoter, a S V40 promoter, a MRP pol III promoter, a constitutive MPSV promoter, a tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), and a constitutive CMV promoter.

[0158] The expression vectors should contain expression and replication signals compatible with the cell in which the chimeric immunogens are expressed. Expression vectors useful for expressing chimeric immunogens include viral vectors such as retroviruses, adenoviruses and adenoassociated viruses, plasmid vectors, cosmids, and the like. Viral and plasmid vectors are preferred for transfecting the expression vectors into mammalian cells. For example, the expression vector pcDNAl (Invitrogen, San Diego, CA), in which the expression control sequence comprises the CMV promoter, provides good rates of transfection and expression into such cells.

[0159] The expression vectors can be introduced into the cell for expression of the chimeric immunogens by any method known to one of skill in the art without limitation. Such methods include, but are not limited to, e.g., direct uptake of the molecule by a cell from solution; facilitated uptake through lipofection using, e.g., liposomes or immuno liposomes; particle-mediated transfection; etc. See, e.g., U.S. Patent No. 5,272,065; Goeddel et al, eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression — A Laboratory Manual, Stockton Press, NY; Sambrook et al, 1989, Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al. , eds. , Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0160] The expression vectors can also contain a purification moiety that simplifies isolation of the protein. For example, a polyhistidine moiety of, e.g., six histidine residues, can be incorporated at the amino terminal end of the protein. The polyhistidine moiety allows convenient isolation of the protein in a single step by nickel-chelate chromatography. In certain embodiments, the purification moiety can be cleaved from the remainder of the chimeric immunogen following purification. In other embodiments, the moiety does not interfere with the function of the functional domains of the chimeric immunogen and thus need not be cleaved.

5.6. Cell for Expressing a Chimeric Immunogen

[0161] In yet another aspect, the invention provides a cell comprising an expression vector for expression of the chimeric immunogens, or portions thereof. The cell is preferably selected for its ability to express high concentrations of the chimeric immunogen to facilitate purification of the protein. In certain embodiments, the cell is a prokaryotic cell, for example, E. coli. As described in the examples, the chimeric immunogens are properly folded and comprise the appropriate disulfide linkages when expressed in E. coli.

[0162] In other embodiments, the cell is a eukaryotic cell. Useful eukaryotic cells include yeast and mammalian cells. Any mammalian cell known by one of skill in the art to be useful for expressing a recombinant polypeptide, without limitation, can be used to express the chimeric immunogens. For example, Chinese hamster ovary (CHO) cells can be used to express the chimeric immunogens.

5.7. Compositions Comprising Chimeric Immunogens, and Uses Thereof [0163] In yet another aspect, the invention provides compositions comprising one or more chimeric immunogens. The compositions are useful for eliciting an immune response against the heterologous antigen, particularly against pathogens or cells expressing the heterologous antigen. A composition of the invention can include one or a plurality of chimeric immunogens. For example, a composition of the invention can include chimeric immunogens with heterologous antigens from several circulating strains of a pathogen. As the pathogen changes, additional chimeric immunogens can be constructed that include the altered antigens, for example, from breakthrough viruses.

5.7.1. Compositions

[0164] The chimeric immunogens of the invention can be formulated as compositions. The compositions are generally formulated appropriately for its immediate intended use. For example, if the composition is not to be administered immediately, it can be formulated in a manner suitable for storage. One such composition is a lyophilized preparation of the chimeric immunogen together with a suitable stabilizer. Alternatively, the composition can be formulated for storage in a solution with one or more suitable stabilizers. Any such stabilizer known to one of skill in the art without limitation can be used. For example, stabilizers suitable for lyophilized preparations include, but are not limited to, sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino acids. Stabilizers suitable for liquid preparations include, but are not limited to, sugars, salts, surfactants, proteins, chaotropic agents, lipids, and amino acids. Specific stabilizers than can be used in the compositions include, but are not limited to, trehalose, serum albumin, phosphatidylcholine, lecithin, and arginine. Other compounds, compositions, and methods for stabilizing a lyophilized or liquid preparation of the delivery constructs may be found, for example, in U.S. Patent Nos. 6,573,237, 6,525,102, 6,391,296, 6,255,284, 6,133,229, 6,007,791, 5,997,856, and 5,917,021.

[0165] Further, the compositions of the invention can be formulated for administration to a subject. The formulation can be suitable for administration to a nasal, oral, vaginal, rectal, or other mucosal surface. Such compositions generally comprise one or more chimeric immunogens of the invention and a pharmaceutically acceptable excipient, diluent, carrier, or vehicle. Any such pharmaceutically acceptable excipient, diluent, carrier, or vehicle known to one of skill in the art without limitation can be used. Examples of a suitable excipient, diluent, carrier, or vehicle can be found in Remington's Pharmaceutical Sciences, 20th Ed. 2000, Mack Publishing Co., Easton.

[0166] The compositions can also include an adjuvant that potentiates an immune response when used in administered in conjunction with the chimeric immunogen. Useful adjuvants, particularly for administration to human subjects, include, but are not limited to, alum, aluminum hydroxide, aluminum phosphate, CpG-containing oligonucleotides (both methylated and unmethylated), bacterial nucleic acids, lipopolysaccharide and lipopolysaccharide derivatives such as monophosphoryl lipid A, oil-in-water emulsions, etc.. Other suitable adjuvants are described in Sheikh et ah, 2000, Cur. Opin. MoI. Ther. 2:37-54. Adjuvants are most useful when the composition is to be injected rather than administered to a mucosal membrane of the subject. However, certain of the above adjuvants are also known in the art to be useful in compositions to be administered to mucosal surface.

[0167] In certain embodiments, the compositions are formulated for oral administration. In such embodiments, the compositions are formulated to protect the chimeric immunogen from acid and/or enzymatic degradation in the stomach. Upon passage to the neutral to alkaline environment of the duodenum, the chimeric immunogen then contacts a mucous membrane and is transported across the polarized epithelial membrane. The chimeric immunogens may be formulated in such compositions by any method known by one of skill in the art, without limitation.

[0168] In certain embodiments, the oral formulation comprises a chimeric immunogen and one or more compounds that can protect the chimeric immunogen while it is in the stomach. For example, the protective compound should be able to prevent acid and/or enzymatic hydrolysis of the chimeric immunogen. In certain embodiments, the oral formulation comprises a chimeric immunogen and one or more compounds that can facilitate transit of the immunogen from the stomach to the small intestine. In certain embodiments, the one or more compounds that can protect the chimeric immunogen from degradation in the stomach can also facilitate transit of the immunogen from the stomach to the small intestine. Preferably, the oral formulation comprises one or more compounds that can protect the chimeric immunogen from degradation in the stomach and facilitate transit of the immunogen from the stomach to the small intestine. For example, inclusion of sodium bicarbonate can be useful in facilitating the rapid movement of intra-gastric delivered materials from the stomach to the duodenum as described in Mrsny et al., 1999, Vaccine 17:1425-1433.

[0169] Other methods for formulating compositions so that the chimeric immunogens can pass through the stomach and contact polarized epithelial membranes in the small intestine include, but are not limited to, enteric-coating technologies as described in De Young, 1989, Int J Pancreatol. 5 Suppl:31-6, and the methods provided in U.S. Patent Nos. 6,613,332, 6,174,529, 6,086,918, 5,922,680, and 5,807,832.

[0170] Accordingly, in certain aspects, the invention provides a composition comprising a chimeric immunogen that comprises a receptor binding domain, a translocation domain, and a heterologous antigen that is ovalubumin, or a portion thereof. In certain embodiments, the chimeric immunogen, when administered to a subject, induces an immune response in the subject that is specific for one or more ovalbumin epitope(s).

[0171] In certain embodiments, the composition further comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier. In certain embodiments, the composition is formulated for nasal or oral administration.

5.7.2. Dosage

[0172] Generally, a pharmaceutically effective amount of the compositions of the invention is administered to a subject. The skilled artisan can readily determine if the dosage of the chimeric immunogen in the composition is sufficient to elicit an immune response by monitoring the immune response so elicited, as described below. In certain embodiments, an amount of composition corresponding to between about 1 μg and about 1000 μg of chimeric immunogen is administered. In other embodiments, an amount of composition corresponding to between about 10 μg and about 500 μg of chimeric immunogen is administered. In still other embodiments, an amount of composition corresponding to between about 10 μg and about 250 μg of chimeric immunogen is administered. In yet other embodiments, an amount of composition corresponding to between about 10 μg and about 100 μg of chimeric immunogen is administered. Preferably, an amount of composition corresponding to between about 10 μg and about 50 μg of chimeric immunogen is administered. Further guidance on selecting an effective dose of the compositions may be found, for example, in Rose and Friedman, 1980, Manual of Clinical Immunology, American Society for Microbiology, Washington, D.C.

[0173] The volume of composition administered will generally depend on the concentration of chimeric immunogen and the formulation of the composition. In certain embodiments, a unit dose of the composition is between about 0.05 ml and about 1 ml, preferably about 0.5 ml. The compositions can be prepared in dosage forms containing between 1 and 50 doses (e.g., 0.5 ml to 25 ml), more usually between 1 and 10 doses (e.g., 0.5 ml to 5 ml)

[0174] The compositions of the invention can be administered in one dose or in multiple doses. A dose can be followed by one or more doses spaced by about 4 to about 8 weeks, by about 1 to about 3 months, or by about 1 to about 6 months. Additional booster doses can be administered as needed. In certain embodiments, booster doses are administered in about 1 to about 10 years.

5.7.3. Administration of Compositions

[0175] The compositions of the invention can be administered to a subject by any method known to one of skill in the art. In certain embodiments, the compositions are contacted to a mucosal membrane of the subject. In other embodiments, the compositions are injected into the subject. By selecting one of these methods of administering the compositions, a skilled artisan can modulate the immune response that is elicited. These methods are described extensively below.

[0176] Thus, in certain embodiments, the compositions are contacted to a mucosal membrane of a subject. Any mucosal membrane known by one of skill in the art, without limitation, can be the target of such administration. For example, the mucosal membrane can be present in the eye, nose, mouth, lungs, esophagus, stomach, small intestine, large intestine, rectum, anus, vagina, or penis of the subject. Preferably, the mucosal membrane is a nasal mucous membrane.

[0177] In other embodiments, the composition is delivered by injection. The composition can be injected subcutaneously or intramuscularly. In such embodiments, the composition preferably comprises an adjuvant, as described above. 5.7.4. Kits Comprising Compositions

[0178] In yet another aspect, the invention provides a kit comprising a vaccine composition of the invention. In certain embodiments, the kit further comprises instructions directing a medical professional to administer the composition to a subject. In further embodiments, the instructions direct the medical professional to administer the composition of a mucous membrane of the subject.

[0179] In still another aspect, the present invention provides a kit comprising packaging material and a composition of the invention contained within the packaging material, said composition in a form suitable for administration to a subject, preferably a human, or in a format that can be diluted or reconstituted for administration to the subject. In one embodiment, the article of manufacture further comprises printed instructions and/or a label directing the use or administration of the composition. The instructions and/or label can, for example, suggest a dosing regimen for induction of an immune response against one or more heterologous antigens. Thus, instructions and/or label can provide informational material that advises the physician, technician or subject on how to appropriately induce, monitor, and optionally boost with repeated administration an immune response induced against one or more heterologous antigens.

[0180] As with any pharmaceutical product, the packaging material and container of the kits of the invention are designed to protect the stability of the product during storage and shipment. More specifically, the invention provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, sprayer, insufflator, intravenous (i. v.) bag, envelope and the like; and at least one unit dosage form of a composition of the invention contained within said packaging material.

5.8. Making and Testing the Chimeric Immunogens

[0181] The chimeric immunogens of the invention are preferably produced recombinantly, as described below. However, the chimeric immunogens may also be produced by chemical synthesis using methods known to those of skill in the art. Alternatively, the chimeric immunogens can be produced using a combination of recombinant and synthetic methods.

5.8.1. Manufacture of Chimeric Immunogens

[0182] Methods for expressing and purifying the chimeric immunogens of the invention are described extensively in the examples below. Generally, the methods comprise introducing an expression vector encoding the chimeric immunogen into a cell that can express the chimeric immunogen from the vector. The chimeric immunogen can then be purified for administration to a subject following expression of the immunogen.

5.8.2. Verification of Chimeric Immunogens

[0183] Having selected the domains of the chimeric immunogen, the function of these domains, and of the chimeric immunogens as a whole, can routinely be tested to ensure that the immunogens can induce the desired immune response. For example, the chimeric immunogens can be tested for cell recognition, cytosolic translocation and immunogenicity using routine assays. The entire chimeric protein can be tested, or, the function of various domains can be tested by substituting them for native domains of the wild-type toxin.

5.8.2.1. Receptor binding/Cell recognition

[0184] Receptor binding domain function can be tested by monitoring the chimeric immunogen' s ability to bind to the target receptor. Such testing can be accomplished using cell-based assays, with the target receptor present on a cell surface, or in cell-free assays. For example, chimeric immunogen binding to a target can be assessed with affinity chromatography. The chimera can be attached to a matrix in an affinity column, and binding of the receptor to the matrix detected, or vice versa. Alternatively, if antibodies have been identified that bind to either the receptor binding domain or its cognate receptor, the antibodies can be used, for example, to detect the receptor binding domain in the chimeric immunogen by immunoassay, or in a competition assay for the cognate receptor. An exemplary cell-based assay that detects chimeric immunogen binding to receptors on cells comprises labeling the chimera and detecting its binding to cells by, e.g., fluorescent cell sorting, autoradiography, etc.

5.8.2.2. Translocation

[0185] The function of the translocation domain can be tested as a function of the chimeric immunogen' s ability to gain access to the interior of a cell. Because access first requires binding to the cell, these assays can also be used to assess the function of the cell recognition domain.

[0186] The chimeric immunogen' s ability to enter the cell can be assessed, for example, by detecting the physical presence of the chimera in the interior of the cell. For example, the chimeric immunogen can be labeled with, for example, a fluorescent marker, and the chimeric immunogen exposed to the cell. Then, the cells can be washed, removing any chimeric immunogen that has not entered the cell, and the amount of label remaining determined. Detecting the label in this fraction indicates that the chimeric immunogen has entered the cell.

5.8.2.3. ER Retention and Trafficking to the Cvtosol

[0187] A related assay can be used to assess the ability of the chimeric immunogen to traffic to the ER and from there into the cytosol of a cell. In such assays, the chimeric immunogen can be labeled with, for example, a fluorescent marker, and the chimeric immunogen exposed to the cell. The cells can then be washed and treated to liberate the cellular contents. The cytosolic fraction of this preparation can then be isolated and assayed for the presence of the label. Detecting the label in this fraction indicates that the chimeric immunogen has entered the cytosol.

[0188] In another method, the ability of the translocation domain and ER retention domain to effect trafficking of the chimeric immunogen to the cytosol can be tested with a construct containing a domain III having ADP ribosylation activity. Briefly, cells expressing a receptor to which the construct binds are seeded in tissue culture plates and exposed to the chimeric protein or to an engineered PE exotoxin containing the modified translocation domain or ER retention sequence in place of the native domains. ADP ribosylation activity can be determined as a function of inhibition of protein synthesis by, e.g., monitoring the incorporation of 3H-leucine.

5.8.2.4. Immunogenicity

[0189] The ability of the chimeric immunogens to elicit an immune response against the heterologous antigen can be assessed by determining the chimeric immunogen's immunogenicity. Humoral, cell-mediated, and secretory immunogenicity can be assessed. For example, a humoral immune response can tested by inoculating an animal with the chimeric immunogen and detecting the production of antibodies specific for at least one of the heterologous antigens with a suitable immunoassay. Such detection is well within the ordinary skill of those in the art. Similarly, a secretory immune response can be tested by detecting in a secreted fluid, for example, saliva, antibodies specific for at least one of the heterologous antigens with a suitable immunoassay. [0190] In addition, cell-mediated immunogenicity can be tested by immunizing an animal with the chimeric immunogen, isolating cytotoxic T cells from the animal, and detecting their ability to kill cells whose MHC Class I molecules bear peptides sharing amino acid sequences with the heterologous antigen. This assay can also be used to test the activity of the cell recognition domain, the translocation domain and the ER retention domain because generation of a cell mediated response requires binding of the chimera to the cell, trafficking to the ER, and translocation to the cytosol.

6. EXAMPLES

[0191] The following examples merely illustrate the invention, and are not intended to limit the invention in any way.

6.1. Construction of a Chimeric Immunogen

[0192] A chimeric immunogen expression vector is generated in a multistep process. A DNA oligonucleotide duplex encoding the desired antigen (i.e., ovalbumin) is digested with appropriate restriction enzymes and gel purified (Qiagen Inc., Valencia, CA). Alternatively, larger DNA fragment encoding ovalbumin, or a portion thereof, is prepared from a previously-obtained source, such as a recombinant plasmid or other cloning vehicle. A DNA fragment of PE encoding amino acids 1-360 is generated by PCR using pPE64pSTΔ553 as a template. See Hertle et al, 2001, Infect. Immun. 69(15): 6962-6969. The PCR fragment is digested with appropriate restriction enzymes and gel purified (Qiagen Inc., Valencia, CA). The purified fragment encoding ovalbumin, or a portion thereof, and PCR-fragment are ligated into an appropriate site of pPE64pSTΔ553 (i.e., the region encoding domain Ib and/or domain III) depending on the restriction enzymes used to prepare the DNA fragments encoding the antigens and the immune response desired to be induced with a chimeric immunogen expressed from the construct.

[0193] In this example, ovalbumin is inserted into domain III of ntPE by cutting the ovalbumin gene and the ntPE plasmid with restriction enzymes BamHl and Agel and ligating together the DNA fragments produced thereby. Alternately, ovalbumin is inserted into the Ib loop of ntPE by cutting both the ovalbumin gene and the ntPE plasmid with Pstl and ligating together the resulting DNA fragments. Preparation of an ntPE chimera containing ovalbumin in both locations simultaneously is performed by sequential application of the ligation steps outlined above. The final construct is verified by restriction enzyme digestion. [0194] In addition, a toxic form of this chimera is constructed by ligating the antigen of interest, e.g., ovalbumin, together with DNA fragments derived from pPE64-PstI. Such constructs are verified by restriction enzyme digestion. Chimeras expressed from this plasmid are useful as positive controls to assess toxicity of the chimeric immunogen.

6.2. Expression of a Chimeric Immunogen

[0195] E. coϊi DH5α cells (Gibco/BRL) are transformed using a standard heat-shock method in the presence of the appropriate plasmid. Transformed cells, selected on antibiotic- containing media, are isolated and grown in Luria-Bertani broth (Difco; Becton Dickinson, Franklin Lakes, NJ.) with antibiotic and induced for protein expression by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG). Two hours following IPTG induction, cells are harvested by centrifugation at 5000 rpm. Inclusion bodies are isolated following cell lysis and proteins are solubilized in 6M guanidine HCl and 2 mM EDTA (pH 8.0) plus 65 mM dithioerythreitol. Following refolding and purification, as previously described (Buchner et aU 1992, Anal. Biochem. 205:263-70; Hertle et al, 2001, Infect. Immiin. 69(15): 6962-6969), proteins are stored in PBS (pH 7.4) lacking Ca²⁺ and Mg²⁺ at -80°C.

6.3. Characterization of a Chimeric Immunogen

[0196] The chimeric immunogen is prepared by genetically grafting the antigens of interest into domain Ib and/or domain III of ntPE (Fig. 1) as described above. Purified proteins used in these studies are assessed by size-exclusion chromatography using a ZORBAX^® GF-450 column (Agilent Technologies, Palo Alto, CA) and demonstrated to be greater than 95% monomeric. Additionally, purified chimeric immunogens used in the experiments described herein are determined to have the anticipated mass and composition using amino acid analysis and SDS-PAGE, the correct N-terminal sequence, about 6.5 ng host cell protein/mg chimeric immunogen, <2 pg host cell DNA/mg chimeric immunogen, and about 6.3 EU endotoxin/mg chimeric immunogen.

[0197] Cytotoxicity due to inhibition of protein synthesis is examined by exposing L929 (ATCC CCL-I) cells to PE as described previously. See Ogata et ah, 1990, J. Biol Chem. 265 :20678-85. Incubation of PE-sensitive L929 cells with either PE or a toxic form of the chimeric immunogen produced as described above result in similar toxicity profiles. This assay is also used to demonstrate a lack of cytotoxicity by the non-toxic form of the chimeric immunogen. 6.4. Chimeric Immunogen Immune Response Assays

6.4.1. Isolation of Secreted Antibodies

[0198] Mouse saliva (typically 50-100 μl) from mice administered a chimeric immunogen is collected over a 10 min period using a polypropylene Pasteur pipette following the induction of hyper-salivation by an intra-peritoneal injection of 0.1 mg pilocarpine per animal. Serum samples (100 μl) are obtained using serum separators with blood collected from periorbital bleeds. Serum and saliva samples are then aliquoted in 10 μl volumes and stored at -70°C until analysis. Secreted antibodies thus obtained were characterized in the assays described below.

6.4.2. ELISA Assays

[0199] Antibodies against one or more antigens present in a chimeric immunogen are measured by enzyme-linked immunosorbent assay (ELISA). Costar 9018 E.I.A./R.I.A. 96- well plates are coated overnight with 0.6μg/well of the chimeric immunogen that is used to induce production of the assayed antibodies in 0.2M NaHCOs-Na₂COa, pH 9.4. Each 96- well plate is washed four times with PBS containing 0.05% Tween 20-0.01% thimerosal (wash buffer); and then blocked for 1 h with PBS/Tween 20 containing 0.5% BSA-0.01% thimerosal (assay buffer). Serum and saliva samples are diluted with assay buffer, loaded onto a 96-well plate, and incubated for 2 h for serum IgG and overnight for saliva and serum IgA. Each 96-well plate is then washed four times with wash buffer, and horseradish peroxidase ("HRP") conjugated goat anti-mouse serum IgG (Pierce Chemical Company, Rockford, IL), to assess humoral immune responses, or serum IgA (Kirkegaard & Perry Laboratories, Gaithersburg, Maryland), to assess secretory immune responses, is added, then the plates are incubated for 1 and 4 h, respectively. All incubation and coating steps are performed at room temperature covered with parafϊlm on a shaker at 4 rpm for the specified times. TMB (3,3',5,5'tetramethylbenzidine), substrate for HRP, is used to quantify bound antibody at 450 nm.

6.4.3. Cell-Mediated Cell Killing Assays

[0200] The following examples describe methods that can be used to assess cell-killing by effector cells of the immune system (e.g., cytotoxic T lymphocytes, natural killer cells, etc.) following induction of a cell-mediated immune response with a chimeric immunogen of the invention. 6.4.3.1. Chromium 51 Release Assay

[0201] First, effector cells are isolated by standard PBMC isolation procedures. For one NK assay, generally 5-10 mL of whole blood are required, or 5 x 10⁶ isolated PBMC. K562 cells (human chronic myelogenous leukemia, ATCC #CCL-243) are used as target cells. Other cells can be used as target cells depending on the nature of the antigen used to induce the cell- mediated immune response if the ability to specifically recognize and kill cells that express that antigen is to be tested. For example, in the case of pathogen-derived antigens, cells infected with the pathogen or that express the antigen are used as target cells. In the case of, for example, cancer antigens, cells that express the antigen can be used as target cells, or, preferably, cancer cells that express the antigen are used as target cells.

[0202] Complete medium (CM): for culture of K562 target cells, preparation of the effector cells and all assay procedures, RPMI 1640 media supplemented with 2% L-glutamine, 1% penicillin/streptomycin, 10% heat-inactivated fetal calf serum and 2.5% Hepes buffer is used. (All reagents can be obtained from Gibco, Gaithersburg, MD or equivalent.) Complete medium should be warmed to 37°C before use in all procedures described below. Sodium Chromate, Na₂ ⁵¹CRO₄ (⁵¹Cr), is obtained from, e.g., NEN, Dupont, Boston, MA or Amersham Life Sciences, Arlington, IL. The concentration is adjusted to 1 mCi/mL in sterile PBS. Magnetic beads, e.g., Dynabeads M-450, are first coated with sheep anti-mouse IgGl- and coated with anti-CD3 (e.g., (cat. nos. 110.03 and cat. no. Ml 11.13; Dynal, Oslo, Norway). CD56 monoclonal antibody can be obtained from, e.g., NCAM, clone 123C3 — cat. no. 18-0152; Zymed Laboratories, CA.

[0203] On the day prior to the assay, 1-3 x 10⁶ K562 cells are placed into a flask with fresh CM. On the day of the assay, the log phase K562 cells are pelleted and resuspended in 1 mL of CM. 1 x 10⁶ K562 cells per three samples are removed and placed in a fresh 15 mL tube. The cells are then repelleted, and 100 mCi of ⁵¹Cr is added per 1 x 10⁶ K562 cells, followed by 10% v/v of fetal bovine serum (FBS). The K562 cells are incubated for 1 h at 37°C, shaking the tube every 15 min to resuspend the cells and ensure uniform labeling. After incubation, 10 mL CM is added, the cells are pelleted and gently resuspended. This step is repeated two more times to wash the cells free of excess ⁵¹Cr. After the final wash, the cells are resuspended in 1 mL of CM, counted using a hemacytometer, the concentration of cells is adjusted to 5 x 10⁴ viable K562 cells/mL. [0204] PBMC (or other suitable effector cells) are separated from whole blood using standard separation techniques, and 5 x 10⁵ PBMC are resuspended in 5 niL of CM. In order to partially purify the PBMC population, adherent macrophages can be removed by placing the PBMC suspension in a 25 cm² tissue culture flask and incubating the cells at least 1 h at 37°C. The PBMC are then collected and dispensed into a 15 mL centrifuge tube, pelleted, and resuspended in 500 μL of CM. The PBMC are then counted, and the concentration of the cells is adjusted to 5 x 10⁶ cells/mL. Stepwise dilutions of the PBMC are performed in CM medium to generate aliquots of cells at 2.5, 1.25, and 0.25 x 10⁶ , respectively for the required E:T ratios.

[0205] In a 96-well, U-bottomed microtiter plate, 100 μL of each effector cell concentration is dispensed in triplicate. 100 μL of target cells, adjusted to 5 x 10⁴ cells/mL, is dispensed to every well containing effector cells. For controls, 100 μL of sterile 10% SDS is used to lyse 100 μL of target cells to release all the ⁵¹Cr from the target cells to calculate the maximum release (max), while 100 μL of CM is added to 100 μL of target cells in order to calculate the amount of CR⁵¹ spontaneously released. The cells are then incubated for 4 h at 37°C.

[0206] The plate is removed from the incubator and the supernatant fluid is harvested. An aliquot (usually 35 μL) is collected from the 96-well plate using a multichannel pipet and transferred to another 96-well plate in which dry scintillant is coated. After drying the plate overnight, radioactivity is measured in a 96-well format liquid scintillation counter (Packard, CT, USA).

[0207] The level of activity as denoted by the percentage specific lysis (% lysis) of labeled targets is determined by the following formula.

mean test cpm - mean spon cpm

% lysis = x 100 mean max cpm - mean spon cpm cpm = counts per minute (mean cpm is usually average of three replicates); test cpm = cpm released by the target cells in the presence of effector cells; spon = cpm released by the target cells in the absence of any effector cells; and max = cpm released by the target cells in the presence of SDS.

[0208] In appropriate assays, to verify that the lysis of K562 is NK cell-mediated, specific cell types are depleted from the isolated PBMCs and the change in % lysis against K562 cells is examined. For example, T-cells or NK cells can be depleted from the PBMC, and run the three populations concurrently in the standard NK cell assay. To do so, PBMCs are resuspended at 5 x 10⁵/mL as described above. The PBMCs are divided equally into three polypropylene tubes with at least 5 x 10⁶ PBMC/tube, then pelleted and resuspended. One tube is the control depletion, another is for T-cell depletion, and the third is for NK cell depletion. The T-cells can be depleted using, e.g., anti-CD3 monoclonal antibody, while NK cells can be depleted using, e.g., anti-CD56 monoclonal antibody. 20 μL diluted antibody is added per 10 cells, then incubated for 45 min at 4°C, shaking occasionally. The cells are then washed twice with cold PBS with calcium and magnesium. Appropriate anti-mouse (or other species) magnetic beads (e.g., Dynabeads M-450 coated with sheep anti-mouse IgGl- cat. no. 110.03) are added sufficient to yield a bead:cell ratio of 10:1. 3 mL PBS is added, and the solution is placed on a magnet (e.g., Dynal MPCl or equivalent) for 2 min. The cells are then resuspended in 100 μL media, incubated at 4°C for 20 min with occasional shaking. These steps are then repeated twice.

[0209] Most gamma and liquid scintillation counters can be programmed to calculate the mean epm and percentage specific lysis values (% lysis). Triplicate cpm values should have a mean SEM of less than 5% and values outside of means should be outliered using standard statistical methods. The % lysis values alone can suffice as a measure of NK (or other cell) activity or non-parametric tests, such as Kruskall-Wallis or Wilcoxon tests can be used to determine significant statistical differences between slopes of response curves at different E:T ratios and different groups of patients and controls NK cytotoxic activity can also be expressed as lytic units (LU). An LU is defined as the number of lymphocytes required to yield a particular % lysis, e.g., the LU_2O is the number of lytic units that yield 20% lysis of targets by a particular number of effector cells. The % specific lysis is plotted versus the log of the effector cell number for each E:T ratio (for example, in the standard NK set up described above, at the 100:1 ratio there are 5 x 10⁵ cells per well, at 50:1 there are 2.5 x 10⁵ effector cells per well, etc.). An NK specific response, for example, is defined as >50% decrease in % specific lysis at two or more E:T ratios in the NK depleted PBMC fraction relative to the whole PBMC. There must be <10% decrease in % specific lysis in the anti- CD3 depleted PBMC relative to the whole PBMC. 6.4.3.2. Flow Cytometry Assay

[0210] This example provides an assay that can be used to test the ability of NK cells, cytotoxic T lymphocytes, etc. to kill target cells. As with the Chromium 51 release assay, the target cell used depends on the type of cell-mediated immune response being tested.

[0211] The isolation of PBMC from buffy coat is performed as described by Schober et al, 1984, Exp. Cell. Res. 152:348-356. NK cells are isolated using, e.g., the MACS-device and the NK-cell-isolation kit 465-2 (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendations. Membrane staining is performed as follows: a stock solution is prepared by dissolving DIOC 18 (Sigma) in DMSO (2mg/ml; Sigma) over night with agitation. The NK cells (10⁶ cells/ml) are incubated in 10 μg/ml DIOC 18 (final concentration) for 1 h at 37 °C. Cells are washed twice and maintained in medium (RPMI 1640 [Bio Whittaker, Boehringer Ingelheim, Germany] supplemented with 120% bovine serum and L-glutamine).

[0212] K562 target cells and all other cell lines are obtained from the ATCC and kept under aseptic conditions in a 5% CO₂-enriched atmosphere in medium. The stained NK cells are incubated with native target cells at different E/T ratios (1:1; 5:1; 10:1; 20:1), whereby the concentration of effector cells is always 10⁶/ml. Samples are taken at the indicated time points and 5 μg/ml (final concentration) 7-AAD (Sigma) is added. The suspension is analyzed using, e.g., a Coulter Epics XL flow cytometer (Coulter, Krefeld, Germany). The scatter gate is set to all cellular events (including dead cells), and the percentage of vital versus necrotic effector and target cells is calculated from an FLl (DIOC 18) versus FL4 (7-AAD) dot-plot statistic. If additional anti-CD34 surface antibodies are used, these antibodies (e.g., clone HPCA-2; Becton-Dickinson, Hamburg, Germany) are added 15 min before the final analysis and analyzed in the FL2 (PE) channel. The events from the scatter plot were transferred to an FLl (DIOC18) histogram, and the DIOCl 8-negative target cells were then transferred to an FL2 (CD34-PE) versus FL4 (7-AAD) plot to calculate the vitality of all cells as well as the CD34-positive target cells specifically.

[0213] All experiments are performed in parallel without effector cells. Such background values (typical below 2%) are subtracted from those obtained with effector cells. 6.4.4. Antibody Dependent Cell Killing Assays

[0214] Antibody-dependent cell killing assays against one or more of the antigens of the chimeric immunogens are assessed according to the following protocol.

6.4.4.1. Complement-dependent cytotoxicity.

[0215] Cell lysis with baby rabbit complement is determined using a 51 Cr-release assay. Cancer cells bearing an antigen of interest or cells infected with a pathogen from which an antigen of interest is derived are labeled with 0.1 mCi 51Cr-sodium chromate (New England Nuclear) at 37⁰C for 1 hour. The cells are then washed three times with RPMI 1640 medium.

-> 1 Cr-labeled cells (1 x 10^ cells) are incubated with various concentrations of antibody obtained as described above or control IgG on ice for 30 minutes. The unbound antibody is removed by washing the cells three times with medium. The cells are then distributed into 96- well plates and incubated with serial dilutions of baby rabbit complement (Cedarlane, Ontario, Canada) at 37°C for 2 hours. After incubation, supernatants from each well (50 μL) are harvested and 51 Cr is measured using a gamma counter. Spontaneous release of 51 Cr is measured after incubating 51Cr-labeled cells with medium alone. The maximum release of

51 Cr is determined after incubation of ->lCr-labeled cells with 1% NP -40. Percentage of cytotoxicity is calculated from the formula: specific cytotoxicity (%) = (A - C)Z(B - C) * 100, where A = experimental 51 Cr release, B = maximum 51 Cr release, and C = spontaneous 51 Cr release.

6.4.4.2. Antibody-dependent cell-mediated cytotoxicity (ADCCV

[0216] ADCC activity is determined by standard 4-hour 51 Cr-release assay. Splenic mononuclear cells from SCID mice are used as effector cells and cultured in RPMI 1640 medium with or without 500 U/mL of recombinant mouse interleukin (IL)-2 (Genzyme,

Cambridge, MA) for 6 days, then washed, and resuspended in medium before use. 51 Cr- labeled target cells expressing an antigen of interest, as described above, are placed in 96- well plates and various concentrations of antibody obtained as described above or control IgG are added to wells. Effector cells are then added to the plates at various effector to target (EIT) ratios. After 4 hours incubation, supernatants are removed and counted in a gamma counter. The percentage of cell lysis is determined as above. 6.4.4.3. Statistical analysis.

[0217] The statistical significance in the data of in vitro experiments is determined by the unpaired Mest. The significant differences in survival data are evaluated using a log-rank test.

6.5. Vaccination using a Chimeric Immunogen

[0218] 8/group BALB/c mice (Charles River Laboratories, Wilmington, MA), 6-8 weeks at initial dosing, are used in these studies since age-related suppression of immune function has been demonstrated in this species. See Linton & Dorshkind, 2004, Nat. Immunol. 5:133-9. Intranasal inoculation is performed to mice lightly anesthetized with isoflurane. All intranasal (IN) administrations are performed under mild anesthesia since fluid introduced into the nares of awake mice that is in excess of its cavity volume is rapidly ingested while suppression of this reflex occurs under anesthesia. Thus, administration to anesthetized mice results in preferential delivery to the trachea rather than the esophagus following IN administration. See Janakova et al, 2002, Infect. Immun. 70:5479-84. Mice receive 10 μl of chimeric immunogen (5 μl/nares) in PBS for each immunization. Variations in concentration from 100 μg/ml to 10mg/ml are prepared for dosing studies to assess immune responses over the range of 1 to 100 μg of chimeric immunogen.

[0219] Mice receiving an IN inoculation dose schedule of 0, 7, 14, and 28 days with 1, 10 or 100 μg chimeric immunogen are evaluated for mucosal and systemic humoral immune responses, with similar IN delivery of PBS to mice serving as a negative control. Animals receiving a subcutaneous (SubQ) injection of 10 μg chimeric immunogen in a standard protocol using Freund's complete/incomplete adjuvant materials serve as a positive control.

[0220] Immune responses induced by the chimeric immunogen are assessed by detecting salivary (secretory immune response) and serum (humoral immune response) antibodies specific for one or more of the antigens present in the chimeric immunogen. Exemplary methods for detecting such antibodies are described above. [0221] The present invention provides, inter alia, chimeric immunogens and methods of inducing an immune response in a subject. While many specific examples have been provided, the above description is intended to illustrate rather than limit the invention. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

[0223] All publications and patent documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent document were so individually denoted. Citation of these documents is not an admission that any particular reference is "prior art" to this invention.

Claims

What is claimed is:

1. A chimeric immunogen, comprising

a)- a receptor binding domain,

b)- a translocation domain, and

c)- ovalbumin, or a portion thereof.

2. The chimeric immunogen of Claim 1 , wherein said chimeric immunogen, when administered to a subject, generates an immune response in said subject that is specific for one or more ovalbumin epitope(s).

3. The chimeric immunogen of Claim 1 , wherein said chimeric immunogen further comprises an endoplasmic reticulum retention domain.

4. The chimeric immunogen of Claim 3, wherein said ovalbumin, or a portion thereof, is located between said translocation domain and said endoplasmic reticulum retention domain.

5. The chimeric immunogen of Claim 3, wherein said endoplasmic reticulum retention domain is an enzymatically inactive domain III of Pseudomonas exotoxin A.

6. The chimeric immunogen of Claim 5, wherein said enzymatically inactive domain III of Pseudomonas exotoxin A is inactivated by deleting a glutamate at position 553.

7. The chimeric immunogen of Claim 3, wherein said endoplasmic reticulum retention domain comprises an amino acid sequence that is selected from the group of RDEL (SEQ ID NO.:2) or KDEL (SEQ ID NO.:3).

8. The chimeric immunogen of Claim 1, wherein said translocation domain is selected from the group consisting translocation domains from Pseudomonas exotoxin A, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin.

9. The chimeric immunogen of Claim 5, wherein said translocation domain is domain II of Pseudomonas exotoxin A.

10. The chimeric immunogen of Claim I₅ wherein said translocation domain comprises amino acids 280 to 364 of domain II of Pseudomonas exotoxin A.

11. The chimeric immunogen of Claim 1 , wherein said receptor binding domain is selected from the group consisting of domain Ia of Pseudomonas exotoxin A; a receptor binding domains from cholera toxin, diptheria toxin, shiga toxin, or shiga- like toxin; a monoclonal antibody, a polyclonal antibody, or a single-chain antibody; TGFα, TGFβ, EGF, PDGF, IGF, or FGF; IL-I, IL-2, IL-3, or IL-6; and MIP-Ia, MIP- Ib, MCAF, or IL-8.

12. The chimeric immunogen of Claim 11, wherein said receptor binding domain is domain Ia of Pseudomonas exotoxin A.

13. The chimeric immunogen of Claim 12, wherein said domain Ia of Pseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.:5.

14. The chimeric immunogen of Claim 1, wherein said receptor binding domain binds to α2-macroglobulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycopolypeptide receptor, CD3, CD4, CD8, chemokine receptor, CD25, CDl IB, CDl 1C, CD80, CD86, TNFα receptor, TOLL receptor, M-CSF receptor, GM-CSF receptor, scavenger receptor, or VEGF receptor.

15. The chimeric immunogen of Claim 14, wherein said receptor binding domain binds to α2-macroglobulin receptor.

16. The chimeric immunogen of Claim 1, wherein said chimeric immunogen comprises more than one ovalbumin, or portion thereof,

17. A polynucleotide that encodes a chimeric immunogen, said chimeric immunogen comprising:

a)- a receptor binding domain,

b)- a translocation domain, and

c)- ovalbumin, or a portion thereof.

18. The polynucleotide of Claim 17, wherein said chimeric immunogen, when administered to a subject, generates an immune response in said that is specific for one or more ovalbumin epitope(s).

19. The polynucleotide of Claim 17, wherein said chimeric immunogen further comprises an endoplasmic reticulum retention domain.

20. The polynucleotide of Claim 19, wherein said ovalbumin, or a portion thereof, is located between said translocation domain and said endoplasmic reticulum retention domain.

21. The polynucleotide of Claim 19, wherein said endoplasmic reticulum retention domain is an enzymatically-inactive domain III of Pseudomonas exotoxin A.

22. The polynucleotide of Claim 21 , wherein said enzymatically inactive domain III of Pseudomonas exotoxin A is inactivated by deleting a glutamate at position 553.

23. The polynucleotide of Claim 19, wherein said endoplasmic reticulum retention domain comprises an amino acid sequence that is selected from the group of RDEL (SEQ ID NO.:2) or KDEL (SEQ ID NO.:3).

24. The polynucleotide of Claim 17, wherein said translocation domain is selected from the group consisting translocation domains from Pseudomonas exotoxin A, diptheria toxin, pertussis toxin, cholera toxin, heat-labile E. coli enterotoxin, shiga toxin, and shiga-like toxin.

25. The polynucleotide of Claim 24, wherein said translocation domain is domain II of Pseudomonas exotoxin A.

26. The polynucleotide of Claim 17, wherein said translocation domain comprises amino acids 280 to 364 of domain II of Pseudomonas exotoxin A.

27. The polynucleotide of Claim 17, wherein said chimeric immunogen comprises more than one ovalbumin, or a portion thereof.

28. The polynucleotide of Claim 17, wherein said receptor binding domain is selected from the group consisting of domain Ia of Pseudomonas exotoxin A; a receptor binding domains from cholera toxin, diptheria toxin, shiga toxin, or shiga-like toxin; a monoclonal antibody, a polyclonal antibody, or a single-chain antibody; TGFα, TGFβ, EGF, PDGF, IGF, or FGF; IL-I, IL-2, IL-3, or IL-6; and MIP-Ia, MIP-Ib, MCAF, or IL-8.

29. The polynucleotide of Claim 28, wherein said receptor binding domain is domain Ia of Pseudomonas exotoxin A.

30. The polynucleotide of Claim 29, wherein said domain Ia of Pseudomonas exotoxin A has an amino acid sequence that is SEQ ID NO.: 5.

31. The polynucleotide of Claim 17, wherein said receptor binding domain binds to α2-macro globulin receptor, epidermal growth factor receptor, transferrin receptor, interleukin-2 receptor, interleukin-6 receptor, interleukin-8 receptor, Fc receptor, poly-IgG receptor, asialoglycopolypeptide receptor, CD3, CD4, CD8, chemokine receptor, CD25, CDI lB, CDI lC, CD80, CD86, TNFα receptor, TOLL receptor, M- CSF receptor, GM-CSF receptor, scavenger receptor, or VEGF receptor.

32. The polynucleotide of Claim 31 , wherein said receptor binding domain binds to α2-macroglobulin receptor.

33. An expression vector comprising the polynucleotide of Claim 17.

34. A cell comprising the expression vector of Claim 33.

35. A composition comprising a chimeric immunogen, wherein said chimeric immunogen comprises:

a)- a receptor binding domain,

b)- a translocation domain, and

c)- ovalbumin, or a portion thereof.

36. The composition of Claim 35, wherein said composition further comprises a pharmaceutically acceptable diluent, excipient, vehicle, or carrier.

37. The composition of Claim 35, wherein said chimeric immunogen, when administered to a subject, induces an immune response in said subject that is specific for one or more ovalbumin epitope(s).

38. The composition of Claim 35, wherein said composition is formulated for nasal or oral administration.

39. The composition of Claim 35, wherein said chimeric immunogen further comprises an endoplasmic reticulum retention domain.