WO2001049867A1 - M cell directed vaccines - Google Patents

Abstract

This invention provides a vaccine that can direct gene transfer 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

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 vaccines directed to follicle-associated epithelium. Even

more specifically, the invention is directed to polycation conjugated M cell ligand (e.g.,

enteric adheins)-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, herein incorporated by reference in its entirety. All 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-l (Boyer), rotavirus (Herrmann et al, Jlnfec Dis (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, Behring Inst

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), transferrin (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

(Batra 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, FEBSLett (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 protem (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 transferrin 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 introducing

DNA into cells using M cell ligands for specific targeting of DNA vaccines 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), DNA 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.

Our presently formulated DNA vaccine induces improved mucosal IgA antibody

responses and promotes 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 as part of

a M cell ligand complex is achievable.

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 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 vaccine compositions comprising a polypeptide (or other complexing agent) linked

electrostatically to (or otherwise associated or complexed with) a DNA structural sequence.

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 immunogenic 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 degradtaion 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.

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.

Also contemplated are nucleotide 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, Schistoma, measles virus, mumps virus, herpes virus,

HIN, cancer and influenza virus. Plasmid vectors in which DΝA sequences encode such an

immunogen and are operably linked to transcription regulatory elements are a preferred

embodiment of the present invention.

The vaccines of the present invention are preferably formulated with a

pharmaceutically acceptable excipient or an adjuvant such as an immunomodulator.

Examples of contemplated immunomodulators include cytokines, lymphokines,

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

iinmune 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 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, lysing of antigen expressing cells is indicative of

mucosal immunity in the vaccinated animal.

Use of the foregoing assay method is contemplated with isolated cells including, for

example mucosal B cells, T cells, lamina propria isolates, mtraepithelial isolates, Peyer's

patches cells, lymph nodes, nasal passages, NALT, adenoids and vaginal epithelium. The

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

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. 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.

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 that our recombinant reovirus protein σl can bind murine nasal M

cells.

Figure 2 shows sustained mucosal IgA responses against the reporter gene product,

luciferase.

Figure 3 shows induced cytolytic T cell responses against the reporter gene product,

β-galactosidase.

Figure 4 shows the mucosal intestinal IgA response of mice immunized with one of

three designated HIN DΝA vaccine constructs presenting gpl60, gpl40(c) or gp 140(s).

Figure 5A and 5B show enhanced cytolytic activity (cell-mediated immunity) against

target cells expressing HIN gpl20 from biopsies from mice immunized mtranasally with an

M cell-formulated HIN DΝA vaccine.

Detailed Description of the Invention

Definitions

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

improve the immune response.

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

immune system and can be an immunogen.

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.

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 "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.

As used herein, the term "immunogen" refers to a 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, polysaccharides, 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 neurofransmitter, that binds to a

receptor.

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. By way of distinction, transferrin 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)

1 1 (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, JBiol Chem (1988) 263(25): 12576-12583). While M cell ligands

(rather than M cell specfic 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 "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 term "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 deliver the compositions of the present invention to their target M cells. 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, the term "reovirus" refers to a genus of the family Reoviridae

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

birds, cattle, monlceys, 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 would be a protein encoded by nucleic acids that is expressed

by a vaccinated host to form an expressed protein or peptide subunit vaccine.

General

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, induces 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.

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 surrounds mucosal inductive tissues referred to as M

cells. Thus, a ligand binds M cells to mediate internalization of the dislcosed DNA vaccine.

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. 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 immunogen 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

protem 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

mdpseudotuberculosis 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; andM17448.

In general, it is the formulation of an appropriate DNA conjugate or 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 nucleic acids that can be used for

eliciting a host response, such a response can be enhanced through effective targeting

mediated by M cell ligands.

In a preferred 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 referred 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 formulations encoding various selected antigens may be administered to

immunize individuals against, but not limited to, diseases such as tuberculosis (e.g., BCG

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

complex: Νaito 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), diptheria (e.g., diptheria 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 promastigotes: Lasri et al, Vet Res

(1999) 30(5):441-449), salmonella (e.g., covalently bound capsular polysaccharide (Ni) 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,

Pept Res (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 (HΝ) viral gene product: Brown et al, J Infect Dis (1996)

174(3):619-622), herpes (e.g., HSN-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., immunodommant peptide from hemagglutinin: Novak

et al, J Clin Invest (1999) 104(12):R63-67) and cancer (see Wang RF., JMol Med (1999)

77(9):640-655). Administration of the formulation to a host results in stimulation of the

host's immune system to produce a protective immune response.

The present invention further provides recombinant DNA molecules (rDNAs) that

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

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

DNA 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 preferred 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 protem

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.

In 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.

Vectors 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 formed by a DNA sequence that permits binding of RNA 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 DNA

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 Pharmacia, Piscataway, N. J.

Expression vectors compatible with eukaryotic cells, preferably those compatible with

vertebrate cells, can also be used to form a rDNA 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 DNA segment. Typical of such vectors are pSVL and pKSV-10

(Pharmacia), pBPV-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 DNA vaccine molecules of the

present invention may further include a selectable marker that is effective in an eukaryotic cell,

preferably a drug resistance selection marker. A preferred 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 drag for the selectable

marker.

The M cell ligand-polybasic conjugates according to the invention may be produced

chemically or by the recombinant method. Coupling by the chemical method can be carried out

in a manner known per 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 Icnown 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 succiriimidyl 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 carried

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 components may vary in terms of their size and amino acid sequence.

Production by genetic engineering has the advantage of allowing the M cell Hgand 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 component is inserted, hi 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 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 appUcation. 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 μg/ml), to ensure that the complexes

are not precipitated, as would be known to persons skilled in the art.

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

complex according to the invention is an immunogen structural gene encoded by the nucleic

acids. 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.

Antibodies against M cell ligand-polybasic moiety protein conjugate or complex may be

prepared by immunizing suitable mammalian hosts using the peptides, polypeptides or proteins

alone or conjugated to suitable carriers. Methods for preparing immunogenic conjugates with

carriers such as BSA, KLH, or other carrier proteins are well Icnown 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 formation.

While the polyclonal antisera produced in this way may be satisfactory for some

applications, for pharmaceutical compositions, use of monoclonal preparations is preferred.

hnmortaUzed cell lines which 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 iinmortalized 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 immuiiologically 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.

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 eUcits 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 eUcits antibodies that crossreact with the

immunogenic protein, and are considered to be functional equivalents.

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, an 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 mterleukin-12 protein (or other

cytokines, e.g. GM-CSF), concurrently with or subsequent to intranasal introduction of the M

cell Ugand-polybasic conjugate-polynucleotide complex of this invention may be advantageous.

The polynucleotide 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 saUne. 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.

hi addition, if desired, the DNA vaccine complexes may contain minor amounts of

auxiUary substances such as wetting or emulsifying agents, pH buffering agents, and/or

adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants wliich may be

effective include but are not limited to: aluminum hydroxide;

N-acetyl-muramyl-L-threonyl-D-isoglutamme (thr-MDP);

N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP);

N-acetylmuramyl-L-alanyl-D-isoglutam^^

-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE); and

RIBI, which contains three components extracted from bacteria, monophosphoryl Upid 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 wliich 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 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., isopropylarnine, trimethylamine, 2-ethylamino-ethanol, histidine, andprocaine.

The M cell ligand-polybasic moiety (or conjugate)-polynucleotide compositions 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 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 adrriinistering

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, ampUfication 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 etal. (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning

Vol. I and It, IRL Press, Oxford, UK; Hames et al. (eds.) (1985) Nucleic Acid Hybridization,

URL 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 σl protein

The cloned protein σl cDNA from reovirus serotype 3 strain in the pST3-S 1

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-S 1 , 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

referred to as recombinant protein σl . Example 2. Preparation of recombinant fusion protein σl-polylysine-DNA complex

The recombinant protein σl was covalently linked to poly-L-lysine (PL) according

to the methods of Wagner et α/(1990). Protein σl was purified and resuspended in phosphate-

buffered saline (PBS), pH 7.3. To generate the ditMopyridine 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 milHgrams 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 σl 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 άithiopyridine 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 σ 1 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 referred 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 Ugand binding assay

To assess the cell-binding capacity of the protein σl and protein σl-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 σl antibody (HB-167,

ATCC) and SA-PE (Southern Biotech. Assoc, Birmingham, AL), and specific binding was then

assessed using flow cytometry. 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

lninimiim 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, 5X 10⁵

cells were added to each well of 6- ell 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

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 DI 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 (+)

(hivitrogen, Carlsbad, CA). The cells were lysed with lx luciferase lysis buffer (Promega,

Madison, WI). Twenty μl of supernatant of cell lysates were mixed with 100 μl of Luc assay

buffer and assayed 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. 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 pCMV-β-Gal (Life Technologies)

were harvested, loaded with 200 μM 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

2.5X10⁵ cells/ml. Flow cytometry analysis was performed using a Becton Dickinson

FACSCalibur.

Example 6. Histochemical determination of fusion protein σl 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 σl in DMEM with gentle rotation on a

GeneMate orbital Shaker (hitermountain Scientific Co., Bountiful, UT) for 45 minutes at 4^°C.

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% normal

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

visuaUzed upon reaction with the precipitable substrate, 3-amino-ethylcarbazole (AEC: Sigma).

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 2).

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 irrelevant 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 3).

Example 10. HJV

Intranasal (i.n.) immunization with protein σl -polylysine (PL) conjugate was conducted

to enhance induced mucosal IgA responses in mice. The mean endpoint liters (± SE) for mice

immunized i.n. with protein σl-PL-pCMVgpl60 and σl-PL-pCMNgpl40 or uncomplexed

pCMNgpl60 and pCMVgpl40 (5 mice/group) was compared. Significant differences between

protein σl-PL-pCMVgρl60 and σl-PL-pCMVgpl40 versus pCMVgpl60 and ρCMNgpl40

only were deteπnined 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 HJV DΝA vaccine

constructs, that is gpl60, gp 140(c) and gp 140(s), as indicated in Fig 4. 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 DΝAvaccine formulation improved mucosal IgA responses when compared to

conventional naked DΝA immunization. Example ll. Brucella

Intranasal (i.n.) immunization with protein σl -polylysine (PL) conjugate enhances

induced mucosal IgA and IgG responses in bison. The mean endpoint liters (± 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-pCMVL7/L12 ribosomal protein versus pCMVL7/L12 ribosomal

protein only were determined by student t-test. Using our mucosal DNA formulation, we

observed increases in serum IgG and vaginal IgA and IgG anti-L7/L12 antibody titer in bison.

Example 12. HIN gp!20

Intranasal immunization with an M cell-formulated HIN DΝA vaccine promotes

enhanced cytolytic activity (cell-mediated immunity) against target cells expressing HIN gpl20

as shown in Figs 5 A and 5B. Mice received a formulated vaccine, naked DΝA version, protein

sigmal by the intranasal route three times at one week intervals or were left imimmunized.

Mice were sacrificed six weeks subsequent to this initial immunization to procure specified

tissues, hi a dose-dependent fashion, the lungs from only mice receiving only the formulated

vaccine showed effector function. These results show that the vaccine as formulated 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 gpl20 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.

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

ClaimsWe claim:

1. A composition comprising:

an M cell specific Hgand;

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 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 further comprises 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, SalmoneUa, Schistoma,

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

9. The composition of claim 1, wherein said M cell specific Hgand is selected from

the group consisting of the protein σl of a reovirus, adhesin derived from Salmonella and

adhesin derived from poUo virus and M cell tropic fragments thereof.

10. The composition of claim 9, wherein said M cell specific ligand is the protein

σl of a reovirus and M cell tropic fragments thereof.

11. The composition of claim 2, wherein said DNA sequence further comprises a

plasmid vector in wliich said DNA sequence encoding an immunogen is operably linked to

transcription regulatory elements.

12. A vaccine comprising the composition of any of claims 1 to 11 and a

pharmaceutically acceptable excipient.

13. The vaccine of claim! 2, which induces a protective immune response in a

vaccinated host against said immunogen.

14. The vaccine of claim 12, further comprising an adjuvant.

15. The vaccine of claim 14, wherein said adjuvant comprises an

immunomodulator.

16. The vaccine of claim 15, wherein said immunomodulator is selected from the

group consisting of cytokines, lymphokines, mterleukins, interferons and growth factors.

17. The vaccine of claim 12, wherein the vaccine is formulated in unit dosage form.

18. The vaccine of claim 12, further packaged with instructions for the use of the

vaccine to mduce an immune response against said immunogen or against the disease with

which said immunogen is associated.

19. The vaccine of claim 12, wherein the vaccine is a therapeutic vaccine.

20. The vaccine of claim 12, wherein the vaccine is formulated for adrninistration

through a route selected from the group consisting of oral, nasal, vaginal, rectal and urethral

routes of administration.

21. A method for immunizing a host against an immunogen, comprising the step of

administeriiig the vaccine of claim 12 to the host.

22. A method for assaying for mucosal immunity comprising the steps of

administering the vaccine of claim 12 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, wherein

lysing of antigen expressing cells is indicative of mucosal immunity in the animal.

23. The method of claim 22, wherein said isolated cells are selected from the group

consisting of mucosal B cells, T cells, lamina propria isolates, intraepithelial isolates, Peyer's

patches cells, lymph nodes, nasal passages, NALT, adenoids and vaginal epithelium.

24. The method of claim 22, comprising the additional step of evaluating the

animal's cytokine profile.

25. An isolated nucleic acid encoding a fusion protein comprising a nucleic acid

binding moiety and an M cell specific Hgand.

26. The nucleic acid of claim 25, wherein said binding moiety comprises a

polymeric chain of basic amino acid residues.

27. The nucleic acid of claim 26, wherein said polymeric chain comprises

polylysine.

28. The nucleic acid of claim 25, wherein said M cell Hgand is selected from the

group consisting of: protem σl of a reovirus, adhesin derived from Salmonella and adhesin

derived from polio virus and M cell tropic fragments thereof.

29. A vector comprising the nucleic acid of any of claims 25 to 28.

30. The vector of claim 29, wherein said vector is an expression vector.

31. A polypeptide comprising the expression product of the vector of claim 30.

32. The vector of claim 29, wherein said nucleic acid is in operable linkage and

wherem the operable linkage is selected from the group consisting of sense and antisense

orientations relative to transcriptional elements comprising the vector.

33. A host cell comprising the vector of claim 29.

34. A method of expressing a fusion protein comprising the step of expressing the

vector of claim 30.

35. An isolated polypeptide comprising a nucleic acid binding moiety and an M cell

specific ligand.

36. The polypeptide of claim 35, wherein said binding moiety comprises a

polymeric chain of basic amino acid residues.

37. The polypeptide of claim 36, wherein said polymeric chain comprises polylysine.

38. The polypeptide of claim 35, wherein said M cell Hgand is selected from the

group consisting of: protein σl of a reovirus, adhesin derived from Salmonella and adhesin

derived from poHo virus and M cell tropic fragments thereof.

39. An isolated antibody that binds to the polypeptide of claim 35.

40. A kit comprising:

an M cell specific ligand; and

a nucleic acid bmding moiety.

41. The kit of claim 40, wherein said binding moiety is a polypeptide.

42. The kit of claim 41, wherein said polypeptide comprises a polymeric cham of

basic amino acid residues.

43. The kit of claim 41, wherein said polymeric chain comprises polylysine.

44. The kit of claim 41, wherein said polypeptide further comprises said M cell

specific Hgand.

45. The kit of claim 40, wherem said M cell specific ligand is selected from the

group consisting of: protein σ 1 of a reovirus, adhesin derived from Salmonella and adhesin

derived from polio virus and M cell tropic fragments thereof.

46. The kit of claim 45, wherein said M cell specific Hgand is the protein σl of a

reovirus and M cell tropic fragments thereof.