WO2002072015A2

WO2002072015A2 - M cell directed vaccines

Info

Publication number: WO2002072015A2
Application number: PCT/US2002/007254
Authority: WO
Inventors: David W. Pascual
Original assignee: Montana State University-Bozeman
Priority date: 2001-03-12
Filing date: 2002-03-12
Publication date: 2002-09-19
Also published as: AU2002254161A1; WO2002072015A3

Abstract

This invention provides a vaccine that can direct gene transfer and the transfer of other immunogens to follicle associated epithelium or M cells to induce mucosal immunity using M cell ligands for receptor-mediated endocytosis. Also provided are polynucleotides sequences encoding M cell ligand-polybasic component fusion proteins, host cells, and methods of producing such proteins recombinantly and chemically. Further, methods are described for immunizing animal and human subjects against bacterial, viral, parasitic, fungal infectious agents or cancer and methods for assaying mucosal immunity using this vaccine.

Description

M CELL DIRECTED VACCINES

Related Applications

This application claims priority to Application Serial No. 60/274,639, and is

related to PCT Application, PCT/US01/00426, filed January 8, 2001, which claims

priority to United States Provisional Application Number 60/174,786, filed January 6,

2000.

Technical Field

The present invention is in the general field of vaccine development. The

present invention provides methods and compositions useful for, among other

purposes, the identification, diagnosis, prevention and treatment of bacterial, viral,

parasitic, fungal infectious agents or cancer for human, livestock, and wildlife. More

specifically, the present invention provides DNA and other vaccines directed to

follicle-associated epithelium. Even more specifically, the invention is directed to

polycation conjugated M cell ligand (e.g., enteric adhesins)-DNA complex vaccine

compositions and diagnostic and therapeutic uses thereof.

Background of the Invention

Aspects of this invention are discussed in Wu et al, Gene Therapy (2000)

7(l):61-69, and in Wu et al, Proc. Natl. Acad. Sci. USA (2001) 98:9318-23, each of

which is herein incorporated by reference in its entirety. AU publications and patent applications mentioned or identified in this specification are

incorporated by reference to the same extent as if each individual publication or patent

application was specifically and individually indicated to be incorporated by reference.

Recent studies have shown the utility of DNA vaccination for inducing protective

immunity in experimental animals exposed to influenza (Fynan et al, Proc Natl Acad Sci

USA (1993) 90:11478-11482 and Robinson et al, Vaccine (1993) 11:957-960), herpes

simplex virus (HSV) (Gillichan et al, JInfDis (1998) 177:1155-1161), HIV-1 (Boyer),

rotavirus (Herrmann et al, JInfecDis (1996) 174(Suppl.l):S93-S97 and Chen et al, J Virol

(1998) 72:5757-5761), and Borrelia burgdorgeri infections (Simon et al, J Immunol (1996)

26:2831 -2840). DNA immunization has a number of attractive features including ease of

preparation for encoding desired protective immunogens, co-expression of immunogens,

co-expression of adjuvant (e.g., cytokines), no requirement for large-scale protein

purifications, and ease of delivery. However, conventional DNA vaccine technology

immunizes the host at peripheral sites, e.g., intradermal or intramuscular sites. While these

methods can elicit systemic cell-mediated and antibody-dependent responses, most

infectious agents infect via a mucosal surface, and such DNA immunizations at peripheral

sites do not result in optimal mucosal immunity (i.e., both antibody, particularly IgA, and

cellular (cytotoxic T lymphocyte (CTL) immunity induction).

This lack of mucosal immunity induction has prompted attempts to deliver DNA

vaccines to mucosal surfaces. For example, successful induction of mucosal immunity has

been accomplished using DNA vaccines by intraoral jet delivery (Chen et al., Vaccine

(1999) 17(23-24):3171-3176); co-administration of a DNA vaccine with a polymer by

intranasal injection (Hamajima et al, Clinical Immunol Immunopathol (1998) 88(2):205-210); co-administration of a DNA vaccine with IL-12 (Okada et al, J Immunol

(1997) 159(7):3638-3647); intravaginal administration of DNA vaccines (Wang et al,

Vaccine (1997) 15(8):821-825); oral delivery of micro-encapsulated DNA vaccines (Jones

et al, Dev Biol Stand (1998) 92: 149-155); and parenteral and mucosal injection of DNA

vaccines (Shroff et al, Vaccine (1999) 18(3-4):222-230). However, these methods

generally lack mucosal surface selectivity. Nevertheless, they illustrate the desire to

observe mucosal immunity as the end-point in determining the efficacy of these vaccines.

Transepithelial transport of antigens and pathogens is the first step in the induction

of a mucosal immune response. Mucosal inductive tissues are sites in the small intestine or

in the nasal passages where vaccine antigens are taken to be processed and presented to

mucosal lymphocytes for the development of mucosal immunity (Frey et al, Behringlnst

Mitt (1997) 98:376-389). In the intestine, the delivery of antigen across the epithelial

barrier to the underlying lymphoid tissue is accomplished by M cells, a specialized

epithelial cell type that occurs only in the lymphoid follicle-associated epithelium (Frey et

al., 1997). Further, such follicle-associated epithelium is found in the nasal lymphoid

tissues (believed to be sites of induction of mucosal immune responses to airborne antigens;

Giannasca et al, Infect Immun (1997) 65(10):4288-4289). Selective and efficient transport

of antigen by M cells is considered an essential requirement for effective mucosal vaccines.

Thus, targeting of M cells by taking advantage of their capacity to endocytose particles,

including those particles comprising gene transfer vehicles and DNA vaccines, has

generated great interest as selective transfer of genes across the follicle-associated

epithelium would be advantageous from both investigational and therapeutic standpoints. Although viruses can be efficient gene transfer vehicles, progress has been made

toward developing non- viral delivery systems. Coupling of a specific ligand to vaccines or

drugs can be a powerful aid to route compounds to a certain target population. One of the

most promising means is by exploiting receptor-mediated endocytosis pathways using

selective ligands. In this method, DNA-ligand complexes are internalized by targeted cells

when the ligand binds to its respective cell-surface receptor. Such receptor-mediated gene

transfer has been accomplished using a variety of receptors by conjugating DNA to their

cognate ligands such as asialo-orosomucoid (Wu et al, JBiol Chem (1989)

264:16985-16987 and Wu et al, JBiol Chem (1994) 269:11542-11546), rransferrin (Lozier

et al, Human Gene Ther (1994) 5:313-322; Wagner et al, Proc Natl Acad Sci USA (1990)

87:3410-3414; and Wagner et al, Proc Natl Acad Sci USA (1992) 89:6099-6103), lectins

(Barra et al, Gene Ther (1994) 1:255-260), folate (Leamon et al, Biochem J

(1993)291:855-860), lung surfactant protein (Ross et al, Human Gene Ther (1995)

6:31-40), insulin (Sobolev et al, JBiol Chem (1998) 273:7928-7933) and would include

receptor specific monoclonal antibodies (Chen et al, FEBS Lett (1994) 338:167- 169 and

Kang et al, J Pharmacol Exp Therapeut (1994) 269:344-350).

Receptor-mediated gene transfer has some advantages over the other methods of in

vivo gene transfer. Compared to attenuated viral vectors, it shares tissue-specificity, but

receptor-mediated gene transfer minimizes the use of viral gene elements, obviating the

concerns regarding genomic integration. Further, it lessens concerns with the

proinflammatory properties often associated with viral vectors (Simon et al, Human Gene

Ther (1993) 4:771-780; Yang et al, J Virol (1996) 70:7209-7212; and van Ginkel et al, J

Immunol (1997) 159:685-693). The DNA-ligand complex is believed to be internalized by receptor-dependent endocytosis rendering transfection to be minimally toxic. The conjugate

carrier complex can be designed for cell-specific targeting by selecting the appropriate

receptor ligand. For example, efficient transfer of DNA to the intestinal epithelial cells by

transferrin-polylysine conjugates and M cell lectins have been used to successfully transfect

gastrointestinal cells in vitro (Batra et al, Cancer Gene Ther (1994) 1(3):185-192 and

Curiel et al, Am JRespir Cell Mol Biol (1992) 6(3):247-252). However, as transferrin

receptors are not restricted to M cells or follicle associated epithelium and as M cell lectins

can potentially bind to any α-linked galactose (Giannasca et al, 1997), the use of these

systems in vivo is limited.

The surface properties of many enteric pathogens are important in the establishment

of the pathogen in the host. For example, enteropathic Escherichia coli (EPEC) induce

characteristic attaching and effacing (A/E) lesions on epithelial cells of Peyer's patches

(Hartland et al, Mol Microbiol (1999) 32(1):151-158). This event is mediated, in part, by

binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir

(translocated intimin receptor), which is exported by the bacteria and integrated into the host

cell plasma membrane. Both of these protein have been shown to bind to host cells in vitro

(Hartland et al, 1999 and DeVinney et al, Cell Mol Life Sci (1999) 55(6-7):961-976).

Reovirus is an enteric pathogen and infects the host following attachment to

intestinal Peyer's patch M cells (Lee et al, Virology (1981) 108:156-63 and Mah et al, J

Virol (1990) 179:95-103). Thus, as with other enteric pathogens, reovirus exploits M cells

as a means to gain entry into the host. Mediating reovirus attachment is the adhesin, σl,

which is expressed as a viral coat protein (Lee et al, 1981). The protein σl is a 45

kilodalton protein that polymerizes via its N-terminus (Mah et al., 1990) to form a tetramer when isolated from reovirus-infected cells or purified as a recombinant protein from E. coli

(Bassel-Assel-Duby et al, J Virol (1987) 61:1834-1841). In vitro analysis has

demonstrated that neutral liposomes comprising σl protein can be taken up by rat Peyer's

patches (Rubas et al., J Microencapsul (1990) 7(3):385-395). Thus, enteric pathogen

adhesins make more effective targeting ligands than either transfemn or M cell lectins

(Batra et al, 1994, Curiel et al, 1992 and Giannasca et al, 1997).

This invention exploits receptor mediated endocytosis as a means of delivering

antigens to mucosal lymphoid tissue and introducing DNA into cells using M cell ligands

for specific targeting of DNA to follicle associated epithelium of nasal or gastrointestinal

origin. We have discovered that, by chemically coupling M cell ligands to a polymeric

chain of basic amino acids (e.g., polylysine) and allowing that construct to associate or

complex with DNA, the DNA (or RNA or other nucleic acid) can be delivered to

appropriate tissue types to obtain enhanced in vivo mucosal IgA antibody and T cell

responses against an encoded antigen.

Further, to demonstrate the efficacy of the present vaccine design, we have applied

this concept to reporter gene products, β-galactosidase and luciferase, as well as vaccine

antigens derived from human immunodeficiency virus (HIV) and Brucella in vivo. Using

these systems, enhanced mucosal IgA antibody responses can be demonstrated between

animals vaccinated with DNA only (that is, DNA not included in our formulation) and those

vaccinated with conjugated DNA complexes.

Summary of the Invention

The present invention is based, in part, on the observation that a DNA vaccine

protected from the mucosal environment can be effectively used to vaccinate a host by targeting the mucosa. Data described herein shows that appropriately formulated DNA

constructs show improved mucosal IgA antibody responses when compared to DNA applied

, directly to a mucosal surface. The present invention is further based on the induced

anti- vaccine antibody and cellular immune responses produced by vaccinated mice, cattle,

and bison. Based on these observations, the present invention provides compositions and

methods for use in a variety of animals, particularly humans, livestock, and wildlife.

It is therefore an object of this invention to provide a method for inducing mucosal

immunity using receptor mediated endocytosis pathways to deliver nucleotide, particularly

DNA, vaccines, as well as other immunogens, to specific cells of the follicle associated

epithelium, preferably M-cells, for example, of nasal and gastrointestinal origin. It is also

an object of this invention to provide DNA and other vaccine compositions comprising a

polypeptide (or other complexing agent) linked electrostatically to (or otherwise associated

or complexed with) a DNA structural sequence or gene or other immunogen. Particularly

contemplated are polypeptide-DNA complexes, in which the polypeptide is comprised of a

polymeric chain of basic amino acid residues and an M cell specific ligand.

The DNA structural sequence preferably encodes an iimnunogenic antigen from an

infectious agent, but also may encode other immunogens, such as a tumor specific antigen,

against which the induction of an immune response is desired, but also including antigens

against which a host might be tolerized. The present invention provides the ability to

produce a previously unknown protein ~ and to elicit an immune response against such

proteins — using the cloned nucleic acid molecules derived, for example, from any given

infectious agent be it bacterial, fungal, viral, protozoan, parasitic or protective molecule

against cancer. Consistent with the foregoing, a preferred embodiment of the present invention

includes an M cell specific ligand, a nucleic acid sequence encoding an immunogen, and a

nucleic acid binding moiety. Preferably, the nucleic acid will be DNA although RNA

vaccines are contemplated. In such vaccines, the binding moiety preferably is a

polypeptide, however, other binding and complexing agents may be utilized so long as they

stabilize or protect the nucleic acid and protein components of the vaccine from degradation

and facilitate their delivery, by various routes of administration, to the target mucosal

tissues. Thus, for example, a polypeptide binding moiety preferably comprises a polymeric

chain of basic amino acid residues and a contemplated polymeric chain would comprise

polylysine.

Another embodiment of the present invention includes an M cell specific ligand

conjugated or complexed to an immunogen via an appropriate linker. Immunogens in this

instance would include a variety of macromolecules such as peptides, proteins, lipoproteins,

lipids, glycoproteins, polysaccharides, carbohydrates, some nucleic acids, and certain of the

teichoic acids, or any other molecule or gene from a pathogen or tumor cell that could be

used to generate a protective immune response. Such immunogens may be conjugated or

complexed with the M cell specific ligand using any means known in the art. For instance,

immunogens may be conjugated to an M cell specific ligand using an appropriate

crosslinker. Cross-linking may be performed with either homo- or heterobifunctional

agents, i.e., SPDP, DSS, SIAB. Alternatively, immunogens may be complexed with an M

cell specific ligand using an appropriate complexing agent. Complexes may be formed

between a 6x His tag on one molecule and a nitrilotriacetic acid-metal ion complex on the

other molecule. Methods for cross-linking are disclosed in PCT/DK00/00531 (WO 01/22995) which is herein incorporated by reference. Alternatively, conjugates and

complexes can comprise the following scenarios: polypeptides with attached immunogens

may be conjugated to M cell specific ligands; liposomes can replace the polypeptide,

wherein the M cell specific ligand may be conjugated to a liposome containing the

immunogens, or conjugated to a liposome with one or several copies of an immunogen or

different immunogens attached/displayed to its surface; and peptide and protein

immunogens may be expressed as fusion proteins operably linked to the M cell specific

ligand.

In general, the M cell specific ligand is selected from the group consisting of the

protein σl of a reovirus, or is (or is derived from) an adhesin of Salmonella or a polio virus.

M cell tropic fragments of the foregoing also are contemplated. In a preferred embodiment

of the invention, a polypeptide binding moiety would further comprise an M cell specific

ligand and may be expressed as a fusion protein.

DNA encoding protective epitopes useful for the development of M cell targeted

vaccines may be obtained from the following sources. Oligonucleotide (gene) sequences

encoding an epitope as described in the scientific literature or derived from genomic data

that contains sequence data for microbial virulence factors may be used as the source.

(Weinstock, G. M., Genomics and Bacterial Pathogenesis. Emerg. Infect. Dis., (2000) 6(5)).

Another source of DNA encoding protective epitopes may be screening of epitope libraries,

such as phage display libraries, as described herein.

Particularly contemplated are nucleotide and other vaccines in which the

immunogen to be delivered to the target mucosal tissue is an immunogen expressed by an

infectious agent such as a microorganism or is a tumor specific antigen. Preferred immunogens are derived from or, like an expressed toxin, are associated with a bacterium,

protozoan, parasite, virus, fungus, prion, tuberculobacillus, leprosy bacillus, malaria

parasite, diphtheria bacillus, tetanus bacillus, Leishmania, Salmonella, Schistosoma,

measles virus, mumps virus, herpes virus, HIN, cancer and influenza virus.

Plasmid vectors in which DΝA sequences encoding such immunogens operably

linked to transcription regulatory elements are a preferred embodiment of the present

invention. Moreover, the vaccines of the present invention are preferably formulated with a

pharmaceutically acceptable excipient or an adjuvant such as an i munomodulator.

Examples of contemplated immunomodulators include cytokines, lymphokines,

interleukins, interferons and growth factors. Preferably, these vaccines induce a protective

immune response in a host vaccinated against the immunogen. In other embodiments of the

invention, contemplated vaccines will tolerize a host vaccinated against appropriate

immunogens.

Vaccines formulated in unit dosage form, and vaccines packaged with instructions

for the use of the vaccine to induce an immune response against said immunogen or disease

with which said immunogen is associated are preferred. Therapeutic as well as prophylactic

vaccines also are contemplated. Moreover, preferred vaccines are formulated for

administration through a route selected from the group consisting of oral, nasal, vaginal,

rectal and urethral routes of administration.

Another preferred embodiment of the present invention provides a method for

immunizing a host against an immunogen by administering the nucleotide vaccines or other

vaccines as described above. In addition, other embodiments of the invention provide a

method for assaying for mucosal immunity comprising the steps of administering the vaccine to an animal which is free of infection of the infectious agent whose antigen is to be

tested; isolating cells from the animal; and co-incubating said isolated cells with

heterologous antigen expressing cells. In this assay, cells expressing the antigen or vaccine

will be lysed. This serves as one indication that mucosal immunity was induced in the

vaccinated animal. The foregoing assay method is performed using CTLs isolated from

lymphoid tissue from the vaccinated animal. For example, CTLs may be isolated from

Peyer's patches cells, lymph nodes, NALT, adenoids, spleens and other organized lymphoid

tissue, as well as from non lymphoid tissue such as nasal passages, intestinal lamina propia,

lungs, liver and vaginal epithelium.

An additional step of evaluating the animal's cytokine profile also is contemplated.

Evaluation of cytokine responses is a means of measuring mucosal immunity and can give

an indication of which types of cells participate in an immune response. For example, T

cells can be either CTLs (CD8⁺) or helper cells (CD4⁺) that assist B cells in making

antibodies. CD4⁺ Th cells may be subdivided further into at least two functionally distinct

subsets, Thl and Th2, based on the unique profiles of cytokines they produce and the major

regulatory functions they play in the host's immune responses. For example, Thl cells

secrete IL-2, IFN-γ, and TNF-β, and function in cell-mediated immunity for protection

against intracellular pathogens such as Listeria monocytogenes, Mycobacterium species, and

Salmonella species. Thl cells may also provide B cell help. For example, Thl cell-derived

IFN-γ favors the development of IgG2a responses in mice. Thl cell activity is promoted by

IL-12 and and IL-18. Th2 cells preferentially secrete IL-4, IL-5, IL-6, IL-10, and IL-13, and

provide effective help for B cell responses, most notably for IgGl, IgE, and IgA. Promotion

of Th2 cells occurs by suppression of IL-12 by 11-4 and TGF-β. Other studies have also shown that T cells and certain cytokines (e.g., IL-5 and IL-6) are of particular importance

for the induction of committed surface (s)IgA⁺ B cells to differentiate into IgA-producing

cells.

A related embodiment of the present invention provides an isolated nucleic acid

encoding a fusion protein comprising a nucleic acid binding moiety and an M cell specific

ligand. In such nucleic acids, the binding moiety encodes a polymeric chain of basic amino

acid residues such as polylysine. Associated vectors comprising these nucleic acids, such as

expression vectors, are expressly contemplated. Moreover, the polypeptide expression

products of such vectors also may be used as immunogens in vaccines. Contemplated

nucleic acids would be in an operable linkage, and would include both sense and antisense

orientations relative to transcriptional elements comprising the vector. Host cells

comprising or transformed with such vectors are also contemplated.

Another embodiment of the invention includes methods of expressing fusion

proteins from such cells. Particularly contemplated are isolated polypeptides comprising a

nucleic acid binding moiety and an M cell specific ligand. Optionally, the immunogen also

may be encoded by such fusion proteins, with or without the presence of a binding moiety

or interim protein sequence. It is also contemplated that antibodies may be generated that

bind selectively or preferentially to such polypeptides, as opposed to the immunogen or to

the M cell specific ligand or nucleic acid binding moiety themselves.

Yet another embodiment of the present invention relates to various kits for assay and

other test purposes that include an M cell specific ligand and a nucleic acid binding moiety

as well as the other constructs and components described above. Preferred kits will be

further packaged with instructions for the use of the vaccine to induce an immune response against the immunogen or against the disease with which the immunogen is associated, for

instance setting forth preferred dosage schemes and formulations as disclosed herein.

Other objects, features and advantages of the present invention will become apparent

from the following detailed description. It should be understood, however, that the detailed

description and specific examples, while indicating preferred embodiments of the invention

are given by way of illustration only, since various changes and modifications within the

spirit and scope of the invention will become apparent to those skilled in the art from this

detailed description.

Brief Description of the Drawings

Figure 1 shows the cell binding capacity of recombinant protein σl and recombinant

protein σl-PL conjugates. Recombinant protein σl binds to (a) mouse L cells, (b) Caco-2

cells and (c) RFL-6 cells, as well as (d) a polylysine (PL) conjugate to mouse L cells.

Figure 2 shows that our recombinant reovirus protein σl can bind murine nasal M

cells.

Figure 3 shows sustained mucosal IgA responses against the reporter gene product,

luciferase.

Figure 4 shows induced cytolytic T cell responses against the reporter gene product,

β-galactosidase.

Figure 5 shows the mucosal intestinal IgA response of mice immunized with one of

three designated HIV DNA vaccine constructs presenting gpl60, gpl40(c) or gp 140(s). Figure 6A and6B show enhanced cytolytic activity (cell-mediated immunity) against

target cells expressing HIV gpl20 from biopsies from mice immunized intranasally with an

M cell-formulated HIV DNA vaccine.

Figure 7 shows that the Candida carbohydrate epitope demonstrates dose dependent

inhibition of antibody-binding to mimitopes discovered through the use of phage display

libraries, and also to a synthetic peptide-carrier protein conjugate.

Detailed Description of the Invention

Definitions

As used herein, the term "adjuvant" refers to a substance added to a vaccine

formulation to improve the immune response, for example, aluminum phosphate (see Singh

et al, Proc. Natl. Acad. Sci. USA, 2000 Jan. 18, 97(2): 811-6). Adjuvants include

immunomodulators including, but not limited to, cytokines, such as IL-1B, TNF , IL-2, IL-

4, GM-CSF, IL-12, IL-18, to name a few, lymphokines, interleukins, interferons and growth

factors. Cytokines, such as interferon-gamma, have found to have particular utility in

tumor-cell vaccines. See van Slooten et al, Int. J. Pharm. (1999 Jun 10), 183(1): 33-6; see

also van Slooten et al, Pharm. Res. (2000 Jan.), 17(1): 42-8). Cytokines have also been

found to enhance antiviral responses. See Barouch et al., Potent CD4⁺ T cell responses

elicited by a bicistronic HIV-1 DNA vaccine expressing gpl20 and GM-CSF. J Immunol.

(2002) 168:562-568; see also Sun et al., Co-expression of granulocyte-macrophage colony-

stimulating factor with antigen enhances humoral and tumor immunity after DNA

vaccination. Vaccine (2002) 20:1466-1474. Adjuvants also include proteins that are toxins. Studies have shown that genetically

modified toxins can enhance immune responses.

Adjuvants also include chemokines, MPL and saponins (i.e., CSL). Suitable

chemokines are disclosed in Ulrich H. von Andrian, M.D., Ph.D. and Charles R. Mackay,

Ph.D. T-Cell Function and Migration: Two Sides of the Same Coin. Ian R. Mackay, M.D.,

and Fred S. Rosen, M.D., eds. The New England Journal of Medicine 343(14), 1020-1034,

October 5, 2000.

Adjuvants can be coadministered with the vaccine delivery system or may be

incorporated into the vaccine complex. In DNA vaccines, for instance, the cDNA for a

particular cytokine may be incorporated into the vaccine under control of a separate

promote. Alternatively, the cytokine may be expressed as a fusion protein together with the

M cell ligand and immunogen.

As used herein, the term "antibody" refers to an immunoglobulin molecule that has

a specific amino acid sequence by virtue of which it interacts only with the antigen that

induced its synthesis in cells of the lymphoid series (especially plasma cells) or with antigen

closely related to it. Antibodies are classified according to their mode of action as

agglutinins, bacteriolysins, haemolysins, opsonins, precipitins, etc. Antibodies would

include not only antibodies raised during an in vivo immune response to antigen, but also

those that are engineered or obtained in vitro, including human, humanized and chimeric

antibodies, Fv and Fab₂ fragments, and immunologically reactive fragments thereof, such as

the Fab and Fab', of F(ab')₂ fragments, etc.

As used herein, the term "antigen" refers to a substance recognized as foreign by the

immune system and can be an immunogen. As used herein, the term "complexed" refers to molecules that are non-covalently

bound to each other through one or more linker molecules.

As used herein, the term "complexing agent" refers to a compound that is capable of

non-covalently binding two molecules together.

As used herein, the term "conjugated" refers to molecules that are covalently bound

to each other through one or more linker molecules.

As used herein, the term "crosslmker" refers to a compound that is capable of

covalently binding two molecules together. After the reaction, the crosslmker, or part of the

crosslinker, forms a part of the linkage between the conjugated molecules.

As used herein, the term "DNA vaccine" specifically refers to a therapeutic or

prophylactic pharmaceutical formulation that contains a nucleic acid that encodes a protein

or peptide against which a vaccinated host is induced to mount an immune response,

preferably a protective immune response. Preferably, such a DNA vaccine contains a

complete eukaryotic expression system encoding the molecular machinery for the

expression of such a protein or peptide subunit vaccine. For example, such a DNA vaccine

may be encoded in plasmid nucleic acids, which comprise promoters, enhancers,

transcriptional terminators, etc. or any other sequences required for gene expression.

As used herein, the term "enteric adhesin" refers to a peptide, protein, carbohydrate

or other class of compound that allows for or facilitates pathogen attachment to M cells as a

means to gain entry to the host. For example, a reovirus σl protein having a molecular

weight of 47 kDa is an enteric adhesin (Nagata et al., Nucleic Acids Res (1984)

12(22):8699-710). As used herein, the term "expression" refers to the expression of peptides or proteins

that are encoded by, for example, the DNA vaccine or associated delivery vector. After

expression of such a peptide or protein by, for example, an M cell to which a DNA vaccine

has been targeted, such expression by the M cell would lead to the induction of an immune

response by a vaccinated host against that encoded immunogen.

As used herein, the term "fusion protein" refers to a protein comprising a first

polypeptide portion, for instance which functions to target such a protein to the mucosal

lymphoid tissue, such as a polypeptide derived from an M cell ligand protein, which is

operably linked to a second polypeptide portion, for instance which functions as a linker to

couple the M cell ligand polypeptide to the immunogen to be targeted to the mucosal

lymphoid tissue. Where the immunogen is a peptide, such a fusion protein may comprise an

M cell ligand polypeptide operably linked to the immunogen peptide itself, without an

intervening linker sequence, hi this context, "operably linked" typically means that the

fusion protein is expressed from a single mRNA that is expressed from a single gene

sequence.

As used herein, the term "immunization" refers to a process that increases or

enhances an organism's reaction to antigen and therefore improves its ability to resist or

overcome infection. Immunization of animals may be used to obtain antibodies against

pathogen epitopes to be used in epitope identification and library screening, for instance.

As used herein, the term "immunogen" refers to an antigen that is capable of

eliciting (inducing) an immune response. For example, an immunogen usually has a fairly

high molecular weight (usually greater than 10,000 daltons). Thus, for example, a variety of

macromolecules such as peptides, proteins, lipoproteins, lipids, glycoproteins, polysaccharides, carbohydrates, some nucleic acids, and certain of the teichoic acids, can act

as immunogens.

As used herein, the term "infectious agent" refers to a microorganism (or associated

substance such as a toxin) that affects or communicates disease through invasion and

multiplication of said substance in body tissues, which may be clinically unapparent or

result in local cellular injury due to competitive metabolism, toxins, intracellular replication

or antigen antibody response.

As used herein, the term "ligand" refers to any molecule that binds to another; in

normal usage a soluble molecule such as a hormone or neurotransmitter, that binds to a

receptor.

As used herein, the term "linker" refers to a moiety that brings two molecules into

close enough association such that the two molecules may effectively be delivered together

to a target cell. Linkers, for example, would include moieties that form complexes,

conjugates, covalent and noncovalent associations as well as those that provide a carrier for

an M cell targeting moiety and immunogen or an immunogen-encoding DNA.

As used herein, the term "M cell(s)" and "follicle associated epithelium" refer to

specialized mucosal cells overlying mucosal associated lymphoreticular tissue (MALT), gut

associated lymphoid tissue (GALT), bronchus associated lymphoid tissue (BALT) and nasal

associated lymphoid tissue (NALT) and any other corresponding mucosal cells that are

known to or become known to persons skilled in the art.

As used herein, the term "M cell specific ligand" refers to a molecule that selectively

binds to a receptor available on the surface of follicle associated epithelial cell

subpopulations, and an M cell specific physiologic effect accompanies that binding (e.g., uptake of pathogen). For example, the enteric adhesin, protein σl of reovirus, is an M cell

specific ligand, as would be any M tropic portion or fragment of σl that retains the ability to

selectively bind to follicle associated epithelial cell subpopulations. M cell tropic portions

of protein σl are known in the art. For instance, Bassel-Duby et al. characterized the amino

acid sequence of protein σl and defined a carboxy terminal portion of the protein as being

responsible for receptor interaction (Nature, 1985 May-Jun, 315(6018): 421-3). Similarly,

by characterizing deletion mutants of protein σl, Nagata et al. defined the receptor binding

domain as being localized to two restriction fragment-generated domains in the carboxy

terminus of the protein (Virology, 1987 Sept., 160(1): 162-8). Nibert and colleagues found

that there were two separate domains that contributed to receptor binding, one in the amino

terminus and one in the carboxy terminus of protein σl (J. Virol., Aug. 1995, 69(8): 5057-

67). Therefore, M cell-tropic variants of protein σl would also include variants with

internal deletions but retaining both the amino and carboxy terminus. An M cell ligand of

the invention would also include a tetramer or trimer of protein σl or variants of protein σl,

as σl has been reported to form tetramers and dimers in binding to cells (see Banerha et al,

Virol. 167: 601-12 (1988); see also Strong et al, Virol. 184(l):23-32 (1991)).

By way of distinction, transfemn and certain other M cell lectins are not considered

M cell specific ligands because: 1) the transferrin receptor is not limited to M cells (e.g.,

neurons express these receptors: Taylor et al, JComp Physiol (1991) 161(5):521-524) and

would not select for follicle associated epithelium subpopulations; and 2) because certain M

cell lectins select for α-linked galactose, and many cells possess carbohydrates with said

linkages which are not follicle associated epithelium cells (e.g., hepatocytes: Oda et al, J

Biol Chem (1988) 263(25): 12576-12583). While M cell ligands (rather than M cell specific ligands) are contemplated for the compositions and methods of certain embodiments of the

present invention, the M cell specific ligands are preferred.

As used herein, the term "minitopes" refers to peptide-size epitopes that are the

"mimimum units" of structure of an antigenic biomolecule. The term "mi igenes" refers to

the correspondingly short oligonucleotide sequences that encode the "minitopes."

As used herein, the term "mucosal" refers to any membrane surface in a host

organism, preferably a mammal such as a human being or agriculturally important animal,

that is covered by mucous.

As used herein, the teπn "nucleic acid" includes DNA and RNA molecules and is

used synonymously with the terms "nucleic acid sequence" and "polynucleotide."

As used herein, the term "nucleic acid binding moiety" refers to compositions and

substances that are capable of binding to or complexing with DNA and serving as a vehicle

to attach the DNA to the M cell ligand-containing compositions of the present invention.

Polybasic chains of amino acids are particularly contemplated for this purpose, as are, for

example, synthetic compounds known to persons skilled in the art that have appropriate

ionic charges to form complexes with DNA.

As used herein, "polymeric chain" refers to compounds formed by the joining of

smaller, usually repeating, units linked by covalent bonds.

As used herein, the term "polymeric chain of basic amino acids" (i.e., polybasic)

refers to a DNA binding sequence that is rich in basic amino acids, such as lysine, arginine,

and ornithine, that is typically about ten to 300 residues long. D-isomers of these basic

amino acids are suitable so long as the length of the stretch of basic amino acids is within the prescribed length. The polymeric chain of basic amino acids can be a homopolymer of a

basic amino acid or it can comprise more than one kind of basic amino acid residue.

As used herein, "polypeptide" refers to an amino acid sequence including, but not

limited to, proteins and protein fragments, naturally derived or synthetically produced.

As used herein, "protective immune response" refers to an immune system reaction

that a human or animal develops in response to a vaccine that protects the human or animal

against subsequent challenge with the pathogen or cancer from which the vaccine was

designed. "Protects" in this sense means that the vaccinated human or animal develops a

"memory" for the immunogen in the vaccine such that a prompt immune response occurs

when the immunogen is later presented on or by a pathogen or tumor cell, and as a

consequence the human or animal does not develop a disease caused by the pathogen or a

malignancy associated with a vaccine tumor antigen.

As used herein, the term "reovirus" refers to a genus of the family Reoviridae

infecting vertebrates only. Transmission is horizontal and infected species include humans,

birds, cattle, monkeys, sheep, swine, and bats. Reovirus 1, reovirus 2, and reovirus 3 infect

mammals, and reovirus 1 is the type species.

As used herein, the term "transcriptional factors" refer to a class of proteins that bind

to a promoter or to a nearby sequence of DNA to facilitate or prevent transcription

initiation.

As used herein, "tumor specific immunogens" refer to immunogens that are

preferentially expressed by tumor cells, more preferably immunogens that are selectively

expressed by tumor cells. As used herein, the term "vaccination" refers to the introduction of vaccine into the

body of an animal (or host) for the purpose of inducing immunity.

As used herein, the term "vaccine" generally refers to a therapeutic or prophylactic

pharmaceutical formulation that' contains a component against which a vaccinated host is

induced to mount an immune response, preferably a protective immune response. For

example, such a component could be a nucleic acid that is expressed by a vaccinated host to

form an expressed protein or peptide subunit vaccine. Alternatively, such a component

could be an immunogenic peptide or any other molecule that induces an immune response.

"Therapeutic" vaccine means that the immune response raised by the vaccine treats or

ameliorates or lessens an ongoing infection or cancer, for instance. "Prophylactic" means

that the vaccine induces a protective immune response that protects the subject against a

future infection or cancer.

General

Targeting of peptide and other epitopes and epitope minigenes to the mucosal

immune compartments of the host is a main object of the invention. Mucosal inductive sites

in humans, such as the Peyer's patches in the intestinal tract and the nasal-associated

lymphoreticular tissue in the oropharyngeal cavity, stand as sentinels to the intestinal and

respiratory tracts and represent the major sites where mucosal immune responses are

initiated. Ghose et al., 1988 Mol. Immunol. 25(3):223-230.

The common cellular feature of these inductive sites are microfold or M cells

scattered about a mucosal surface. (Neutra. et al; Cell (1996) 86: 345-348; Kermeis. et al,

Gastroenterology (1993) 277: 949-952; van Ginkel et al, Merg. Infect. Dis. (2000) 6: 123- 132). M cells appear to function in the uptake, transport, processing and presentation of

microbial antigens. Thus, vaccine constructs that target epitopes and epitope DNA to

respiratory or intestinal M cells represent the basic formulation for the development of new

mucosal vaccines that can be administered by oral, rectal and nasal vaccines or by

inhalation.

This invention provides DNA vaccines, preferably polybasic-M cell ligand

conjugate-polynucleotide complexes, which, when directly introduced into a vertebrate in

vivo, including mammals such as humans, induce the expression of encoded proteins within

the animal. Prior to the present invention, the art had taught that DNA vaccines represent an

efficient method of inducing immunity against a given pathogen if the responsible gene for

eliciting protection is identified. As described below, the present inventors have found that

the described DNA vaccine formulations improve the targeting of DNA to mucosal

inductive tissues. The present invention is based, in part, on the ability of such vaccine

formulations to selectively and preferentially target mucosal inductive tissues. Mucosal

inductive tissues are sites within the mucosa that support the development of B and T

lymphocytes to become stimulated against a specific pathogen or vaccine component or

subunit. If the antigen or vaccine can reach this site, there is a strong likelihood that a

mucosal immune response will be induced.

The invention also provides other immunogen complex vaccines, preferably M cell

ligand conjugate-immunogen complexes, where the immunogen is conjugated to the M cell

ligand by any suitable moiety. Such vaccine formulations are also designed to selectively

and preferentially target mucosal inductive tissues by virtue of the M cell ligand portion of the complex. Once the complexed immunogen is delivered to the target mucosal tissue, a

mucosal immune response will be induced.

To specifically induce such a mucosal immune response, the compositions and

methods of the present invention employ ligands formulated to preferentially or specifically

target the specialized epithelium that suπounds mucosal inductive tissues refeπed to as M

cells. Thus, with regard to DNA vaccines, an M cell ligand binds M cells to mediate

internalization of the attached DNA. With regard to other types of vaccines including those

comprising complexed peptide and carbohydrate immunogens, an M cell ligand binds M

cells to mediate localization of the immunogen to the mucosal lymphoid tissue. In one

embodiment, the M cell ligand is an adhesin of a pathogen, preferably an enteric adhesin of

a pathogen, such as a σl protein of a reovirus. Additionally, adhesins from Salmonella and

poliovirus, as well as other infectious agents having the same tissue tropism would be

appropriate. See Frey et al, Behringlnst. Mitt. (1997 Feb.) 98: 376-89; Sansonetti &

Phalipon, M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction,

consequences for the disease process, Semin. Immunol. (1999) 11 : 193-203; Wilson et al,

Salmonella enterica serovars gallinarum and pullorum expressing Salmonella enterica

serovar typhimurium type 1 fimbriae exhibit increased invasiveness for mammalian cells,

Infect Immun. (2002) 68:4782-4785; Neutra, Interactions of viruses and microparticles with

apical plasma membranes of M cells: implications for human immunodeficiency virus

transmission, J Infect Dis. (1999) 179 Suppl 3:S441-S443. For example, the nucleotide

sequences encoding said proteins include but are not limited to polynucleotides comprising

nucleotide sequences as set forth in GenBank accession numbers: J02325; M10491;

AF059719; AF059718; AF059717; AF059716; U74293; and U74292. In another embodiment, the M cell ligand may be an enteric adhesin of a pathogen

such as an intimin of an enteropathic Escherichia coli. For example, the nucleotide

sequences encoding said intimin protein include but are not limited to polynucleotides

comprising nucleotide sequences as set forth in GenBank accession numbers: U38618;

AJ223063; Y13111; Y13112; AF043226; and U62657. In another embodiment, the

immunogen is an enteric adhesin receptor of a pathogen such as an Tir of an enteropathic

Escherichia coli. For example, the nucleotide sequences encoding the intimin receptor

protein include but are not limited to polynucleotides comprising nucleotide sequences as

set forth in accession number: AF113597. In another embodiment, the immunogen is an

enteric adhesin of a pathogen such as an invasin of Salmonella typhimurium, Yersinia pestis

and pseudotuberculosis and enteropathic Escherichia coli. For example, the nucleotide

sequences encoding said invasin proteins include but are not limited to polynucleotides

comprising nucleotide sequences as set forth in accession numbers: AF140550; Z48169;

X53368; U25631; and M17448.

In another embodiment, the immunogen may be a peptide mimetic, or a minitope, of

an infectious agent or a tumor specific antigen, or the nucleotide sequence encoding the

minitope being termed a minigene. The invention relates to novel technology for

developing vaccine constructs capable of eliciting long-term systemic and mucosal immune

responses to diverse epitope structures including conformational epitopes embedded in

complex structures of infectious agents. This method emphasizes, in a prefeπed

embodiment, the selection of peptide mimics and code (nucleotide sequence) for antibody

binding domains displayed in a library of modularly coded biomolecules in a phage display

format. (Burritt. et al.Anal. Biochem. 238: 1-13). Display technology has become a routine tool for enriching molecular diversity and producing novel types of proteins or peptides (Li.

Nat. Biotechnol (2000) 12: 1251-1256). The use of display technology circumvents the

need for genomic analysis and the difficulty, indeed, the impossibility in some cases, of

deducing from gene sequence analyses the structure and antigenic properties of epitopes

containing protein, lipid and carbohydrate or formed as a discontinuous sequence of amino

acids or monosaccharides. (Scott. Et al, Science (1990) 249: 386-390).

Particularly contemplated are nucleotide and other vaccines in which the

infectious agent such as a microorganism or is a tumor specific antigen. Prefeπed

immunogens are derived from or, like an expressed toxin, are associated with a bacterium,

measles virus, mumps virus, herpes virus, HIV, cancer and influenza virus.

Exemplary bacterial disease organisms include: Group A streptococci, Group B

streptococci, Streptococcus faecalis, Staphylococcus aureus, Listeria monocytogenes,

Helicobacter pylori, Bacillus anthracis, Brucella abortus, Brucella melitensis, Neisseria

gonorrhoeae, Neisseria meningitidis, Hemoplilus influenzae, Mycobaderium tuberculosis,

Bordetella pertussis, Vibrio cholerae, Salmonella typhi, Salmonella enteritidis, Shigella

dysenteriae, Shigella flexneri, Escherichia coli 0157 :H7, Escherichia coli, Escherichia coli

(bovine scouring strains), Chlamydia pneumoniae and Chlamydia trachomatis.

Exemplary bacterial toxins and microorganisms include: A/B bacterial toxins, such

as Shiga toxin-Shigella, Shiga-like toxins-Enterohemoπhagic E. Coli, Diptheria

toxin-Corynebacterium diptheriae, Botulinum toxin-Clostridium botulinum, Tetanus toxin-Clostridium tetani, Cholera toxin- Vibrio cholerae, A toxin-Pseudomonas aeruginosa,

IPT-ΕIEC-Escherica coli; a, β, χ, δ, ε, η, θ, K, γ toxin-Clostridium perfringes; Dick

(Erythrogenic) toxin-Streptococcus pyrogenes; Lethal toxin-Bacillus anthracis; Alpha

toxin-Staphylococcus aureus; and Plague toxin- Yersinia pestis. Fungal diseases include:

Candida albicas; Aspergillus fumigatus; Cryptococcus neoformans; Coccidioides immitis;

and Histoplasma capsulatum.

Exemplary viral diseases and causative agents include: Rhinoviruses-polio, cold

viruses; Alphaviruses-yellow fever, encephalitis; Lyssavirus-rabies; Calcivirus-norwalk

virus; Prthopox virus-smallpox; Papillomavirus-warts; HIV; HPV; Herpesvirus-genital

herpes, simplex, shingles, chickenpox; Bunyavirus-hentavirus; Coronavirus-respiratory

infections; Mobillivirus-mumps, measles; Reovirus-respiratory infections;

Enterovirus-intestinal infections; Influenzavirus-influenza.

Exemplary spirochetal diseases and organisms include Treponema pallidum

(syphilis) and Boπelia recuπentis (Recurring fever). Exemplary protozoan diseases and

causative agents include: Entamoeba histolytica; Giardia lamblia; Taxoplamsa gondii;

Plasmodium species (Plasmodium); Trypanosoma cruzi; Trypanosoma gambiiense;

Leishmaniasis donovani; Pneumocystis carinii; Cryptosporidium; Trichomonas vaginalis;

Schistosoma mansoni and Tritrichomonas faetus.

Exemplary antigens to be included in whole or in part as suitable immunogens, or to

be encoded by the nucleotide vaccines of the invention, and the diseases with which they are

associated include, but are not limited to: tuberculosis (e.g., BCG antigen: Kumar et al,

Immunology (1999) 97(3):515-521), leprosy (e.g., antigen 85 complex: Naito et al, Vaccine

(1999) 18(9-10):795-798), malaria (e.g., surface antigen MSA-2: Pye et al, Vaccine (1997) 15(9):1017-1023), diphtheria (e.g., diphtheria toxoid: U.S. Patent No. 4,691,006 ), tetanus

(e.g., tetanus toxin: Fairweather et al, Infect Immun (1987) 55(11):2541-2545), leishmania

(e.g., Leishmania major promasxigotes: Lasri et al, Vet Res (1999) 30(5):441-449),

salmonella (e.g., covalently bound capsular polysaccharide (Vi) with porin, both isolated

from S. typhi.: Singh et al, Microbiol Immunol (1999) 43(6):535-542), schistomiasis (e.g.,

major antigen of Schistosoma mansoni (Sm28 GST): Auriault et al, PeptRes (1991)

4(1):6-11), measles (e.g., the surface glycoprotein and fusion protein of measles virus:

Machamer et al, Infect Immun (1980) 27(3):817-825), mumps (e.g.,

hemagglutinin-neuraminidase (HN) viral gene product: Brown et al, J Infect Dis (1996)

174(3):619-622), herpes (e.g., HSV-2 surface glycoproteins (gB2 and gD2): Corey et al,

JAMA (1999) 282(4):331-340), AIDS (e.g., gpl60: Pontesilli et al, Lancet (1999)

354(9182):948-949), influenza (e.g., immunodominant peptide from hemagglutinin: Novak

et al, JClin Invest (1999) 104(12):R63-67), Group A streptococcus: (extracellular cysteine

protease, Lukomski, S. et al, Infect. Immun. 1999. 67(4): 179-1788, Streptococcal inhibitor

of complement (Sic) (Lukomski, S., et al, Infect. Immun. 2000. 68(2): 535-542, Hyaluronic

acid capsule, Schrager, H. et al, J. Clin. Invest. 1998. 101: 1708-1716), Group B

streptococcus (capsular polysaccharide, Type I, II, II, IV and V, Pincus, S. H, et al, J.

Immunol. 1998. 160: 293-298.), Shigella species (Lipopolysaccharide (0 somatic antigen)

Phalipon, A., et al, Eur. J. Immunol. 1997. 27: (10), 2620-2625), Brucella abortus

(Lipopolysaccharide (antigen A) Montaraz, J, et al, Immun. 1986. 51: 961-963),

Escherichia coli (EPEC) (intimin,and/or Hp90 protein, Hartland, et al, Mol. Microbiol.

1999. 32 (1): 151-158, Kenny, B., et al, Cell. 1997. 91: 511-520), Escherichia coli (EHEC)

0157-H7 (lipopolysaccharide (LPS), Konadu, E., et al, Infect. Immun. 67:6191-6193), Salmonella typhi (Vi capsular polysaccharide, Singh, et al, Microbiol. Immunol. 1999.

43(6): 535-542), Vibrio cholerae (cholera toxin B subunit, Liljeqvist, S., et al, Appl

Environ. Micro. 1997. 63(7): 2481-2488), Helicobader pylori (Urease A and B, Lee, C. et

al. J. Infect. Dis. 1995. 172: 161-172, Le ^b binding adhesin, Iver, D. et al ., Science, 1998.

279: 373-377), Bordetella pertussis (Filamentous hemagglutinin (FHA), Breiman, M. and

R. Shahin. Am. J. Respir. Crit. Care Med. 1996, 154: 145-149), Haemophilus influenze

(HMW1/HMW2 adhesin, St. Geme, J. The Finnish Med. Soc. DUODECIM. Ann. Med.

1996, HifE pilus (adhesin), Hia adhesin, Barenkamp, S and J. St. Geme, Mol. Microbiol.

1996. 19: 1215-1223), Chlamydia peumoniae (Major outer membrane protein (MOMP),

Peterson, E., et al, Infect. Immun., 1996. 64(8): 3354-3359), HIV (Fusion-dependent

immunogen, LaCasse, R. A., et al, Science. 1999. 283: 357-362, 5-Helix protein, Root, M.

et al, Sciencexpress Report, January 11, 2001), Poliovirus (M cell ligand, Frey, A. et al,

Behring Inst. Mitt. 1997. 98: 376-389), Measles virus (surface glycoprotein, fusion protein,

Machamer et al, Infect. Immun. 1980. 27(3): 817-825), Cryptococcus neoformans

(Capsular polysaccharide-glucuronoxylomannan, Blackstock, R. and A. Casadevall. 1997.

Immunol. 92:334-339), and Schistosoma mansoni (9B antigen peptides,Arnon, R. et al.,

Immunology. 101(4): 555-562). Administration of such antigens in formulations according

to the invention to a host results in stimulation of the host's immune system to produce a

protective immune response.

Exemplary tumor specific antigens may be derived from cancers including:

leukemia- lymphocytic, granulocytic, monocytic or myelocytic; Lymphomas; basal cell

carcinoma; squamous cell carcinoma; breast, colon, endometrial, pancrecatic, lung, etc.

carcinoma; and uterine, vaginal, prostatic, testis, ostogenic or pulmonary sarcoma (see Wang RF., J Mol Med (1999) 77(9):640-655). Tumor antigens according to the invention

include 707-AP (707 alanine proline), AFP (alpha (α)-fetoprotein), ART-4 (adenocarcinoma

antigen recognized by T cells 4), BAGE (B antigen), β-catenin/m (β-catenin/mutated), Bcr-

abl (breakpoint cluster region- Abelson), CAMEL (CTL-recognized antigen on melanoma),

CAP-1 (carcinoembryonic antigen peptide - 1), CASP-8 (caspase-8), CDC27m (cell

division-cycle 27 mutated), CDK4/m (cycline-dependent kinase 4 mutated) CEA

(carcinoembryonic antigen), CT (cancer/testis antigen), Cyp-B (cyclophilin B), DAM

((differentiation antigen melanoma) (the epitopes of DAM-6 and DAM- 10 are equivalent,

but the gene sequences are different; DAM-6 is also called MAGE-B2 and DAM- 10 is also

called MAGE-B 1 ), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene

6/acute myeloid leukemia 1 gene ETS), G250 (glycoprotein 250), GAGE (G antigen), GnT-

(N-acetylglucosaminyltransferase V), GplOO (glycoprotein 100 kD), HAGE (helicose

antigen), HER 2/neu (human epidermal receptor-2/neurological), HLA-A*0201-R170I

(arginine (R) to isoleucine (I) exchange at residue 170 of the - helix of the α2-domain in

the HLA-A2 gene), HPV-E7 (human papiUoma virus E7), HSP70-2M (heat shock protein

70 - 2 mutated), HST-2 (human signet ring tumor - 2), hTERTox hTRT (human telomerase

reverse transcriptase), iCE (intestinal carboxyl esterase KIAA0205 (name of the gene as it

appears in databases), LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP -L-

fucose: β-D-galactosidase 2-α-L-fucosyltransferase), MAGE (melanoma antigen), MART-

1/Melαn-A (melanoma antigen recognized by T cells- 1 /Melanoma antigen A), MC1R

(melanocortin 1 receptor), Myosin/m (myosin mutated), MUC1 (mucin 1), MUM-1, -2, -3

(melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NY-

ESO-1 =New York - esophageous 1), P15 (protein 15), pl90 minor bcr-abl (protein of 190 KD bcr-abl), Pml/RAR (promyelocytic leukaemia/retinoic acid receptor ), PRAME

(preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSM

(prostate-specific membrane antigen), RAGE (renal antigen), RUl or RU2 (renal ubiquitous

1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1

or 3), TEL/AMLl (translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m

(triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein 1, or gp75), TRP -2

(tyrosinase related protein 2), TRP-2/INT2 (TRP-2/intron 2), WT1 (Wilms' tumor gene).

These antigens are disclosed in references that are cited in Cancer Immunology

Immunotherapy 50:3-15 (2001), which is herein incorporated by reference. The cited

references may be consulted for methods of isolating the specific antigens or genes

encoding the specific antigens for use in the vaccines of the invention.

In general, it is the formulation of an appropriate DNA conjugate or immunogen

complex (or other delivery vector) to deliver the DNA to a target M cell that improves host

immune responses against a specific pathogen or other immunogen. For example, such a

vaccine may be comprised of a polybasic conjugate/DNA complex by incorporating an M

cell ligand. Thus, for any given immunogen encoded by a nucleic acid or mimicked by a

peptide encoded by a nucleic acid that can be used for eliciting a host response, such a

response can be enhanced through effective targeting mediated by M cell ligands.

DNA Conjugate Vaccines

M cell-directed-DNA vaccine formulations of the present invention have been

demonstrated to be a robust epitope DNA delivery technology. In one embodiment, the

formulations employ plasmid DNA (pDNA) constructs (Shroff et al, Pharm. Sci. Tech. Today (1999) 2: 205-212), that can be designed to contain either a single epitope or multiple

epitopes (polytopes). Thus, many different formulations of epitope DNA-M cell directed

vaccines which target epitope DNA and immunogenic peptides to key compartments of the

immune system can be used to elicit potent cellular as well as humoral immune responses to

infectious agents. There is every expectation that the same technology could be applied to

the development of vaccines against many forms of cancer (Kieber-Emmons et al., J.

Immunol. (2000) 165:623-627; Qui et al., Hybridoma (1999) 18:103-112), parasitic

diseases, (Arnon et al, Immunol. (2000) 101:555-562; de la Cruz, et al.J. Biol. Chem.

(1988) 283: 4318-4322) viral diseases, (Frey. et al, Behringlnst. Mitt. (1997) 98: 376-389:

Prince, et al, Vaccine. (1991) 15: 916-919) fungal diseases, (Glee, et al, Ann. Meeting Am.

Soc. Micro. (1997), and bacterial infections (Lowrie. et al, Springer Semin.

Immunolpathol, (1997) 19: 161-173; Wu et al, Gene Therapy (2000) 7:61-69).

The observation that mammalian cells can express genes encoded by plasmid DNA

(pDNA) injected intramuscularly has led to experiments using gene immunization.

(Donnelly, et al Ann. Rev. Immunol (1997) 15: 617-648; Shroff, et al, Pharm./ Sci. Tech

Today. (1999) 2: 205-212; Lee. et al, Ann. Med. (1998) 30: 460-468; Jones, et al, Vaccine.

(1997) 115: 814-817). The pDNA of vaccine constructs has not been found to integrate into

host chromosomal DNA (unlike retrovirus and adenovirus vectors) thereby averting the

activation of oncogenes or disruption of normal gene function, nor do pDNA preparations

(if free of protein and RNA) stimulate an anti-DNA humoral response or generate tolerance

in neonatal animals.

DNA vaccines can be delivered by any route but the most promising appears to be

those that target key cells (antigen-presenting/processing cells) in immune compartments of the mucosal membrane. To be expressed in a host cell, plasmids must cross the plasma

membrane, escape endosomal degradation pathways and access the cytoplasm. By any

method of administration, pDNA must finally enter the nucleus before gene expression can

commence, but, once there, noπnal cellular transcriptional and translational pathways are

exploited for the production of gene products. The enormous potential of DNA vaccines lies

in their versatility, ease of manufacture and safety. Of paramount importance is their ability

to induce a cytotoxic T lymphocyte (CTL) response, which is necessary for the eradication

of most viral diseases, certain bacterial diseases (tuberculosis, Brucella), parasitic infections

and tumors. (Jones, et al., Vaccine. (1997) 15: 814-817; Offit. et al., J. Virol. (1991) 65:

1318-1324; Gallichan. et al, J. Exp. Med. (1996) 184: 1879-1890; Hilleman, M. et al,

Vaccine (1998) 16: 778-793; Van Ginkel et al, Emerg. Infect. Dis. (2000) 6(2)123-132).

The ability to stimulate a CTL response coupled with the capacity for repeated

(endogenous) immunizations also raises the possibility of using these vaccines for therapy.

Our presently formulated DNA vaccines induce improved mucosal IgA antibody

responses and promote sustained CTL responses, demonstrating efficacious vaccination via

the mucosa. Further, as the present invention shows the ability of the protein σl to mediate

efficient gene transfer to the nasal-associated lymphoid tissue (NALT) in vivo, we have

demonstrated that systemic and mucosal immunity to intranasally delivered DNA or

peptides or other antigens as part of an M cell ligand complex is achievable.

In a prefeπed embodiment, a contemplated polynucleotide is a nucleic acid which

contains essential regulatory elements such that upon introduction into a living vertebrate

cell, it is able to direct the cellular machinery to produce translation products encoded by the

structural gene sequence component of the polynucleotide. In one embodiment of the invention, the polynucleotide is a polydeoxyribonucleic acid comprising immunogen (or

antigen) structural genes or fragments thereof operatively linked to a transcriptional

promoter(s). In another embodiment of the invention the polynucleotide comprises

polyribonucleic acid encoding antigen structural genes or fragments thereof which are

amenable to translation by the eukaryotic cellular machinery (ribosomes, tRNAs, and other

translation factors).

Where the protein encoded by the polynucleotide is one which does not normally

occur in that animal except in pathological conditions, (i.e. an heterologous protein) such as

proteins associated with human immunodeficiency virus (HIN) and Brucella, the animals'

immune system is activated to launch a protective immune response. Because these

exogenous proteins are produced by the animals' own tissues, the expressed proteins are

processed by the major histocompatibility system (MHC) in a fashion analogous to when an

actual infection occurs. The result, as shown in this disclosure, is induction of immune

responses against an antigen.

Polynucleotides for the purpose of generating immune responses to an encoded

protein are refeπed to herein as polynucleotide or DΝA vaccines. The described vaccine

works by inducing the vaccinated animal to produce antibodies or cell-mediated immune

responses specific for the vaccine. The production of these antibodies or cell-mediated

immune responses will^' protect the host upon subsequent exposure to the infectious agent.

The present invention further provides recombinant DΝA molecules (rDΝAs) that

contain a coding sequence. The vaccines are produced using conventional eukaryotic

plasmid expression systems for the encoded gene. As used herein, a rDΝA molecule is a

DΝA molecule that has been subjected to molecular manipulation in situ. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al,

Molecular Cloning (1989). In the prefeπed rDNA molecules, a coding DNA sequence is

operably linked to expression control sequences and/or vector sequences.

The choice of vector and/or expression control sequences to which one of the protein

encoding sequences of the present invention is operably linked depends directly, as is well

known in the art, on the functional properties desired, e.g., protein expression, and the host

cell to be transformed. A vector contemplated by the present invention is at least capable of

directing the replication or insertion into the host chromosome, and preferably also

expression, of the structural gene included in the rDNA molecule.

Expression control elements that are used for regulating the expression of an

operably linked protein encoding sequence are known in the art and include, but are not

limited to, inducible promoters, constitutive promoters, secretion signals, and other

regulatory elements. Preferably, the inducible promoter is readily controlled, such as being

responsive to a host cell's environment.

hi one embodiment, the vector containing a coding nucleic acid molecule will

include a prokaryotic replicon, i.e., a DNA sequence having the ability to direct autonomous

replication and maintenance of the recombinant DNA molecule extrachromosomally in a

prokaryotic host cell, such as a bacterial host cell, transformed therewith. Such replicons

are well known in the art. In addition, vectors that include a prokaryotic replicon may also

include a gene whose expression confers a detectable marker such as a drug resistance.

Typical bacterial drug resistance genes are those that confer resistance to ampicillin or

tetracycline. Nectors that include a prokaryotic replicon can further include a prokaryotic or

bacteriophage promoter capable of directing the expression (transcription and translation) of

the coding gene sequences in a bacterial host cell, such as E. coli. A promoter is an

expression control element foπned by a DΝA sequence that pemiits binding of RΝA

polymerase and transcription to occur. Promoter sequences compatible with bacterial hosts

are typically provided in plasmid vectors containing convenient restriction sites for insertion

of a DΝA segment of the present invention. Typical of such vector plasmids are pUC8,

pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA), pPL and

pKK223 available from Phannacia, Piscataway, Ν. J.

Expression vectors compatible with eukaryotic cells, preferably those compatible

with vertebrate cells, can also be used to form a rDΝA molecules that contains a coding

sequence. Eukaryotic cell expression vectors are well known in the art and are available

from several commercial sources. Typically, such vectors are provided containing

convenient restriction sites for insertion of the desired DΝA segment. Typical of such

vectors are pSNL and pKSN-10 (Pharmacia), pBPN-l/pML2d (International

Biotechnologies, Inc.), pTDTl (ATCC, #31255), the vector pCDM8 described herein, and

the like eukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the DΝA vaccine molecules of

the present invention may further include a selectable marker that is effective in an

eukaryotic cell, preferably a drag resistance selection marker. A prefeπed drug resistance

marker is the gene whose expression results in neomycin resistance, i.e., the neomycin

phosphotransferase (neo) gene. (Southern et al, J. Mol. Anal. Genet. 1:327-341, 1982.)

Alternatively, the selectable marker can be present on a separate plasmid, and the two vectors are introduced by co-transfection of the host cell, and selected by culturing in the

appropriate drug for the selectable marker.

The M cell ligand-polybasic conjugates employed in the DNA vaccines of the

invention may be produced chemically or by the recombinant method. Coupling by the

chemical method can be carried out in a manner known er se for the coupling of peptides

and if necessary the individual components may be provided with linker substances before

the coupling reaction (this procedure is necessary when there is no functional group suitable

for coupling available at the outset, such as a mercapto or alcohol group).

Depending on the desired properties of the conjugates, particularly the desired

stability thereof, coupling may be carried out by means of various techniques known to

persons skilled in the art, including but not limited to the following techniques. For

example, the use of disulphide bridges, which can be cleaved again under reductive

conditions (e.g., using succinimidyl pyridyl dithiopropionate, are contemplated. See Jung et

al, Biochem Biophys Res Comm 101:599-606 (Jul. 30, 1981). Also contemplated is the use

of compounds which are largely stable under biological conditions (e.g., thioethers, by

reacting maleimido linkers with sulfhydryl groups of the linker bound to the second

component). Further comtemplated is the use of bridges that are unstable under biological

conditions, e.g., ester bonds, or using acetal or ketal bonds which are unstable under weakly

acidic conditions.

The production of the conjugates according to the invention by the recombinant

method offers the advantage of producing precisely defined, uniform compounds, whereas

chemical coupling produces conjugate mixtures which then have to be separated. The recombinant preparation of the conjugates according to the invention can be

caπied out using methods known for the production of chimeric polypeptides. The present

invention further provides methods for producing a protein of the invention using nucleic

acid molecules herein described. In general terms, the production of a recombinant form of

a protein typically involves the following steps:

First, a nucleic acid molecule is obtained that encodes an M cell ligand protein of the

invention. If the encoding sequence is uninterrupted by introns, it is directly suitable for

expression in any host. The nucleic acid molecule is then preferably placed in operable

linkage with suitable control sequences, as described above, to form an expression unit

containing the protein open reading frame. The expression unit is used to transform a

suitable host and the transformed host is cultured under conditions that allow the production

of the recombinant protein. Optionally the recombinant protein is isolated from the medium

or from the cells; recovery and purification of the protein may not be necessary in some

instances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. For example, the

desired coding sequences may be obtained from genomic fragments and used directly in

appropriate hosts. The construction of expression vectors that are operable in a variety of

hosts is accomplished using appropriate replicons and control sequences, as set forth above.

The control sequences, expression vectors, and transformation methods are dependent on

the type of host cell used to express the gene and were discussed in detail earlier. Suitable

restriction sites can, if not normally available, be added to the ends of the coding sequence

so as to provide an excisable gene to insert into these vectors. A skilled artisan can readily adapt any host/expression system known in the art for use with the nucleic acid molecules

of the invention to produce recombinant protein.

The polybasic and other binding or linker moiety components may vary in terms of

their size and amino acid sequence. Production by genetic engineering has the advantage of

allowing the M cell ligand component of the conjugate to be modified, by increasing the

ability to bind to the receptor, by suitable mutations, for example, or by shortening the M

cell ligand component to the part of the molecule which is responsible for the binding to the

receptor. It is particularly expedient for the recombinant preparation of the conjugates

according to the invention to use a vector which contains the sequence coding for the M cell

ligand component as well as a polylinker into which the required sequence coding for the

polybasic or binding moiety component is inserted. In this way, a set of express plasmids

can be obtained, of which the plasmid containing the desired sequence can be used as

necessary in order to express the conjugate according to the invention.

The nucleic acids which are to be transported into the cell may be DNAs or RNAs,

with no restrictions as to the nucleotide sequence. The nucleic acids may be modified,

provided that this modification does not affect the polyanionic nature of the nucleic acids;

these modifications include, for example, the substitution of the phosphodiester group by

phosphorothioates or the use of nucleoside analogues.

With regard to the size of the nucleic acids the invention again permits a wide range

of uses. There is no lower limit brought about by the transporting system according to the

invention; thus, any lower limit which might arise would be for reasons specific to the

particular intended use or target specificity. It is also possible to convey different nucleic

acids into the cell at the same time using the conjugates according to the invention. Within the scope of the present invention it has been possible to demonstrate that M

cell ligand-polybasic conjugates can be efficiently absorbed into living cells and

internalized. The disclosed conjugates or complexes according to the invention are not

apparently harmful to cell growth. This means that they can be administered repeatedly and

thus ensure a constantly high expression level of the genes and nucleotide sequences

inserted into the cell.

The ratio of nucleic acid to conjugate can vary within a wide range, and it is not

absolutely necessary to neutralize all the charges of the nucleic acid. This ratio will have to

be adjusted for each individual case depending on criteria such as the size and structure of

the nucleic acid which is to be transported, the size of the polybasic component and the

number and distribution of its charges, so as to achieve a ratio of transportability and

biological activity of the nucleic acid which is favorable to the particular application. This

ratio can first of all be adjusted coarsely, for example by using the delay in the speed of

migration of the DNA in a gel (e.g., using the mobility shift on an agarose gel) or by density

gradient centrifugation. Once this provisional ratio has been obtained, it may be expedient

to carry out transporting tests with the radioactively labeled complex with respect to the

maximum available activity of the nucleic acid in the cell and then reduce the proportion of

conjugate if necessary so that the remaining negative charges of the nucleic acid are not an

obstacle to transportation into the cell.

The preparation of the M cell ligand-polybasic conjugate/nucleic acid complexes,

which are also a subject of the invention, can be carried out using methods known per se for

the complexing of polyionic compounds. One possible way of avoiding uncontrolled

aggregation or precipitation is to mix the two components together first of all at a high (about 1 molar) concentration of common salt and subsequently to adjust to physiological

saline concentration by dialysis or dilution. Preferably, the concentrations of DNA and

conjugate used in the complex forming reaction are not too high (more than 100 g/ml), to

ensure that the complexes are not precipitated, as would be known to persons skilled in the

art.

A prefeπed nucleic acid component of the M cell ligand-polybasic moiety-nucleic

acid complex according to the invention is an immunogen structural gene. The invention

further relates to a process for introducing nucleic acid or acids into human or animal cells,

preferably forming a complex which is soluble under physiological conditions.

There are many embodiments of the instant invention which persons skilled in the

art can appreciate from the specification. Thus, different transcriptional promoters,

terminators, carrier vectors or specific gene sequences may be used successfully. Various

methods are known for such constructs which, upon introduction into mammalian cells,

induces the expression, in vivo, of the polynucleotide thereby producing the encoded

protein. It is readily apparent to those skilled in the art that variations or derivatives of the

nucleotide sequence encoding a protein can be produced which alter the amino acid

sequence of the encoded protein.

It is well known in the biological arts that certain amino acid substitutions can be

made in protein sequences without affecting the function of the protein. Generally,

conservative amino acids are tolerated without affecting protein function. Similar amino

acids can be those that are similar in size and/or charge properties, for example, aspartate

and glutamate, and isoleucine and valine are both pairs of similar amino acids. Similarity

between amino acid pairs has been assessed in the art in a number of ways. For example, Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3,

Chapter 22, pages 345-352, which is incorporated by reference herein, provides frequency

tables for amino acid substitutions which can be employed as a measure of amino acid

similarity. Dayhoff et al. 's frequency tables are based on comparisons of amino acid

sequences for proteins having the same function from a variety of different evolutionary

sources. The altered expressed protein may have an altered amino acid sequence, yet still

elicits immune responses which react with the antigen protein, and are considered functional

equivalents. In addition, fragments of the full length genes which encode portions of the full

length immunogenic protein may also be constructed. These fragments should encode a

protein or peptide which elicits antibodies that cross-react with the immunogenic protein,

and are considered to be functional equivalents.

Other M Cell-Immuno en Vaccines

Another embodiment of the present invention includes an M cell specific ligand or

M cell tropic fragment or portion thereof conjugated or complexed to an immunogen,

optionally via an appropriate linker. Immunogens in this instance would include a variety

of macromolecules such as peptides, proteins, lipoproteins, lipids, glycoproteins,

polysaccharides, carbohydrates, some nucleic acids, genes, and certain of the teichoic acids,

or any other molecule from a pathogen or tumor cell that could be used to generate a

protective immune response. Particularly prefeπed immunogens are as provided above.

Immunogens may be conjugated or complexed with the M cell specific ligand using

any means known in the art. For instance, peptide and protein immunogens may be

comprised in fusion proteins where they are operably linked to the M cell specific ligand or fragment. Fusion proteins are expressed from a single open reading frame encoding both

the M cell specific ligand and the immunogen in such a manner that the M cell specific

ligand portion retains its capability to target M cells and the immunogen retains its

immunogenic potential. Fusion proteins can optionally contain an intermediate peptide

region or linker comiecting the M cell binding portion to the immunogen portion. Genes

encoding the fusion proteins of the invention are also encompassed, as are plasmid vectors

and host cells comprising and expressing the same.

Alternatively, immunogens may be conjugated or complexed with an M cell specific

ligand using an appropriate linker. Such linkers may include chemical cross-linkers or

fusion mediators. Cross-linking may be performed with either homo- or heterobifunctional

agents, i.e., SPDP, DSS, SIAB. Methods for cross-linking are disclosed in

PCT/DK00/00531 (WO 01/22995) which is herein incoφorated by reference. Such

methods may generally include the steps of:

a) reacting an antigen or immunogen with a first crosslinker thereby producing a

mixture of crosslinker derivatives of the immunogen;

b) isolating the antigen derivatised with a single crosslinker residue,

c) activating the isolated crosslinker derivative of the antigen,

d) reacting an M cell ligand with a second crosslinker thereby producing a mixture

of crosslinker derivatives of the M cell ligand component,

e) reacting the activated crosslinker derivative of the antigen with the mixture of

crosslinker derivatives of the M cell ligand, thereby producing conjugates between the

antigen and the M cell ligand. In one embodiment, the first crosslinker is a bifunctional crosslinker (i.e., with two

functional groups), preferably a heterobifunctional crosslinker (i.e., with two different

functional groups). In another embodiment, the second crosslinker is a bifunctional

crosslinker, preferably a heterobifunctional crosslinker. In a further embodiment, the first

and/or second crosslinker is selected from the non-limiting group consisting of N-

succinimidyl (4-iodoacetyl) aminobenzoate (SIAB), N-succinimidyl-3-(2-

pyridylthio)propionate (SPDP), N-succinimidyl S-acetylthioacetate (SATA), m-

maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and N-g-maleimidobutyryloxy-

succinimide ester (GMBS). In a further embodiment, the first and/or second crosslinker is

Traut's Reagent 2-iminothiolane in combination with SPDP. In still a further embodiment

the first and/or second crosslinker is succinimidyl dicarbonyl pentane or disuccinimidyl

suberate. In a further embodiment, the first and/or second crosslinker is selected among

those disclosed in The Pierce Products Catalogue (Pierce Chemical Company, USA) and the

Double Agents™ Cross-Linking Reagents Selection Guide (Pierce Chemical Company),

which are herein incorporated by reference.

In the general method presented above, any suitable method may be used to purify

the crosslinker derivatised immunogen. For instance, the crosslinked immunogen may be

purified by preparative reverse phase HPLC (RP-HPLC). In another embodiment, the

crosslinked immunogen may be purified by membrane filtration, such as ultrafiltration or

diafiltration. Unreacted crosslinker may be removed by size exclusion chromatography,

such as gel filtration. The final conjugate may also be purified using any suitable means,

including for instance gel filtration, membrane filtration, such as ultrafiltration, or ion

exchange chromotography, or a combination thereof. Molar ratios to be used in crosslinking methodology may be readily optimized by

those ₍of skill in the art, but generally will vary between about 1 : 1 to about 5 : 1 crosslinker to

immunogen or ligand depending on the crosslinker and the efficiency of crosslinking. The

ratio of crosslinked immunogen to crosslinked M cell ligand to be admixed may also be

readily optimized by those of skill in the art, but will generally range from about 1 : 1 to

about 10:1 immunogen to M cell ligand.

Conjugates and complexes can comprise the following scenarios: polypeptides with

attached immunogens may be conjugated to M cell specific ligands; liposomes can replace

the polypeptide, wherein the M cell specific ligand may be conjugated to a liposome

containing the immunogens, or conjugated to a liposome with one or several copies of an

immunogen or different immunogens attached/displayed to its surface; and peptide and

protein immunogens may be expressed as fusion proteins operably linked to the M cell

specific ligand. Fusion proteins are known in the art, such as those disclosed in Yu et al,

The biologic effects of growth factor-toxin conjugates in models of vascular injury depend

on dose, mode of delivery, and animal species, J Pharm Sci. (1998) Nov;87(ll): 1300-4;

McDonald et al, Large-scale purification and characterization of recombinant fibroblast

growth factor-saporin mitotoxin, Protein Expr Purif. (1996) Aug;8(l):97-108; Lappi et al,

Expression and activities of a recombinant basic fibroblast growth factor-saporin fusion

protein, J Biol Chem. (1994) Apr 29;269(17):12552-8; and Prieto et al, Expression and

characterization of a basic fibroblast growth factor-saporin fusion protein in Escherichia

coli. AnnN Y Acad Sci. (1991) 638:434-7. By way of example, fusion-derived

immunogen conjugates include K99 fimbrial protein from bovine enterotoxigenic E. coli fused to protein σl, colonization factor antigen 1 fimbrial protein from human

enterotoxigenic E. coli fused to protein σl or myelin basic protein fused to protein σl .

Liposomes are one means by which M cell ligands may be attached to immunogens.

Liposomes may be made by means that are well known in the art, and may be polymerized

or unpolymerized, depending on the desired characteristics for the liposome. In general,

polymerized lipid compositions may be produced according to techniques described in U.S.

Patent 5,962,422 to Nagy et al, "Inhibition of Selectin Binding", utilizing the materials and

methods disclosed therein. Other suitable methods are disclosed in U.S. Patent 6,342,226 to

Betbeder et al, "Method for Increasing Immunogenicity, Product Obtained and

Pharmaceutical Compositions"; U.S. Patent 6,090,406 to Popescu et al, "Potentiation of

Immune Responses with Liposomal Adjuvants"; and U.S. Patent 6,225,445 to Shen et al.,

"Methods and Compositions for Lipidization of Hydrophilic Molecules." In liposomal

formulations, protein σl or another M cell targeting ligand is attached covalently or by

other means to the liposome. The immunogen being delivered to the M cells may be either

encapsulated within the liposome, such as for delivery of an immunogen which needs to be

protected from interaction in vivo prior to the destination of choice, or displayed on the

surface of the liposome. Adjuvants such as the immunomodulators described herein may be

readily included in the liposome formulation.

Non-DNA vaccine formulations may work by receptor-mediated endocytosis

pathways, wherein immunogens are associated with MHC molecules and displayed on the

cell surface of follicle-associated epithelial cells. Non-DNA immunogens may also mount a

mucosal immune response by virtue of being targeted to the mucosal lymphoid tissue by

means of an M cell ligand. The success reported herein with targeting of DNA vaccines to mucosal surfaces indicates that any antigen can be targeted similarly so long as the means

exist to couple the antigen to the M cell ligand.

With regard to the size of the immunogen, the invention again permits a wide range

particular intended use or target specificity. It is also possible to convey different

immunogens and types of immunogens into the cell at the same time using the conjugates

according to the invention.

The ratio of immunogen to M cell specific ligand in the complex or conjugate can

vary within a wide range. This ratio will have to be adjusted for each individual case

depending on criteria such as the size and structure of the immunogen to be targeted, the

size of the linker if required or used, so as to achieve a ratio of transportability and

biological activity of the immunogen which is favorable to the particular application.

between amino acid pairs has been assessed in the art in a number of ways. For example,

Dayhoff et al. (1978) in Atlas of Protein Sequence and Structure, Volume 5, Supplement 3,

similarity. Dayhoff et al. 's frequency tables are based on comparisons of amino acid sequences for proteins having the same function from a variety of different evolutionary

equivalents. In addition, fragments of the full length immunogenic proteins may also be

constructed. These fragments should comprise an epitope of a protein or peptide which

elicits antibodies that cross-react with the immunogenic protein, and are therefore

considered to be functional equivalents.

Preparation of Antibodies

Antibodies against M cell ligand-polybasic protein conjugate or other complexes

described herein may be prepared by immunizing suitable mammalian hosts using the

peptides, polypeptides or proteins alone or conjugated to suitable carriers. It may also be

desirable to isolate antibodies to immunogenic epitopes of molecules derived from

pathogens, for screening of the phage display minitope libraries described herein. Methods

for preparing immunogenic conjugates with carriers such as BSA, KLH, or other carrier

proteins are well known in the art. In some circumstances, direct conjugation using, for

example, carbodiimide reagents may be effective; in other instances linking reagents such as

those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide

accessibility to the hapten. The hapten peptides can be extended at either the amino or

carboxy terminus with a Cys residue or interspersed with cysteine residues, for example, to

facilitate linking to a carrier. Administration of the immunogens is conducted generally by

injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to

determine adequacy of antibody foπnatiόn.

While the polyclonal antisera produced in this way may be satisfactory for some

applications, for pharmaceutical compositions, use of monoclonal preparations is prefeπed.

Immortalized cell lines wliich secrete the desired monoclonal antibodies may be prepared

using the standard method of Kohler and Milstein or modifications which effect

immortalization of lymphocytes or spleen cells, as is generally known. The immortalized

cell lines secreting the desired antibodies are screened by immunoassay in which the antigen

is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell

culture secreting the desired antibody is identified, the cells can be cultured either in vitro or

by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant

or from the ascites supernatant. Fragments of the monoclonals or the polyclonal antisera

which contain the immunologically significant portion can be used as antagonists, as well as

the intact antibodies. Use of immunologically reactive fragments, such as the Fab, Fab', of

F(ab')₂ fragments is often preferable, especially in a therapeutic context, as these fragments

are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced, using current technology, by

recombinant means. Regions that bind specifically to the desired regions of the gene

products can also be produced in the context of chimeras with multiple species origin.

Alternatively, antibodies specific for the M cell ligand polybasic moiety conjugate

can be humanized antibodies or human antibodies, as described in U. S. Patent No.

5,585,089 by Queen et al. See also Riechmann et al, Nature (1988) 332: 323-27. Use of Phage Display to Identify Immunogenic Epitopes

The immunogen of the M-cell vaccine may be a peptide mimetic, or a minitope, of

an infectious agent or a tumor specific antigen, with the nucleotide sequence encoding the

minitope being termed a minigene. The method emphasizes the selection of peptide

mimetics displayed in a library of modularly coded biomolecules in a phage display format

using antibodies or antibody binding domains, and isolation of the nucleotide sequences

encoding the same (Burritt et al, Anal. Biochem. (1996) 238:1-13). Display technology has

become a routine tool for emiching molecular diversity and producing novel types of

proteins or peptides (Li, Min. Nat. Biotechnol 12: 1251-1256). The use of display

technology circumvents the need for genomic analysis and the difficulty, indeed, the

impossibility in some cases, of deducing from gene sequence analyses the structure and

antigenic properties of epitopes containing protein, lipid and carbohydrate or formed as a

discontinuous sequence of amino acids or monosaccharides. (Scott et al, Science (1990)

249: 386-390).

Regardless of the foπnat, a display library consists of modularly coded molecules,

each of which contains three components: displayed entities, a common linker and the

coπesponding individualized codes. One of the most important characteristics of display

technologies is the ability to determine the structure of a desired compound rapidly after

initial screening. Structural (or sequence) characterization is often accomplished by a

process commonly known as coding and decoding, which can be achieved via a coupled

amplification and purification process. There are two requirements for coding strategies:

the availability of adequate information space to cover the diverse entities to be displayed and the presence of a highly sensitive and/or amplifiable property that permits rapid

decoding. Phage display is one of most commonly used format because of its ease of

coding or amplification, but it is limited by the lack of building blocks (only D-and L-

amino acids) and structural space for certain desired properties.

The advantage of phage display is recognized in its ability to select peptides that

recognize not only conformational epitopes but epitopes consisting of or containing

carbohydrates or proteins or conjugates of both (i.e, glycoproteins) (Grobowska et al,

Virology (2000) 269: 47-53). It is expected that peptide mimetics for lipid-rich and nucleic

acid epitopes may also be selected from a display library with an appropriate antibody

probe. For non-biological and some cellular displays, there is virtually no specific limit in

terms of what building blocks may be chosen for the generation of diverse molecules, but

the coding and decoding strategies are laborious and expensive.

The use of display technology simplifies and reduces the cost of the epitope

discovery process for vaccine development because many research reagents, especially

antibodies directed against antigenic determinants on pathogens, are already available to

select peptide sequences with specific antigenic properties. Because antigenic structures

embedded in the cell wall of an infectious agent or capsid of a viral agent have been

previously identified by the immune system as immunological targets, a significant amount

of guesswork in choosing an effective immunological target is eliminated.

An essential criterion for an antibody to be used for selection of peptides is that it

has been demonstrated to bind to specific virulence factors and exert a protective effect in

vivo. Monoclonal antibodies are the desired reagent for peptide selection because they bind

to a limited number of peptide species. However, in general, polyclonal antibodies are capable of selecting a surprisingly limited number of important prominent antigenic

determinants. For example, the selection of sequences from the J404 phage-display library

with unfractionated immune serum directed toward filamentous actin in neutrophils yielded

only two overlapping consensus sequences (Burritt. et al, J. Biol. Chem. 270: 16974-

16980). These two peptides could be traced over the actin crystal structure, which together

defined a single epitope. This selection took place in spite of the less dominant antibodies

of much broader specificity that would exist in any unfractionated serum. Recovery of

epitope mimetics using polyclonal antibodies is therefore driven by the predominantly

recognized determinants that are amplified most rapidly from the library. As a practical

matter, the use of display technology rapidly and inexpensively defines antigenic domains

of complex molecules without having to engage in a sophisticated and expensive genomic

discovery process or even to consult gene expression data.

The isolation of putatively immunogenic peptide epitopes bound by protective

antibody reagents from phage display peptide libraries may thus aid in preparing the

protective vaccines of the invention (Arnon et al, Immunol. (2000) 101:555-562; Wu et al,

Gene Therapy (2000) 7:61-69; Qui et al, Hybridoma (1999) 18:103-112; Kieber-Emmons

et al, J. Immunol. (2000) 165:623-627). Because most current efforts in functional

proteomics seek to deduce the structure of epitopes from analyses of gene expression

products (only proteins), a large and relatively unexplored area of epitope identification is

ready for exploitation. Thus, by probing a phage display peptide library with an antibody

specific for a pathogen's epitope, a peptide that delineates or mimics a continuous or, more

importantly, a discontinuous or conformational epitope ~ an antibody binding determinant composed of residues distant in the primary sequence but adjacent in the folded protein

structure ~ can be isolated.

Because the oligonucleotide sequence encoding a given peptide mimetic is derived

from the sequence analysis of phage DNA, minigenes for epitope expression in vivo can be

readily prepared for use in DNA-based vaccine development, or for use in fusion protein

vaccines, or to recombinantly produce peptide epitopes to be complexed to vaccines, from

stocks of phage identified as expressing relevant mimetics. Minigene expression (of

immunogenic peptides) facilitated by promoter genes of eucaryotic cells can yield protective

immune responses following vaccine delivery or targeting to compartments of the immune

system.

Because the antigenicity of the phage-derived peptide epitopes is immediately

established (it was, after all, selected by an antibody) and their immunogenicity can be

assessed by immunizing animals with appropriate carrier formulations (and by other

techniques known to persons skilled in the art), the minigene encoding the peptide can

predictably be expected to elicit immune responses if the peptide expressed in vivo

appropriately conforms to the structure of the native epitope. Minigenes, comprised for

example of 27 to 45 nucleotide base pairs, and encoding nine-mer to fifteen-mer minitopes

can be incorporated into plasmid constructs and are able to elicit protective immune

responses to an infectious agents. Biological display exploits the cellular biosynthesis

machinery to assemble biopolymers, the sequence of which ultimately specifies structure

and distinct properties.

Phage display formats are most commonly employed where the coding sequence is

embedded in the viral genome and the displayed molecule is part of the viral coat protein. In utilizing phage display techniques as described herein, the peptide epitopes preferably are

displayed as sequences of nine amino acids as an amino-terminal fusion with the minor coat

protein pill. These are "linked" to codes that have chemical and physical properties that can

be readily determined (e.g. the sequence of nucleic acids).

Minigenes or minitopes are predicted to have significant advantages as subunit

vaccine candidates when compared to conventional vaccine components. In particular, it is

the ability of antigen DNA encoding epitopes or peptide mimetics of epitopes, given the

proper delivery system, to elicit T-dependent immune responses that is advantageous (Cox

et al, Science (1994) 254: 716-719; Celis et al, Proc. Natl. Acad. Sci. (1994) 91: 2105-

2109; Wang et al. Science. (1998) 282: 476-480). Peptides can induce Thl responses

(CTL, IgG2a. DH etc.) when minigenes encoding the peptide and appropriate eucaryotic

expression or promoter genes (Th, CMV) are incorporated into DNA plasmids for

vaccination (Grobowska et al, Virology (2000) 269: 47-53; Kawabata et al, Infect. Immun.

(1999) 61: 5863-5869). Several studies have shown that a cell-mediated immunity is an

important feature of the host's response to peptide or DNA vaccines. (Shikhman. et al, Nat.

Biotech. (1997) 15: 512-516; Ulmer. et al, Science (1993) 259: 1745-1749; Kieber-

Emmons. et al, J. Immunol. (2000) 165: 623-627, Kawabata et al., Infect. Immun. (1999)

67:5863-5869; Van Ginkel et al, Emerg. Infect. Dis. (2000) 6(2): 123-132; Ghose et al,

Mol. Immunol, 25(3): 223-230).

Peptides that mimic carbohydrate epitopes also have significant advantages as

vaccines compared with carbohydrate conjugate vaccines. (Qui et al, Hybridoma (1999)

18:103-112; Kieber-Emmons et al, J. Immunol. (2000) 165:623-627 Pincus. et al, J.

Immunol. (1998) 160: 293-298; Phalipon. et al, Eur. J. Immunol. 10: 2621-2625). Carbohydrate antigens are classed as T cell-independent antigens which gives them

immunological properties much different from those associated with protein antigens.

Because pure carbohydrate vaccines provoke naπowly defined immune responses,

predominantly serum IgM, their use in vaccines have limited effectiveness in combating

disease. Conjugate vaccine technology has overcome some of the limitations of

carbohydrate antigens because of the T-dependent help confeπed by the carrier protein.

However, carbohydrate conjugate vaccines induce immune responses that are deficient in

many respects, including the lack of induction of the Thl -associated IgG2a isotype and

cell-mediated immune responses to pathogens and tumor cells.

The immunogenicity of carbohydrate and conformational minitopes associated with

bacterial and fungal pathogens has been demonstrated by the inventors. For example, it has

been demonstrated that minitopes of carbohydrate antigens of Candida albicans and group

B streptococci are both antigenic and immunogenic when administered as conjugates or, as

in the case of group B streptococcoal minitopes, as purified peptides.

Vaccine Formulation

The amount of expressible DNA or transcribed RNA to be introduced into a vaccine

recipient will have a very broad dosage range and may depend on the strength of the

transcriptional and translational promoters used as well as subject size, e.g., human versus

bison (i.e., in bison, 5 mg of DNA can be an effective dose). In addition, the magnitude of

the immune response may depend on the level of protein expression and on the

immunogenicity of the expressed gene product. In general, effective dose ranges of about 1

ng to 5 mg, 100 ng to 2.5 mg, 1 μg to 750 μg, and preferably about 10 μg to 300 μg of DNA is administered intranasally. It is also contemplated that booster vaccinations may be

provided. Following vaccination with M cell ligand-polybasic conjugate-polynucleotide

complexes, boosting with the encoded antigen products is also contemplated. Parenteral

administration, such as intravenous, intramuscular, subcutaneous or other means of

administration of interleukin-12 protein (or other cytokines, e.g. GM-CSF), concurrently

with or subsequent to intranasal introduction of the M cell ligand-polybasic

conjugate-polynucleotide complex of this invention may be advantageous.

The polynucleotide and other immunogens of the invention may be associated with

adjuvants or other agents which affect the recipient's immune system. In this case, it is

desirable for the formulation to be in a physiologically acceptable solution, such as, but not

limited to, sterile saline or sterile buffered saline. The active immunogenic ingredients can

be mixed with excipients or carriers which are pharmaceutically acceptable and compatible

with the active ingredient. Suitable excipients include but are not limited to water, saline,

dextrose, glycerol, ethanol, or the like and combinations thereof.

In addition, if desired, the DNA and other vaccine complexes may contain minor

amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents,

and/or adjuvants wliich enhance the effectiveness of the vaccine. Examples of adjuvants

which may be effective include but are not limited to: aluminum hydroxide;

N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);

N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, refeπed to as nor-MDP);

N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(r-2'-dipalmitoyl-sn

-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, refeπed to as MTP-PE); and

RTJBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80

emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of

antibodies directed against the immunogen resulting from administration of the immunogen

in vaccines which are also comprised of the various adjuvants. Such additional

formulations and modes of administration are known in the art and can also be used.

The DNA and other vaccines of the present invention may be formulated into

compositions as neutral or salt forms. Pharmaceutically acceptable salts include but are not

limited to the acid addition salts (formed with free amino groups of the peptide) which are

formed with inorganic acids, e.g., hydrochloric acid or phosphoric acids; and organic acids,

e.g., acetic, oxalic, tartaric, or maleic acid. Salts formed with the free carboxyl groups may

also be derived from inorganic bases, e.g., sodium, potassium, ammonium, calcium, or

ferric hydroxides, and organic bases, e.g., isopropylamine, trimethylamine,

2-ethylamino-ethanol, histidine, and procaine.

The M cell ligand-polybasic moiety (or conjugate)-polynucleotide compositions and

other vaccine compositions of the present invention are administered in a manner

compatible with the dosage formulation, and in such amount as will be prophylactically

and/or therapeutically effective. The quantity to be administered, which is generally in the

range of about 100 to 1,000 μg of protein per dose, more generally in the range of about 5 to

500 μg of protein per dose, depends on the subject to be treated, the capacity of the

individual's immune system to synthesize antibodies, and the degree of protection desired.

Precise amounts of the active ingredient required to be administered may depend on the

judgment of the physician and may be peculiar to each individual, but such a determination

is within the skill of such a practitioner. The DNA and other vaccines of the present invention may be given in a single dose

or multiple dose schedule. A multiple dose schedule is one in which a primary course of

vaccination may include 1 to 10 or more separate doses, followed by other doses

administered at subsequent time intervals as required to maintain and or reinforce the

immune response, e.g., at 1 to 4 months for a second dose, and if needed, a subsequent

dose(s) after several months.

Immunization by DNA injection allows the ready assembly of multicomponent

subunit vaccines. Simultaneous immunization with multiple influenza genes has recently

been reported. (Donnelly et al, Vaccines (1994) pp 55-59). The inclusion in a DNA

vaccine of genes whose products activate different arms of the immune system may also

provide thorough protection from subsequent challenge.

The vaccines of the present invention are useful for administration to domesticated

or agricultural animals, as well as humans. Vaccines of the present invention may be used to

prevent and/or combat infection of any agricultural animals. The techniques for

administering these vaccines to animals and humans are known to those skilled in the

veterinary and human health fields, respectively.

Except as may be noted hereafter, contemplated techniques for cloning, DNA

isolation, amplification and purification, for enzymatic reactions involving DNA ligase,

DNA polymerase, restriction endonucleases and the like, and various separation techniques

are those well known and commonly employed by those skilled in the art. A number of

standard techniques are described in Sambrook et al. (1989) Molecular Cloning: A

Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.;

Maniatis et al (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth Enzymol. 218, Part I; Wu (ed.) (1979)

Meth Enzymol 68; Wu et al. (eds.) (1983) Meth Enzymol 100 and 101; Grossman et al.

(eds.) Meth Enzymol 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold

Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old et al. (1981) Principles of Gene

Manipulation, University of California Press, Berkeley; Schleif et al. (1982) Practical

Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press,

Oxford, UK; Hames et al. (eds.) (1985) Nucleic Acid Hybridization, LRL Press, Oxford,

UK; Setlow et al. (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum

Press, New York. Abbreviations and nomenclature, where employed, are deemed standard

in the field and commonly used in professional journals such as those cited herein.

The following examples are provided to illustrate the present invention without,

however, limiting the same thereto.

Examples

Example 1. Production of recombinant reovirus 1 protein

The cloned protein σl cDNA from reovirus serotype 3 strain in the pST3-Sl

Banjerjea et al, Virology (1988) 167:601-612) was kindly provided by Dr. Wolfgang K.

Jolik from Duke University Medical Center. For its expression in E. coli, using PCR, a 1.4

kb cDNA fragment containing the restriction endonuclease sites, EcoR 1 and Pst 1, was

inserted into the polylinker site of an E. coli expression plasmid, pMAL-C2 (New England

Biolabs, Beverly, MA). The resultant, pMAL-C2-Sl, was used to transform E. coli, strain

BL21 (DE3; Novagen, Madison, WI). Upon induction with IPTG, the maltose-binding

protein (MBP): :protein σl fusion protein was induced in the cytoplasm of E. coli. The clear lysate of E. coli containing the fusion protein was purified by affinity chromatography using

amylose resin according to manufacturer's directions (New England Biolabs). This

MBP: :protein σl fusion protein is refeπed to as recombinant protein 1.

Example 2. Preparation of recombinant fusion protein 1-polylysine-DNA complex

The recombinant protein σl was covalently linked to poly-L-lysine (PL) according

to the methods of Wagner et <a/(1990). Protein σl was purified and resuspended in

phosphate-buffered saline (PBS), pH 7.3. To generate the dithiopyridine linker, both

protein σl and PL were each modified with succinimidyl 3-(2-pyridyldithio)propionate

(SPDP; Sigma Chemical Co., St. Louis, MO). Briefly, in separate vessels, ten milligrams of

protein σl in 5 ml PBS, pH 7.3, and twenty milligrams of PL (Sigma), with an average

chain length of 270 lysine monomers, in 1 ml of 75 mM sodium acetate were each

vigorously mixed to react with SPDP in 15 mM ethanolic solution for one hour. The

resulting SPDP modified protein 1 was then dialyzed against PBS, pH 7.3, and the modified

PL was then dialyzed against 20 mM sodium acetate to remove unbound SPDP. To

generate the mercaptopropionate linker, the resultant PL with dithiopyridine linker was

further mixed with 23 mg dithiothreitol (DTT) in sodium bicarbonate solution, pH 7.5, for

one hour under argon. The mercaptopropionate PL was dialyzed against 20 mM sodium

acetate to remove free DTT. The 10 mg of dithiopyridine-modified protein σl was then

mixed with the 20 mg of mercaptopropionate-modified PL under argon at room temperature

for 18 hours. The resultant reaction generated what is refeπed to as protein σl-PL

conjugate. This conjugate was dialyzed to remove unreacted mercaptopropionate-PL using

a membrane with an exclusion of 100 kilodaltons, against HEPES buffered saline (20 mM

HEPES, 100 mM sodium chloride, pH 7.4; HS). Protein σl-PL concentration was determined using a Bradford assay (Pierce, Rockford, IL). For control transfections,

MBP-PL conjugates were similarly generated. For the formation of conjugate-DNA

complex, the protein σl-PL conjugate in 125 μl of HS was added dropwise into an equal

volume of HS containing the plasmid DNA and incubated at room temperature for 30

minutes to form conjugate-DNA complex.

Example 3. Cell ligand binding assay

To assess the cell-binding capacity of the protein 1 and protein 1-PL conjugates, an

immunofluorescent binding assay was performed. The protein σl and σl-PL conjugates

were incubated with mouse L cells (CCL-1, American Type Culture Collection, Manassas,

VA), RFL-6 fibroblast cells (CCL-192, ATCC), and Caco-2 cells (HTB-37, ATCC) and

binding was assessed using 20 μg/ml of biotinylated monoclonal anti-reo virus protein 1

antibody (HB-167, ATCC) and SA-PE (Southern Biotech. Assoc, Birmingham, AL), and

specific binding was then assessed using flow cytometry. Protein 1 was able to bind to all

three cell types (Figure 1). No staining was obtained with normal rabbit serum or in the

presence of SA-PE only.

Example 4. Cell culture and transfection with plasmid DNA

The mouse L cells, RFL-6 cells, and Caco-2 cells were used for targeting gene

transfer by protein σl-PL conjugate. The mouse L cells have been used as the in vitro

model for reovirus protein σl binding studies. Cells were maintained in complete media:

Dulbecco's minimum essential medium (DMEM; BioWhittaker, Walkersville, MD),

supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY) at 37°C under 5% CO₂. ^• For Luc assay, 2.5 X 10⁵ cells were added to each well of the 12-well plate and

allowed to adhere overnight. The conjugate-DNA complexes were added and incubated for

another 24 hours in complete media. For chloroquine treatment, the cells were incubated

with protein σl-PL-DNA complexes and 100 μM chloroquine for 4 hours at 37°C. Four

hours after incubation, the conjugate-DNA complexes were removed, and cells were

incubated with complete media for another 24 hours. The cells were lysed to assay reporter

gene activity.

For β-Gal assay, 5 X 10⁵ cells were added to each well of 6-well plate and allowed

to adhere overnight. The conjugate-DNA complexes containing 8 μg σl-PL and

pCMVβ-gal (Life Technologies), with or without chloroquine, were added and incubated

for 24 hours. The cells were then incubated with fresh media for another 24 hours prior to

flow cytometry analysis.

Example 5. Assays for reporter gene detection

Luciferase

The Luc gene was used as a reporter gene to assay protein σl-PL

conjugate-mediated transfection. A 1.4 kb Luc gene fragment flanked with Hind III and

EcoR V was extracted from pSPKuci(+) (Promega, Madison, WI). The pCMVLuciferase

(pCMVLuc) was constructed by ligating the 1.4 kb luciferase gene into the polylinker site in

pcDNA3.1(+) (Invitrogen, Carlsbad, CA). L cells were exposed to 2 g pCMVLuc

complexed with 8 g 1 -PL and 24 hours later cells were assessed for expression of Luc by

lysing the cells with lx luciferase lysis buffer (Promega, Madison, WI), and mixing twenty

μl of supernatant of cell lysates with 100 μl of Luc assay buffer. Luc levels were

quantitated with a luminometer (LUMAT LB 9507, EG&G Berthold, Germany). The relative light units from the total lysates were used to express the Luc activities produced

from each transfection.

Luc activity (486940 ± 43954) could be detected in several independent experiments

as a consequence of σl-PL-pCMVLuc transfection. Significant expression of Luc was also

achieved with RFL-6 and Caco-2 cells, of which Luc activities were 40684 ± 6633 (n=6)

and 40703 ± 6225 (n=6), respectively. The transfection did appear to be mediated by

protein σl when compared to the various control transfections. The background activity of

Luc is 193 ± 29 (n=6). Minimal detectable Luc activity could be measured when L cells

were transfected with pCMVLuc only, protein σl associated, but not covalently attached

with PL-pCMVLuc, or PL complexed with pCMVLuc without any protein σl . Thus,

optimal transfection required the covalent attachment of protein σl for optimal cellular

transfection.

To show specificity of protein σl -mediated gene transfer, transfection of L cells was

performed using protein σl-PL-pCMVLuc in the presence of excess, unconjugated

recombinant protein σl or in the presence of an antireoviras polyclonal antibody. If indeed,

the cell transfection was receptor-mediated, these molecules should inhibit transfection. As

such, the presence of increasing concentrations of unconjugated protein σl resulted in the

attenuation of gene transfer with an IC₅₀ of approximately 75 g/ml for protein σl . Between

100 and 500 g/ml of uncomplexed protein σl resulted in nearly 100% inhibition of cell

transfection.

As for Caco-2 cells, 100 and 500 g/ml of uncomplexed protein σl caused a

respective 89% and 94% inhibition of Luc expression (data not shown). Protein σl-

mediated transfection was receptor-specific since bovine serum albumin (BSA) could not inhibit protein σl -PL-mediated gene transfer. Gene expression in the presence of BSA at

0.5 mg/ml was 899000 ± 145720 light units (n=3).

As with the recombinant protein σl, the polyclonal anti-reo virus 3 antibody showed

inhibition of the protein σl -PL-pCMVLuc gene transfer to mouse L cells in a dose-

dependent fashion. Maximal inhibition was obtained with 10-fold diluted anti-reovirus 3

antibody resulting in a 98% decrease in Luc gene expression. Normal rabbit IgG did not

inhibit protein σl -PL-mediated gene transfer. Interestingly, dilute anti-reovirus 3 antibody

(1 : 100) appeared to slightly enhance transfection with this DNA complex, possibly due to a

prozone effect from antibody and protein σl concentrations. Collectively, these findings

demonstrate that protein σl-PL mediated gene transfer is accomplished via ligand binding

to target cells.

β-Galactosidase

Expression of β-Gal was visualized by incubating the transfected cells with PBS

solution containing 1 mg/ml of 5-boromo-4-chloro-3-indolyl-β-galactopyranoside (X-Gal,

Boeringer Mannheim, Indianapolis, IN) at 37°C for 16 hr. To quantify the transfection

efficiency, cells having been transfected with the constructs PL-pCMV-β-Gal (Life

Technologies) or protein σl-PL-pCMV-β-Gal were harvested, loaded with 200 M

fluorescein-mono-β-D- galactopyranoside (FDG; Molecular Probe, Eugene, OR) for 30

minutes at 37°C and diluted with cold PBS to a final concentration of 2.5 X 10⁵ cells/ml.

Flow cytometry analysis was performed using a Becton Dickinson FACSCalibur.

Transfection with protein σl-PL-pCMV-β-Gal complexes produced a statistically

significant higher level of β-Gal expression than that observed with protein PL-

pCMV-β-Gal in both L cells and RFL-6 cells. Preliminary staining showed that Caco-2 cells exhibit endogenous β-Gal activity, therefore, these cells were not evaluated further.

Transfection with protein PL-pCMV-β-Gal (lacking protein σl) was ineffective and levels

of β-Gal expression did not differ from background levels.

Example 6. Histochemical determination of fusion protein 1 binding to NALT

NALT tissues were collected as previously described (Asanuma et al, J Immunol

Methods (1997) 202:123-131 and Heritage et al, Am JRespir Crit Care Med (1997) 156(4

Pt 1): 1256-1262). Palates with visible NALT were washed in DMEM, and prior to binding

with biotinylated protein σl (following standard procedures), NALT were first incubated in

DMEM alone or in the presence of 500 μg/ml of protein 1 in DMEM with gentle rotation on

a GeneMate orbital Shaker (hitermountain Scientific Co., Bountiful, UT) for 45 minutes at

4°C. Recombinant protein σl was able to bind to NALT. See Figure 2. Control sections,

incubated with SA-Horseradish peroxidase (HRP) only, failed to stain. NALT were

incubated with excess unmodified protein σl in order to inhibit biotinylated protein σl

binding, and thus, show specificity of binding to the NALT.

NALT were then washed gently in DMEM and incubated in 50 μg/ml biotinylated

protein σl in DMEM, and were again rotated gently for 45 min at 4°C. Following

incubation, NALT were removed, rinsed gently in PBS, and then arranged in 15 mm by 15

mm Tissue Tek® Cryomold (Miles Inc., Elkhard, IN) with their ventral surfaces oriented

toward the bottom of the mold. The palates were then frozen in Tissue Tek® O.C.T.

compound embedding media and stored at -80°C until use. For immunoperoxidase staining, frozen NALT sections, previously treated with biotinylated protein σl, were cut at 5 mm,

air dried, fixed in acetone at 4°C, and air dried before rehydration.

Frozen sections were rehydrated in Dulbecco's PBS (DPBS) containing 0.2%

noπnal goat serum (NGS). A 1 :250 dilution of SA-HRP conjugate (BioSource

International, Camarillo, CA) was added for 45 min at room temperature. The location of

the HRP was visualized upon reaction with the precipitable substrate, 3-amino-

ethylcarbazole (AEC: Sigma). Results showed that binding of biotinylated fusion protein

σl was competitively inhibited by excess unlabeled fusion protein σl.

Example 7. In vivo analysis of intranasal immunization with σl conjugates:

Luciferase

Intranasal (i.n.) immunization with protein σl-polylysine (PL) conjugate enhances

induced mucosal IgA responses in mice. Data depicts the mean endpoint titers (± SE) for

mice immunized i.n. with protein σl-PL-pCMVLuciferase (Luc) or uncomplexed

pCMVLuc (5 mice/group). Significant differences between protein σl-PL-pCMVLuc and

pCMVLuc only were determined by student t-test. *p<0.05. **p<0.005. (See Figure 3).

Example 8. β-galactosidase

Intranasal (i.n.) immunization with protein σl-PL-pCMVβ-galactosidase (βgal)

stimulates βgal-specific CTL responses in mice. BALB/c mice received three i.n.

immunizations with either protein σl-PL-pCMVβgal or pCMVβgal. Immune splenocytes

were able to lyse ^5ICr loaded βgal-expressing fibroblasts (BC-βgal), but not iπelevant

BC-envelope (BC-env) targets. The mucosally formulated DNA was as efficient in

stimulating βgal-specific CTLs as those mice receiving naked DNA. (See Figure 4).

Example 9. HIV Intranasal (i.n.) immunization with protein σl-polylysine (PL) conjugate was

conducted to enhance induced mucosal IgA responses in mice. The mean endpoint titers (±

SE) for mice immunized i.n. with protein σl-PL-pCMNgpl60 and σl-PL-pCMNgpl40 or

uncomplexed pCMNgp 160 and pCMNgpl40 (5 mice/group) was compared. Significant

differences between protein σ 1 -PL-pCMNgp 160 and σ 1 -PL-pCMNgp 140 versus

pCMNgpl60 and pCMVgpl40 only were determined by student t-test. Using the mucosal

DΝA formulation, the same magnitude of IgG antibody response is observed as was

observed for the anti-reporter gene responses.

Experimentally, mice were immunized with one of three designated HJN DΝA

vaccine constracts, that is gpl60, gp 140(c) and gp 140(s), as indicated in Fig 5. Each group

(5 mice/group) received three intranasal immunizations either of naked DΝA or of the

identified M cell DΝA vaccine formulation. As indicated, the mucosal intestinal IgA

response was elevated 10 weeks after the initial immunization when compared to intranasal

naked DΝA immunization. Thus, the DΝA vaccine formulation improved mucosal IgA

responses when compared to conventional naked DΝA immunization.

Example 10. Brucella

induced mucosal IgA and IgG responses in bison. The mean endpoint titers (+ SE) for

bison immunized i.n. with protein σl-PL-pCMVL7/L12 ribosomal protein or uncomplexed

pCMVL7/L12 ribosomal protein (5 bison/group) was compared. Significant differences

between protein σl-PL-pCMNL7/L12 ribosomal protein versus pCMVL7/L12 ribosomal

protein only were determined by student t-test. Using our mucosal DΝA formulation, we observed increases in serum IgG and vaginal IgA and IgG anti-L7/L12 antibody titer in

bison.

Example 11. HIN gp 120

Intranasal immunization with an M cell-formulated HIV DΝA vaccine promotes

enhanced cytolytic activity (cell-mediated immunity) against target cells expressing HIV

gpl20 as shown in Figs 6 A and 6B. Mice received a fonnulated vaccine, naked DΝA

version, protein sigmal by the intranasal route three times at one week intervals or were left

unimmunized. Mice were sacrificed six weeks subsequent to this initial immunization to

procure specified tissues. In a dose-dependent fashion, the lungs from only mice receiving

only the formulated vaccine showed effector function. These results show that the vaccine

as fonnulated is superior to naked DΝA in stimulating gpl20-specific immunity.

Data also indicated that antigen restimulation specifically enhances CTL responses

from mice i.n.-immunized with the formulated vaccine as opposed to mice immunized with

the naked DΝA alone. Pulmonary lymph nodes (LRLΝ) and splenocytes from immunized

mice were restimulated in vitro with cells expressing gp 120 or beta-galactosidase (neg.

control), and were subsequently examined for cytolytic activity. The observed killing was

specific since negative targets were not lysed, and other mechanisms of vaccination failed to

stimulate cytolytic activity.

Example 12. Method for selection of antigenic peptide and minigene

The J404 nonapeptide library was employed and expressed on the surface of a new

vector M13KBst (1) for defining polynucleotide sequences and the nonapeptides they

encode. The library has a complexity of 5 X 10⁸ unique phage. The display technology has

been refined and is practiced as described below. Briefly, affinity purification of phage bearing epitopes bound by antibody reagents are performed as follows: 1 x 10¹² phage (75

μl) from the nonapeptide J404 library are combined with 300 μl of Sepharose beads

conjugated with 1.50 mg of the monoclonal reagent or polyclonal antiserum. Alternatively,

if the selecting antibody is an IgG, protein A or G or anti-IgG-coated, sepharose beads may

be coated with selecting antibody for the interaction with phage. The beads are then mixed

with the phage at 4° C for 16 hours by gentle rotation. The mixture is then loaded into a 5

ml plastic column baπel (Evergreen) and unbound phage removed by washing with 50 ml

phage buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% Tween 20 (v/v), 1 mg/ml BSA).

Bound phage are eluted from the column with 2.0 ml of eluting buffer (0.1 M glycine, pH

2.2), and the pH of the eluate neutralized immediately with four drops of 2 M Trizma base.

The titer of the eluate phage is determined for each column eluate by plaque assay according

to standard procedures.

The column matrices are preserved for reuse in second and third round affinity

purifications by washing with 10 ml PBS pH 7.0, followed by 3.0 ml PBS containing 0.02%

sodium azide. The column is stored at 4° C until the next affinity purification, and prepared

for reuse by rinsing with 20 ml of phage buffer prior to mixing with amplified phage.

In order to select strongly binding sequences from the library, three rounds of

selection, alternated with two rounds of amplification are carried out as has been previously

described in Burritt. et al, Anal. Biochem. ("1996) 238: 1-13; Burritt. et al, J. Biol. Chem.

(1995) 16974-16980. Utilization of one or two of these sequences requires identification of

those most strongly bound by the lnAb. Typically, this is done with a plaque lift to select

clones from the third round eluate that give strong signals with the selecting antibody.

Individual clones are picked and amplified and confirmation of specific activity can then b e done by ELISA. Specifically, phage at 10⁹ to 10 ^π plaque forming units are exposed to

wells of an ELISA plate which had been coated with 75 μl of antibody (nιAb-114 or KPL

polyclonal, for example), diluted to 200 mg/ml in PBS, pH 7.4. After incubation of the

phage pool on the antibody, the wells are washed and then probed with a rabbit anti-phage

polyclonal antibody (1 :20,000 dilution in PBS) for one hour at 25° C. Following additional

washing, the wells are exposed to goat anti-rabbit polyclonal secondary antibody tagged

with HRP for one hour at 25° C. After a final wash, the color is developed with a standard

o-phenylenediamine in citrate buffer system and absorbances recorded at 490 nm.

Peptide sequences can be determined by sequencing of the N-terminal chirneric

plll-peptide gene using automated sequencing methodology or using techniques which are

well-known in the art. For reference in this regard, see Current Protocols in Molecular

Biology. Wiley Interscience, 1989. These analyses yield the polynucleotide (minigene)

sequence used for preparing synthetic oligonucleotide minigenes for ligation to plasmid

DNA. Minigenes encoding peptides with greatest binding properties for the antibody probe

are selected for vaccine development.

Example 13. Preparation of minitopes and minigenes for Candida vaccine

development

Infections with the pathogenic fungus, Candida albicans, are represented by

disseminated as well as mucocutaneous forms of candidiasis. In disseminated forms of

candidiasis, the organism infects many tissues and organs often with fatal outcomes

especially in patients with inmpaired immune systems. As part of the normal flora of the

skin, intestine, nose and throat, the organism may produce mucocutaneous disease such as

oral candidiasis (thrush) and candidal vaginitis. There are no effective vaccines available for human use. Studies (Knabe et al, Infect. Immun. (1994) 62: 1662-1668) have identified

and characterized a protective epitope chemically defined as β-1,2 trimannose which is

embedded in the cell wall of C. albicans. Cell wall fractions containing a phosphomannan

complex (PMC) or purified trimannose preparations administered with adjuvants or on

liposomes elicit antibody responses and protect mice against challenge with C. albicans.

A family of peptides recognized by the monoclonal antibody MAb B6.1, an

antibody of the IgM that binds to PMC or β-ϊ,2-trimannose, has been previously identified.

Mab B6.1 was obtained from mice immunized with a phosphomannan protein complex

(PMC) wliich contains an adhesin domain used by Candida for attachment to target cells of

the host. B6.1 is specific for a β-l,2-trimannose carbohydrate moiety that is phosphodiester

within the other mannan complexes expressed on or near the surface of yeast cells.

Mab B6.1 protects against disseminated and mucocutaneous candidiasis in murine

models of infection and has been shown to react with a cell wall of Candida albicans.

Probing the J404 phage display library with MAb B6.1 selected five peptides rich in

aromatic and hydroxylated amino acids with a consensus of Ar-X-X-Ar-Z-Z-Z-Ar-Ar (Ar =

W, F, or Y; X = any amino acid; and Z = S, T, or preferentially G). Each of these peptides

is nine amino acids in length and they bind preferentially to B 6.1. Both phage-displayed

peptides and synthetic peptide-carrier protein conjugates showed specific reactivity with

MAb B6.1. Figure 7 demonstrates that the Candida carbohydrate epitope demonstrates dose

dependent inhibition of antibody binding to all five phage-displayed minitopes and to a

synthetic peptide-carrier protein conjugate. Some of the phage-displayed minitopes (PS76,

PS2, and PS31) demonstrated dose-dependent inhibition of antibody binding to the B6.1

carbohydrate epitope. Balb/c mice were immunized subcutaneously with phage-displayed peptides (clone numbers PS76, PS2, PS31 PS28 and PS55), synthetic peptide (PS76p), with

Ribi RS-700 adjuvant and boosted at day 21 and 35. Immune serum samples were screened

by agglutination against C. albicans cells and by ELISA against carbohydrate extracts with

enzyme conjugated secondary antibody to detect binding of mouse IgG plus IgM. The

results shown in Figure 7 indicate that the selected peptide mimetics act antigenically and

immunologically.

A Candida minigene as described above was constructed with the polynucleotide

sequence, 5'-TAT,CGT,CAG,TTT,GTG,ACG,GGT,TTT,TGG-3' (SEQ ID NO: 1),

encoding the PS76 peptide (YRQFVTGFW ) (SEQ ID NO: 2) for incorporation into a

DNA vaccine construct. The minigene is incorporated into a plasmid as described above

and administered to a mammalian host in order to elicit an immune response. Such vaccine

constructs also include mammalian promoters controlling transcription, preferably the CMV

promoter, a T cell epitope and a mucosal adjuvant preferably cholera toxin (CT) to

stimulate both arms (Thl and Th2) of the immune system.

Example 14. Preparation of minitopes and minigenes for Group B streptooccal

vaccine development

Group B streptococci (GBS) are a major cause of neonatal sepsis and meningitis.

GBS are one of many examples of microbial polysaccharides that are notably poor

immunogens. Efforts to prevent this disease using GBS carbohydrates are marginally

effective in eliciting antibody or a protective immune response. Immunogenic minitopes of

the type III capsular carbohydrate of group B Streptococcus (GBS) have been developed

(Pincus. et al, J. Immunol (1998) 160: 293-298). The murine mAb S9, a protective

antibody against the type III capsular polysaccharide of group B streptococci, was used to select epitope analogues from the J404 peptide display phage library. Two populations of

phage were identified with displayed sequences of WENWMMGNA (SEQ ID NO: 3) and

FDTGAFDPDWPA (SEQ ID NO: 4). The binding of anti-GBS antibody to GBS was

inhibited by the free peptide and significant antibody responses to GBS and purified

capsular polysaccharide was elicited by a single immunization with peptide mimetics

Moreover, a single dose of peptide mimetic induced a greater anti-GBS antibody response

than that seen following infection with 108 colony forming units of GBS.

M celkDNA vaccines for GBS have been prepared by ligating the polynucleotide

sequence 5'-TTT,GAT,ACG,CTG,GCT,TTT,GAT,CCT,GAT,TGG,CCT,GCT-3' (SEQ ID

NO: 5) encoding peptide FDTGAFDPDWPA (SEQ LD NO: 4) into either plasmid

pcDNA3.1 (Invitrogen, Carlsbad, CA) or pCMV-SPORT-bgal (GIBCO) under a CMV

promoter. To assess the efficacy of vaccine constructs, adult BALB/c mice immunized

immunized intranasally with three doses of M cell vaccine have been shown to produce high

titers of antibody against GBS.

Example 15. Preparation of minitopes and minigenes for Brucella a vaccine

development

Numerous studies have clearly demonstrated that protective immunity against

brucellosis coπelates with antibodies administered passively or elicited by immunization

against the 0-antigenic domain of S-LPS (Cloeckaert, A. et al, Infect. Immun. (1992) 60:

312-315; Dubray, G., Annales de l 'Institut Pasteur. Microbiologie (1987) 138: 84-87).

The lipopolysaccharide of smooth Brucella (S-LPS) has been found to contain two

distinct epitopes designated A and M (Corbel, M., 1st Int. Conf. Emerg. Zoonosis. (1997) 3:

213-221). The relative amounts of the two epitopes vary among smooth Brucella stπains, and these epitopes are absent on rough strains. The S-LPS structure has been defined as

homopolymers of 4,6-dideoxy-4-formamido-a-D-mannopyranose residues. The A antigen

contained in S-LPS is a linear -l,2-linked polymer with about 2% α-l,3-linkages, while the

M antigen is a linear polymer ofpentasacchari.de repeating units containing one α-l,3-linked

and four α-l,2-linked monosaccharide residues. Apparently, the α-l,3-linkage is the major

part of the structure recognized by anti-M mAbs in S-LPS of B. abortus, B. melitensis, and B.

suis which express the M epitope in variable amounts. The structure of the common epitope

is unknown. The J404 phage library has been probed with an anti-A antibody, YsT9-l (a

IgGl mAb obtained from the Canadian Research Council) to identify peptide mimetics with

high conformational similarity to the α-1,2 linked carbohydrate residues peculiar to the A

antigen. Minitope and minigene vaccine candidates were prepared as previously described.

Probing the J404 phage display library with mAb YsT9-l which binds to B. abortus

A antigen yielded a nonapeptide consensus sequence of VSWCSSCSL (SEQ ID NO: 6) as

determined by an analysis of the DNA from amplified phage clones. A minigene sequence of

5'-GTT,TCT,TGG,TGT,TCT,TCT,TGT,TCT,CTT-3' (SEQ ID NO: 7) was derived from

the analysis and serves as the minigene construct for the development of the Brucella M cell

vaccine. The polynculeotide shown above, encoding the selected VSWCSSCSL peptide

sequence, was ligated into a plasmid together with immunomodulatory sequences including

a CMV promoter for controlling transcription, T cell epitopes (K99) and enhancer sequences.

The peptide mimetic for the Brucella LPS epitope was expressed as a trimer to improve

immunogenicity. Likewise the K99 fimbrial subunit was expressed as a repetitive trimer on

the N-terminus of protein si. The expression of the K99 fimbrial subunit was included to provide a scaffolding for the LPS minitope and to improve the immunogenicity of the

peptide epitope.

Example 16. Preparation of minigenes and minitopes for the development of

Norwalk virus vaccines

The Norwalk-like viruses (NLV) are the most important cause of epidemic outbreaks

of foodborne and waterborne gastroenteritis. Sequence analyses of viral capsid protein

coding regions have identified two major genetic groups of NLV, with many genetic

subtypes (and likely antigenic subtypes) within each group (Frankhauser, R., et al, J. Infect.

Dis. (1998) 178: 1571-1578.). The NLV are difficult to study because these viruses are

refractory to growth in cell culture and small animal models. Thus, the early steps in

infection including cell binding and entry have been focused on.

Cuπent studies on the molecular interactions between NLV capsids and cellular

receptors were designed to reveal or identify conformational epitope(s) that occur concuπent

with the binding event or fusion of viral epitope with the cell receptor. Monoclonal

antibodies specific for the fusion-induced conformational epitope are used to select

minitopes and prepare minigenes for incorporation into M cell vaccines as described above.

Example 17. Preparation of minitopes and minigenes for the development of an

anthrax vaccine

Anthrax produced by the bacterium Bacillus anthracis is an infectious disease

resulting from contact with endospores in contaminated animal products or their dusts.

Cutaneous anthrax, which accounts for 95% of cases in the world, results from

contamination of a lesion in the skin and progresses to fatal septicemia in 10-20% of

untreated cases. Inhalation anthrax is nearly always lethal without early, aggressive intervention. The results of a field study with the U.S. military Anthrax Vaccine Adsorbed

(AVA) suggested that it prevented cutaneous infection in humans (Demicheli, V. et al,

Vaccine (1998) 16: 880-884). Its effectiveness in preventing inhalation disease in humans is

questionable in view of failure to protect animals against inhalation anthrax.

The inventors have obtained monoclonal antibodies against two major B. anthracis

antigens, PA and poly-γ- D-glutamyl capsular material and protective antigen (PA), a cell

surface binding unit of both anthrax edema toxin and lethal toxin. In addition, toxin genes

(PA) cloned into E. coli hosts serve as the source of epitope DNA for ligation plasmids and

incorporation into M cell vaccine constracts as described above.

Example 18. The generation of mucosal tolerance using M cell delivery of peptides

that promote anergy to self antigens

Autoimmune diseases such as arthritis (7 different types), multiple sclerosis, uveitis,

myasthenia gravis, type 1 diabetes, thyroiditis and colitis respond favorably to the oral

delivery of native proteins, sometimes peptides, associated with the tissue under attack by the

immune system (Cohen, I., Behring, Inst. Mitt. (1985) 77: 88-94) This phenomenon,

refeπed to as oral tolerance, interrupts and suppresses the autoimmune disease process by

stimulating the natural mucosal immune mechanisms in the gut associated lymphoid tissues

(Gait) of the small intestine (Hanninen . Scand. J. Immunol. ( 2000) 52 (3): 217-225;Sbi. et

al, J. Immunol. (1999) 162 (10): 5757-5763; Haflter., Ann. NY. Acad. Sci. (1997) 835:

120- 131). Mucosal oral tolerance can be induced by three different mechanisms : active

suppression, clonal anergy and clonal deletion. Antigen dose is the primary factor

determining the form of peripheral tolerance that develops. The generation of tolerance due

to regulatory T cells (active suppression) is favored by administration of low doses of antigen, whereas administration of high doses of antigen biases toward development of

tolerance due to anergy or deletion.

The distinction between these mechanisms of oral tolerance are not mutually

exclusive and they may occur concunently. In low dose tolerance, regulatory T cells are

stimulated to secret suppressive cytokines, such as TGF-b, IL-4 and IL-10 which function to

down-regulate the activiated inflammatory Thl cells. Clonal anergy may result when high

doses of oral antigen induce unresponsiveness in the immunoreactive Thl cell function. The

cells are not deleted but are rendered intrinsically incapable of responding to a specific

antigen in the context of their T cell receptor and peptide associated with MHC. Anergy may

be overcome with high concentrations of IL-2. Clonal deletion results in the elimination of

antigen response T cells. In the presence of high concentrations of protein, deletion of cells

specific for that protein can occur directly within the Peyer's patch as well as in the thymus.

Most cuπent therapeutics for autoimmune disorders are administered orally as native

proteins or peptide residues from target tissues or secretion products of cells.

The oral delivery of tissue specific antigens (tolerogens) has generally been

accomplished with large or intact proteins which are broken down to fragments by the

normal digestive processes (Rosen, A., et al., 1999. Cell. Death Differ. 6: 6-11; Kweon. et

al, Digestion 63 suppl SI: 1-11; Lipkowski. Et al. Biofactors (2000) 12: 147-150). Specific

fragments or peptides are taken up by antigen-presenting cells (M Cells) and processed for

presentation to undifferentiated T cells. These regulatory T cells release cytokines which

suppress inflammation (Marth. et al, Gastroenterol. (1999) 37 (2): 165-185: Hafler. et al,

Ann. NY. Acad. Sci (1997) 835: 120-131). An alternate strategy which is contemplated

involves the oral delivery of DNA encoding protein fragments or peptides representative of self tissue antigens using the M cell delivery system. The processing of tolerogenic peptide

DNA (with the appropriate regulatory T cell epitope or none at all) by M cells, the synthesis

of tolerogenic peptides in situ and the subsequent presentation to regulatory T cells in the

Peyer's patch will lead to mucosal as well as systemic tolerance. The various self-antigens

or tolerogenic peptides that may be represented or presented in DNA coding by way of an M

cell directed vaccine as described above include Type II collagen (arthritis) (Weiner. et al,

Springer Semin Immunopathol, (1998) 20 (1-2): 289-308), myelin protein MBP, PLP, MOG

9 (multiple sclerosis) (Hafler. et al, Ann. NY. Acad. Sci (1997) 835: 120-131), S-Ag, IRBP

(uveitis), ArchR (myasthenia gravis) (Sempowski, G. et al, J. Immunol. (2001) 166:

2808-2817), insulin, GAD (type 1 diabetes) (Bach, J., Endocr. Rev. (1994) 15: 516-523),

thyroglobulin (thyroiditis), basement membrane antigen (glomeralonephritis ) (Wilson, C. et

al., In The Kidney (1991) Brenner and Rector, eds. W. Saunders, Philadelphia) or colonic

proteins (colitis). Such DNA materials may be obtained by PCR with human tissue or, by

decoding displayed peptides from phage display using auto-immune antibody directed

against the tissue proteins.

Example 19. Tumor vaccines: DNA coding minitopes of carbohydrate tumor

antigens

There are ongoing Phase 1/11 and III clinical trials with DNA vaccines for treating

melanoma, lymphoma, prostate cancer, kidney cancer, and colon cancer. The key to the

success of any of these and future DNA vaccines rests with the use of highly immunogenic

tumor epitopes in combination with appropriate accessory molecules (cytokines, decoy

ligands, targeting molecules) into a carrier system that elicits either a cell mediated or

humoral response or both. The physical characteristics of tumor antigens that would make the most ideal targets

for antibody therapeutics or vaccine constructs include cell surface expression; high, stable

expression levels in tumor cells; low or absent expression in normal tissues; lack of a soluble

form of the antigenic target; and lack of intemalization of a antigen/antibody complex. In

addition, the type of immune response, either Thl or Th2-mediated responses, provoked by

the epitope is key to the successful eradication of a tumor mass. With the recent progress in

molecular biology and gene technology, many new cancer-specific antigens have been

identified (UrCarrilho, C. et al, Virchows Arch (2000) 437(2): 173-9).

A variety of current and past tumor vaccine formulations are predicated on cell-based

technology using either autologous cells (cytokine gene-transduced or hapten-modified, for

example) or allogeneic whole cells consisting of single or pooled cell lines. Early efforts to

evaluate responses to tumor vaccines have focused on humoral responses. The result was the

identification of many antigens that could be defined serologically on tumor cells including

glycoproteins, glycolipids, gangliosides and complex proteins. Adoptive therapies directed

against these antigens have been disappointing but there is renewed interest in humoral

immune responses and in tumor vaccines directed against carbohydrate epitopes.

The dominant thrust of cuπent research in tumor immunobiology has focused on

defining antigens recognized by human T cells and on augmenting the cellular immune

response to tumors. Consequently, the focus of these efforts have been on protein or

oligopeptide tumor antigens. Recent studies have focused on the use vaccines containing

oligopeptides or peptides representative of key regions in the tumor cell epitope. ( Qiu, J. et

al, Hybridoma (1999) 18: 103-112; Kieber-Emmons, T. et al, J. Immunol. (2000) 165:

623-627). T cell responsiveness to an epitope is effected both by its affinity for the presenting

MHC molecule and the affinity of the MHC-peptide complex for the thymus cell receptor

(TCR) (Nagorsen, D., et al, Cancer Res. (2000) 60(17):4850-4). One limitation of cancer

immunotherapy is that natural tumor antigens, especially carbohydrate epitopes, elicit

relatively weak T cell responses, in part because high-affinity T cells are rendered tolerant to

these antigens (Slantsky, J. et al, : Immunity (2000) 13(4):529-538). Many studies have

demonstrated the role of HLA class I-restricted cytotoxic T lymphocytes (CTLs) in cancer

specific-immunotherapies (Mond. et al, Ann. Rev. Immunol. (1995) 13: 655-667) . The

engineering of MHC class I-restricted tumor peptide epitopes that increase the stability of the

MHC-peptide-TCR complex are significantly more potent as tumor vaccines (Nagorsen, D.,

et al, Cancer Res. (2000) 60(17):4850-4). The improved immunity results from enhanced in

vivo expansion of T cells specific for the natural tumor epitope. These results indicate that

peptides that stabilize the MHC-peptide-TCR complex may provide superior antitumor

immunity through enhanced stimulation of specific T cells, and such peptides may be

delivered to M cells or coded by DNA minigenes as described above.

Example 20. Tumor vaccines: Increasing immunogenicity with the use of

immunomodulatory cytokines

Cytokines may be used as immunomodulatory adjuvants to be administered in

formulations with the tumor vaccines and other vaccines described herein. For instance,

liposomes incorporating interferon gamma have been shown to increase the residence time of

the cytokine at the vaccination site as compared to cytokine gene transfection of tumor cells

(van Slooten et al, Pharm. Res. 2000 Jan., 17(1): 42-8). Thus, vaccines may be formulated

to include cytokine-containing liposomes admixed with the M cell ligand complexes of the invention. Alternatively, M cell ligand complexes of the present invention may be included

together in liposome formulations along with adjuvant cytokines. Also, constructs may be

made according to the invention wherein cytokines are otherwise associated with or attached

to the M cell ligand and immunogen complexes of the invention.

Liposomes may be made by means that are well known in the art, and may be

polymerized or unpolymerized, depending on the desired characteristics for the liposome. In

general, polymerized lipid compositions may be produced according to techniques described

in U.S. Patent No. 5,962,422 to Nagy et al, "Inhibition of Selectin Binding", utilizing the

materials and methods disclosed therein. Other references are U.S. Patent No. 6,342,226 to

Betbeder et al, "Method for Increasing Immuiiogenicity, Product Obtained and

Pharmaceutical Compositions", U.S. Patent No. 6,090,406 to Popescu et al., "Potentiation of

Responses with Liposomal Adjuvants", and U.S. Patent 6,225,445 to Sen et al, "Methods

and Compositions for Lipidiziation of Hydrophilic Molecules." Such vaccine formulations

would contain a ratio of cytokine to vaccine complex that is optimized to produce the desired

response.

The foregoing detailed description has been given for clearness of understanding only

and no unnecessary limitations should be understood therefrom as modifications will be

obvious to those skilled in the art. While the invention has been described in connection

with specific embodiments thereof, it will be understood that it is capable of further

modifications and this application is intended to cover any variations, uses, or adaptations of

the invention following, in general, the principles of the invention and including such

departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features

hereinbefore set forth and as follows in the scope of the appended claims.

Claims

Claims What is claimed:

1. A composition comprising:

an M cell specific ligand;

a nucleic acid sequence encoding an immunogen; and

a nucleic acid binding moiety.

2. The composition of claim 1 , wherein said nucleic acid sequence is a DNA sequence.

3. The composition of claim 1, wherein said nucleic acid binding moiety is a polypeptide.

4. The composition of claim 3, wherein said polypeptide comprises a polymeric chain of

basic amino acid residues.

5. The composition of claim 4, wherein said polymeric chain comprises polylysine.

6. The composition of claim 3, wherein said polypeptide is a fusion protein further

comprising said M cell specific ligand.

7. The composition of claim 1, wherein said immunogen is selected from the group

consisting of immunogens expressed by infectious agents and tumor specific antigens.

8. The composition of claim 7, wherein said infectious agent is selected from the group consisting of bacterium, parasite, virus, fungus, prion, tuberculobacillus, leprosy bacillus,

malaria parasite, diphtheria bacillus, tetanus bacillus, Leishmania, Salmonella, Schistosoma,

measles virus, mumps virus, herpes virus, HIV, cancer and influenza virus.

9. The composition of claim 1, wherein said M cell specific ligand is selected from the

group consisting of the protein 1 of a reovirus, adhesin derived from Salmonella and

adhesin derived from polio virus and M cell tropic fragments thereof.

10. The composition of claim 9, wherein said M cell specific ligand is the protein 1 of a

reovirus, a tetramer or trimer thereof, or an M cell tropic fragment thereof.

11. The composition of claim 2, wherein said DNA sequence further comprises a plasmid

vector in which said DNA sequence encoding an immunogen is operably linked to

transcription regulatory elements.

12. A composition comprising:

an M cell specific ligand

an immunogen and

a linker molecule.

13. The composition of claim 12, wherein said linker is a crosslinker and said M cell

specific ligand is conjugated to an immunogen.

14. The composition of claim 13, wherein said crosslinker is selected from the group

consisting of SPDP, DSS, SIAB, SATA, MBS and GMBS.

15. The composition of claim 12, wherein said linker is a complexing moiety and said M

cell ligand is complexed to said immunogen.

16. The composition of claim 15, wherein said complexing moiety is selected from the

group consisting of nitrilotriacetic (NTA)-metal complex and iminodiacetic acid (IDA)-

metal.

17. A composition comprising:

an M cell specific ligand;

an immunogen; and

a liposome.

18. The composition of claim 17, wherein said immunogen is encapsulated within said

liposome and said M cell specific ligand is conjugated to the liposome.

19. The composition of claim 17, wherein said immunogen is surface-displayed on said

liposome and said M cell specific ligand is conjugated to the liposome.

20. A composition comprising:

an M cell specific ligand; an immunogen; and

a polypeptide.

21. The composition of claim 20, wherein said M cell specific ligand and said immunogen

are conjugated to said polypeptide.

22. A composition of claim 21, wherein said M cell specific ligand and said immunogen are

comprised in a fusion protein or polypeptide.

23. The composition of any of claims 12, 17, 20 and 22, wherein said immunogen is

selected from the group consisting of molecules associated with infectious agents and tumor

specific antigens.

24. The composition of any of claims 12, 17, 20 and 22, wherein said infectious agent is

selected from the group consisting of bacterium, parasite, virus, fungus, prion,

tuberculobacillus, leprosy bacillus, malaria parasite, diphtheria bacillus, tetanus bacillus,

Leishmania, Salmonella, Schistosoma, measles virus, mumps virus, herpes virus, HIV,

cancer and influenza virus.

25. The composition of any of claims 12, 17, 20 and 22, wherein said M cell specific ligand

is selected from the group consisting of the protein 1 of a reovirus, adhesin derived from

Salmonella and adhesin derived from polio virus and M cell tropic fragments thereof.

26. The composition of claim 25, wherein said M cell specific ligand is the protein 1 of a

reovirus, a tetramer or trimer thereof, or an M cell tropic fragment thereof.

27. A vaccine comprising the composition of any of claims 1 to 26 and a pharmaceutically

acceptable excipient.

28. The vaccine of claim 27, which induces a protective immune response in a vaccinated

host against said immunogen.

29. The vaccine of claim 27, further comprising an adjuvant.

30. The vaccine of claim 29, wherein said adjuvant comprises an immunomodulator.

31. The vaccine of claim 30, wherein said immunomodulator is selected from the group

consisting of cytokines, lymphokines, interleukins, interferons and growth factors.

32. The vaccine of claim 27, wherein the vaccine is formulated in unit dosage fonn.

33. The vaccine of claim 27, further packaged with instructions for the use of the vaccine to

induce an immune response against said immunogen or against the disease with which said

immunogen is associated.

34. The vaccine of claim 27, wherein the vaccine is a therapeutic vaccine.

35. The vaccine of claim 27, wherein the vaccine is formulated for administration through a

route selected from the group consisting of oral, nasal, vaginal, rectal and urethral routes of

administration.

36. A method for immunizing a host against an immunogen, comprising the step of

administering the vaccine of claim 27 to the host.

37. A method for assaying for mucosal immunity comprising the steps of

administering the vaccine of claim 27 to an animal which is free of infection of the

infectious agent whose antigen is to be tested;

isolating mucosal immune cells from the animal; and

co-incubating said isolated cells with heterologous antigen expressing or presenting cells,

wherein lysing of antigen expressing cells is indicative of mucosal immunity in the animal.

38. The method of claim 37, wherein said mucosal immune cells are isolated from tissues

selected from the group consisting of lamina propria tissue, intraepithelial tissue, Peyer's

patches, lymph nodes, nasal passages, NALT, adenoids and vaginal epithelium.

39. The method of claim 37, comprising the additional step of evaluating the animal's

cytokine profile.

40. An isolated nucleic acid encoding a fusion protein comprising a nucleic acid binding moiety and an M cell specific ligand.

41. The nucleic acid of claim 40, wherein said binding moiety comprises a polymeric chain

of basic amino acid residues.

42. The nucleic acid of claim 41, wherein said polymeric chain comprises polylysine.

43. The nucleic acid of claim 40, wherein said M cell ligand is selected from the group

consisting of: protein 1 of a reovirus, adhesin derived from Salmonella and adhesin derived

from polio virus and M cell tropic fragments thereof.

44. A vector comprising the nucleic acid of any of claims 40 to 43.

45. The vector of claim 44, wherein said vector is an expression vector.

46. A polypeptide comprising the expression product of the vector of claim 45.

47. The vector of claim 45, wherein said nucleic acid is in operable linkage and wherein the

operable linkage is selected from the group consisting of sense and antisense orientations

relative to transcriptional elements comprising the vector.

48. A host cell comprising the vector of claim 44.

49. A method of expressing a fusion protein comprising the step of expressing the vector of

claim 45.

50: An isolated polypeptide comprising a nucleic acid binding moiety and an M cell

specific ligand.

51. The polypeptide of claim 50, wherein said binding moiety comprises a polymeric chain

of basic amino acid residues.

52. The polypeptide of claim 51, wherein said polymeric chain comprises polylysine.

53. The polypeptide of claim 50, wherein said M cell ligand is selected from the group

from polio virus and M cell tropic fragments thereof.

54. An isolated antibody that binds to the polypeptide of claim 50.

55. A kit comprising:

an M cell specific ligand; and

a nucleic acid or immunogen binding moiety.

56. The kit of claim 55, wherein said nucleic acid or immunogen binding moiety is a

polypeptide.

57. The kit of claim 56, wherein said polypeptide comprises a polymeric chain of basic

amino acid residues.

58. The kit of claim 57, wherein said polymeric chain comprises polylysine.

59. The kit of claim 56, wherein said polypeptide is a fusion protein further comprising said

M cell specific ligand.

60. The kit of claim 55, wherein said M cell specific ligand is selected from the group

from polio virus and M cell tropic fragments thereof.

61. The kit of claim 60, wherein said M cell specific ligand is the protein 1 of a reoviras, a

tetramer or trimer thereof, or an M cell tropic fragment thereof.

62. The kit of claim 55 further comprising instructions for the use of said M cell specific

ligand and said nucleic acid or immunogen binding moiety to deliver a nucleic acid vaccine

or other vaccine to mucosal lymphoid tissue.

63. The kit of claim 62 further comprising instructions for measuring a mucosal immune

response raised against said nucleic acid vaccine or other vaccine.

64. A pharmaceutical composition formulated for mucosal delivery comprising

an M cell specific ligand and an immunogen.