WO1996006115A1

WO1996006115A1 - Product and method for detecting internalin

Info

Publication number: WO1996006115A1
Application number: PCT/US1995/010509
Authority: WO
Inventors: Priscilla A. Campbell; Terry A. Potter; Richard T. Sawyer; Douglas A. Drevets; John H. Freed
Original assignee: National Jewish Center For Immunology And Respiratory Medicine
Priority date: 1994-08-18
Filing date: 1995-08-18
Publication date: 1996-02-29
Also published as: AU3582795A

Abstract

Products and methods for detecting internalin are disclosed wherein antibodies to internalin are used for screening samples such as food, clinical specimens and environmental samples for the presence of internalin. The present invention is useful for detecting the presence of pathogenic Listeria microorganisms in samples. Further embodiments relate to therapeutic compositions and methods, test kits, antibodies, vaccines, methods useful in the diagnosis and treatment of listeriosis, and methods for removing pathogenic microorganisms from samples.

Description

PRODUCT AND METHOD FOR DETECTING INTERNALIN

FIELD OF THE INVENTION

The present invention relates to a product and method for detecting intemalin and to an antibody-based assay for screening samples for the presence of intemalin. The present invention also relates to a delivery vehicle that comprises intemalin, as well as a vaccine that is capable of targeting antigens to an MHC Class I pathway using a carrier vehicle having intemalin.

BACKGROUND OF THE INVENTION Particular microorganisms have long been recognized as a source of disease. Pathogenic microorganisms cause disease by disrupting the normal functions of a host. Listeria monocytogenes is a gram positive, motile, bacterium found in soil, water, foods and animal products. Listeria monocytogenes has been responsible for periodic outbreaks of disease and death primarily as a result of contaminated food products such as meat and milk. In adults, listeriosis can occur as a gastrointestinal disease, sepsis and, in severe cases, as a rapidly fatal meningitis. Listeriosis is often associated with immunocompromised individuals, such as pregnant women, individuals suffering from neoplastic diseases, genetic disorders of the immune system and patients undergoing various forms of chemotherapy. For example, it has been found that the occurrence of listeriosis in individuals with Acquired Immunodeficiency Syndrome (AIDS) is approximately 300 times greater than that found in the general population. Listeria monocytogenes also causes spontaneous abortion, still birth, and perinatal and neonatal infections. Neonatal early onset of Listeria monocytogenes infection has a 26% fatality rate, most commonly arising from intrauterine infection occurring at or just after birth. Neonatal late onset of Listeria monocytogenes infection has a 38% fatality rate and often involves the central nervous system with symptoms occurring within several days to several weeks after birth.

Due to the harmful and sometimes deadly consequences of Listeria infection, prior investigators have sought to develop reliable screening procedures to identify Listeria in samples, such as medical and food samples. The techniques currently available for the detection of Lxsteria monocytogenes , however, are unsatisfactory. Conventional culture methods are slow, taking at least 48 hours and typically involve enrichment of the pathogenic microorganisms in selective media. Biochemical tests are then necessary to confirm the identity of a contaminating microorganism. Depending upon the initial population of microorganisms, the co-contamination by other microorganisms and the type of sample being screened, the time required for final confirmation of Listeria contamination may exceed several weeks. Due to the rapid onset of listeriosis the time required to perform conventional culture assays is inadequate for disease diagnosis and effective treatment. Moreover, the procedures presently used take too long in situations where samples to be tested are perishable.

Particular screening methods using antibody-based assays for Listeria have been suggested in U.S. Patent No. 4,950,589 by Butman et al., issued August 21, 1990 and PCT Publication No. WO 93/19372 by Basboll et al., published September 30, 1993. Prior investigators, however, have only disclosed the use of antibodies that do not specifically detect pathogenic Listeria capable of invading a host cell. Since only two species amongst numerous species of Listeria have been shown to be pathogenic, existing screening methods for Listeria in general lack efficiency and specificity for detecting pathogenic microorganisms. As such, there remains a need to develop an assay capable of detecting pathogenic microorganisms, including pathogenic Listeria.

One of the factors that influences the disease-causing properties of a microorganism include the invasiveness of the microorganism. Invasiveness refers to the ability of a microorganism to enter a host cell. Successful invasion of a host cell by a microorganism can result in the propagation of the microorganism, thus leading to disease. Prior investigators have shown that a protein referred to as intemalin is present on Listeria cells capable of invading a host cell. Gaillard et al., pp. 1127-1141, 1991, Cell , Vol. 65 disclose the DNA sequence encoding intemalin protein. Dramsi et al., pp. 931-941, 1993, Molecular Microbiology, Vol. 9 disclose the production of an intemalin protein using the inlA gene of Listeria monocytogenes , and immunizing rabbits with the intemalin protein to produce a polyclonal antiserum capable of binding to intemalin. The polyclonal antiserum was used in Western blot experiments to visualize intemalin protein from Listeria monocytogenes cell lysates. Dramsi et al. use the anti-internalin polyclonal antibody to correlate the appearance of intemalin protein on the surface of Listeria monocytogenes with the expression of intemalin mRNA during the growth cycle of Listeria monocytogenes and the ability of the bacteria to invade epithelial cells.

Prior investigators have not disclosed how to make or use a screening assay capable of specifically detecting pathogenic microorganisms, and specifically Listeria in samples. There remains a need to develop an assay capable of detecting intemalin in samples, such as food samples and clinical samples. Similarly, prior investigators have not taught a test kit for testing samples suspected of being contaminated with pathogenic microorganisms such as Listeria . Thus, there remains a need to develop a kit comprising an anti-intemalin antibody. Moreover, prior investigators have not disclosed a treatment for Listeria infection that specifically attacks the ability of a Listeria cell to invade a host cell. Thus, there remains a need to develop a therapeutic composition comprising intemalin protein and/or an anti-intemalin antibody. Furthermore, prior investigators have not taught a vaccine capable of inducing a Class I response by specifically utilizing the mechanism by which Listeria invade a host cell. Thus, there remains a need to develop a vaccine capable of entering an antigen presenting cell, escaping the endocytic pathway of the cell and entering the endoplas ic reticulum of the cell to bind to a Class I protein. In addition, prior investigators have not disclosed a product and method for targeting desired compounds to cells capable of binding to intemalin. For example, cells in the intestinal tract expressing an intemalin receptor could be affected by a drug bound to intemalin. Thus, there remains a need to develop a target vehicle comprising intemalin joined to a heterologous compound. Finally, there is a need for a method to remove pathogenic microorganisms from samples, such as meat and dairy products, susceptible to contamination by pathogenic Listeria .

SUMMARY The present invention is directed, in one embodiment, to a method for screening a sample, such as food, clinical or environmental samples, for the presence of intemalin, such method comprising immunoreacting a sample with an antibody capable of selectively binding to intemalin and determining the presence of the immunoreaction. The presence of intemalin can be measured by using a detectable signal associated with the immunoreaction. The present invention is particularly useful for detecting the presence of microorganisms of the genus listeria, and particularly Listeria monocytogenes and Listeria ivanovii . The present invention is also directed to antibodies to intemalin that are capable of substantially inhibiting the ability of internalin-bearing microorganisms, such as listeria, to invade a host cell, preferably by forming an immunocomplex with intemalin, thereby inhibiting the ability of intemalin to bind to a host cell's intemalin receptor. In a preferred embodiment, the present antibody comprises a monoclonal antibody which has substantially the same binding characteristics as antibodies produced by particular hybridoma cell lines developed by the present inventors.

The present invention can be used in a method to screen a sample for listeria by immunoreacting a sample with an antibody capable of selectively binding to intemalin and determining the presence of the immunoreaction. Moreover, the present invention can be used to inhibit the propagation of listeria by administering to an animal a reagent capable of inhibiting the uptake of listeria, such reagent comprising an antibody capable of binding to intemalin or an inhibitory compound identified by its ability to inhibit the binding of intemalin protein to a host cell.

Moreover, the present invention is directed to a method for delivering a reagent to a cell having an intemalin receptor thereon by administering to an animal a target vehicle that has intemalin protein joined to a heterologous compound selected from the group consisting of a protein, a peptide, a toxin, a microbial agent and an inert particle. In preferred embodiments, intemalin protein is encoded by a nucleic acid sequence that is capable of hybridizing under stringent conditions to at least a portion of seq. ID No:l. The present invention further relates to a therapeutic composition to treat animals infected with cells having intemalin. In one embodiment, the therapeutic composition comprises intemalin, an antibody capable of binding intemalin and/or a compound that inhibits binding of intemalin to a host cell.

A further embodiment of the present invention relates to a test kit for detecting intemalin, such test kit including an antibody to intemalin and a means for determining an immunoreaction between the antibody and intemalin. Preferably, the test kit includes a detectable tag that indicates immunoreactions between intemalin and an antibody. Additionally, the present invention relates to a target vehicle comprising an intemalin protein joined to a heterologous compound such as an antigen, a drug, a marker, an antibody, a cytokine or a growth factor. Further embodiments include a vaccine capable of inducing a Class I restricted immune response comprising a cell expressing intemalin, hemolysin and a heterologous antigen where the cell is capable of entering and escaping a surface-bound cytoplasmic vesicle and where the cell does not comprise Listeria monocytogenes . Yet a further embodiment of the present invention relates to an intemalin receptor identified by forming an intemalin protein colon: internalin receptor complex by combining intemalin with a sample having an intemalin receptor and isolating the intemalin receptor from the complex. Finally, the present invention relates to a method for removing listeria from a food product by contacting the food product with an antibody capable of binding intemalin and separating the immunocomplex formed from the food product.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 illustrates the fluorescence staining of MP-10 cells using secondary antibody alone, or rabbit anti-ACrlNL antibodies and secondary antibody.

Fig. 2 illustrates the fluorescence staining of MP-10 cells using secondary antibody alone, or mouse anti- intemalin 292 or 1630 antibodies.

Fig. 3 illustrates the fluorescence staining of Listeria ivanovii cells using secondary antibody alone, or mouse anti-intemalin 292 or 1630 antibodies.

Fig. 4 illustrates the number of MP-10 cells associated with Caco-2 cells in the absence and in the presence of either rabbit anti-ACrlNL antibodies or rabbit anti-KLH3054 antibodies.

Fig. 5 illustrates the percent of MP-10 cells associated with J774 cells in the absence and in the presence of either rabbit anti-ACrlNL antibodies or rabbit anti-KLH3054 antibodies. Fig. 6 illustrates the percent of MP-10 cells associated with HUVEC cells in the absence and in the presence of mouse anti-intemalin monoclonal antibodies.

Fig. 7 illustrates the detection of Listeria monocytogenes cells by ELISA using rabbit anti-ACrlNL antibodies.

Fig. 8 illustrates the detection of Listeria monocytogenes cells by ELISA using rabbit anti-ACrlNL antibodies. Fig. 9 illustrates the detection of Listeria monocytogenes cells by ELISA using rabbit anti-ACrlNL antibodies.

Fig. 10 illustrates the detection of intemalin in hot dog juice and hot dog meat by ELISA using rabbit anti- ACrlNL antibodies.

Fig. 11 illustrates the detection of intemalin in hot dog juice by ELISA using rabbit anti-ACrlNL antibodies.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel product and method for detecting the protein intemalin. The present invention includes a novel assay comprising an antibody which selectively binds to intemalin. In particular, the novel product and method of the present invention is capable of detecting particular microorganisms that invade host cells, such as Listeria monocytogenes and Listeria ivanovii microorganisms that have intemalin associated therewith. The present invention is, therefore, particularly advantageous for screening samples for the presence of intemalin which indicates the presence of disease-causing microorganisms.

A pathogenic Listeria cell is a facultative intracellular parasite able to escape destruction within host cells and capable of replication within host cells. In permissive cells, pathogenic Listeria initially attach to the host cell and are phagocytized within an endocytic vesicle termed a phagosome. The Listeria escape from the phagosome by secreting listeriolysin O which acts on cholesterol within the phagosome membrane and forms a hole which disrupts the phagosome. Once Listeria escape the phagosome, the Listeria is able to replicate within the cell cytoplasm and can invade other host cells, thereby completing a life cycle. The pathogenicity of Listeria is, therefore, dependent upon the ability of the Listeria to invade a host cell. As used herein, "pathogenicity" refers to the ability of a microorganism to cause disease. Invasiveness refers to the ability of a microorganism to invade a host cell or tissue and to multiply within such cell or tissue. Invasiveness differs from toxigenicity which refers to the ability of a microorganism to produce toxins that disrupt the normal function of host cells or are generally destructive to host cells and/or tissues. A Listeria cell attaches to a host cell in a specific manner via interactions between a bacterial surface protein called intemalin and a receptor on the surface of the host cell. Intemalin is a bacterial cell-surface associated protein that has been found on the surface of Listeria monocytogenes and Listeria ivanovii . Listeria monocytogenes is responsible for essentially all human listeriosis. Listeria ivanovii is responsible for pathogenesis in non- human animals. Other species of Listeria known as saprophytic Listeria do not have intemalin and are not pathogenic. As such, intemalin is a marker for invasive pathogenic Listeria . Without being bound by theory, it is believed that many cells have a specific intemalin binding molecule on their surfaces, herein referred to as an "intemalin receptor." It is also believed that the binding of Listeria to the intemalin receptor results in receptor mediated endocytosis which assists in the escape of Listeria from the phagosome. According to the present invention, a host cell includes any cell capable of binding to intemalin, in particular host cells include cells having an intemalin receptor. The distribution of cells capable of binding intemalin is not known but it is believed by the inventors that the intemalin receptor may be a relatively ubiquitous molecule found on a large variety and number of cells. A preferred host cell of the present invention includes, but is not limited to, macrophages, endothelial cells, epithelial cells, fibroblasts and blood leukocytes. A more preferred host cell includes a J774 cell, a CaCo-2 cell, or a human umbilical vein endothelial cell (HUVEC) .

One embodiment of the present invention is an intemalin receptor identified by the method comprising the steps of: (1) combining intemalin with a sample having an intemalin receptor to form an intemalin protein:intemalin receptor complex; and (2) isolating the intemalin receptor portion of the intemalin protein:intemalin receptor complex. Following isolation of a protein:intemalin receptor complex, the intemalin receptor portion of the complex is characterized using standard protein characterization methods, such as N- terminal amino acid sequencing, molecular weight determination, glycosylation studies, or mass spectrometry. Suitable intemalin protein for identifying an intemalin receptor includes an intemalin protein described herein. Preferred intemalin protein includes intemalin protein encoded by the nucleic acid sequence shown in Table 1 (SEQ ID N0:1), as well as smaller proteins that are encoded by nucleic acid sequences that are able to hybridize with the nucleic acid sequence represented by SEQ ID NO:l under stringent conditions. SEQ ID N0:l encodes for the protein of SEQ ID NO:2.

Table 1. Intemalin Nucleic Acid Sequence.

1 GGGATCCTGA AGACGGTCTT AGGAAAAACG AATGTAACAG ACACGGTCTC GCAAACAGAT

61 CTAGACCAAG TTACAACGCT TCAGGCGGAT AGATTAGGGA TAAAATCTAT CGATGGATTG

121 GAATACTTGA ACAATTTAAC ACAAATAAAT TTCAGCAATA ATCAACTTAC GGATATAACG 181 CCACTTAAAG ATTTAACTAA GTTAGTTGAT ATTTTGATGA ATAATAATCA AATAGCAGAT

241 ATAAC CCGC TAGCTAATTT GACGAATCTA ACTGGTTTGA CTTTGTTCAA CAATCAGATA

301 ACAGATATAG ACCCGCTTAA AAATCTAACA AATTTAAATC GGCTAGAACT ATCTAGTAAC

361 ACGATTAGTG ATATTAGTGC GCTTTCAGGT TTAACTAATC TACAGCAATT ATCTTTTGGT

421 AATCAAGTGA CAGATTTAAA ACCATTAGCT AATTTAACAA CACTAGAACG ACTAGATATT 481 TCAAGTAATA AGGTGTCAGA TATTAGTGTT CTGGCTAAAT TAACCAATTT AGAAAGTCTT

541 ATCGCTACTA ACAACCAAAT AAGTGATATA ACTCCACTTG GGAT TTAAC AAAT TGGAC

601 GAATTATCCT TAAATGGTAA CCAGTTAAAA GATATAGGCA CATTGGCGAG TTTAACAAAC

661 C TACAGATT TAGATTTAGC AAATAACCAA ATTAGTAATC TAGCACCACT GTCGGGTCTA

721 ACAAAACTAA CTGAGTTAAA ACTTGGAGCT AACCAAATAA GTAACATCAG TCCCCTAGCA 781 GGTTTAACCG CACTCACTAA CTTAGAGCTT AATGAAAATC AGCTGGAAGA TATTAGCCCA

841 ATTTCTAACC TGAAAAATCT CACATATTTA ACGTTGTACT TTAATAATAT AAGTGATATA

901 AGCCCAGTTT CTAGTTTAAC AAAGCTTCAA AGATTATTTT TCTATAATAA CAAGGTAAGT

961 GACGTAAGCT CACTTGCGAA CTTAACCAAT ATTAATTGGC TTTCAGCTGG GCATAACCAA

1021 ATTAGCGATC TTACACCATT GGCTAATTTA ACAAGAATCA CCCAACTAGG GTTGAATGAT 1081 CAAGCATGGA CAAATGCACC AGTAAACTAC AAAGCAAATG TATCCATTCC AAACACGGTG

1141 AAAAATGTGA CTGGCGCTTT GATTGCACCT GCTACTATTA GCGATGGCGG TAGTTACGCA

1201 GAACCGGATA TAACATGGAA CTTACCTAGT TATACAAATG AAGTAAGCTA TACCTTTAGC

1261 CAACCTGTCA CTATTGGAAA AGGAACGACA ACATTTAGTG GAACCGTGAC GCAGCCACTT

1321 AAGGCAATTT TTAATGCTAA GTTTCATGTG GACGGCAAAG AAACAACCAA AGAAGTGGAA 1381 GCTGGGAATT TATTGACTGA ACCAGCTAAG CCCGTAAAAG AAGGTCACAC ATTTGTTGGT

1441 TGGTTTGATG CCCAAACAGG CGGAACTAAA TGGAATTTCA GTACGGATAA AATGCCGACA

1501 AATGACATCA ATTTATATGC ACAATTTAGT ATTAACAGCT ACACAGCAAC CTTTGAGAAT

1561 GACGGTGTAλ CAACATCTCA AACAGTAGAT TATCAAGGCT TGTTACAAGA ACCTACACCA

1621 CCAACAAAAG AAGGTTATAC TTTCAAAGGC TGGTATGACG CAAAAACTGG TGGTGACAAG 1681 TGGGATTTCG CAACTAGCAA AATGCCTGCT AAAAACATCA CCTTATATGC CCAATATAGC

1741 GCCAATAGCT ATACAGCAAC GTTTGATGTT GATGGAAAAT CAACGACTCA AGCAGTAGAC

1801 TATCAAGGAC TTCTAAAAGA ACCAAAGGCA CCAACGAAAG CCGGATATAC TTTCAAAGGC

1861 TGGTATGACG AAAAAACAGA TGGGAAAAAA TGGGATTTTG CGACGGATAA AATGCCAGCA

1921 AATGACATTA CGCTGTACGC TCAATTTACG AAAAATCCTG TGGCACCACC AACAACTGGA 1981 GGGAACACAC CGCCTACAAC AAATAACGGC GGGAATACTA CACCACCTTC CGCAAATATA

2041 CCTGGAAGCG ACACATCTAA CACATCAACT GGGAATTCAG CCAGCACAAC AAGTACAATG

2101 AACGCTTATG ACCCTTATAA TTCAAAAGAA GCTTCACTCC CTACAACTGG CGATAGCGAT

2161 AATGCGCTCT ACCTTTTGTT AGGGTTATTA GCAGTAGGAA CTGCAATGGC TCTTACTAAA

2221 AAAGCACGTG CTAGTAAATA G

A suitable sample from which an intemalin receptor can be isolated comprises a cellular sample, preferably a cell lysate. Preferred cell lysates are made from a host cell of the present invention. Particularly preferred cell lysates are made from J774 cells, CaCo-2 cells and/or human umbilical vein cells (HUVEC) . More preferred cell lysates are made from cells treated with protein labeling isotopes and/or compounds including, but not limited to, ^!25I, ³⁵S, ³²P and biotin. An intemalin protein:intemalin receptor complex of the present invention is isolated using the steps of: (1) i munoreacting an intemalin protein:intemalin receptor complex with an antibody capable of binding to the intemalin to form an immune complex; (2) recovering the immune complex; and (3) isolating the intemalin protein:intemalin receptor complex by separating the intemalin protein:intemalin receptor complex from the immune complex. The intemalin protein can then be separated from the intemalin receptor by charge or size fractionation by electrophoresis or column chromatography; affinity chromatography; or incubation in a dissociation buffer having high or low pH.

A preferred intemalin receptor of the present invention is identified by the method comprising: (1) lysing and extracting membrane preparations of CaCo-2 and J774 cells; (2) resolving the cellular protein by SDS-PAGE gel electrophoresis and transferring the protein to a filter; (3) contacting the filter with intemalin; (4) contacting the filter with intemalin antibody; and (5) identifying a protein on the filter which binds to the antibody. A preferred intemalin receptor protein comprises a polypeptide of about 180,000 MWr.

As illustrated in the above stated procedure, some embodiments of the present invention are directed to the use of intemalin to form i munocomplexes useful in the treatment and diagnosis of disease. A detailed discussion of various embodiments of the present invention involving intemalin protein and its ho ologues is provided below. One embodiment of the present invention comprises an isolated intemalin protein or a mimetope thereof (i.e., a mimetope of a intemalin protein) . According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated intemalin protein can be obtained from its natural source and herein is referred to as "natural intemalin" protein. An isolated intemalin protein can also be produced using recombinant DNA technology or chemical synthesis and herein is referred to as "recombinant intemalin" protein. As used herein, an intemalin protein can be a full-length intemalin protein or any homologue of such a protein such as a intemalin protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide) , inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol) such that the homologue enables a cell expressing such homologue to invade a host cell of the present invention and/or includes at least one epitope capable of eliciting an immune response against intemalin protein which results in the production of antibodies capable of binding to intemalin. The ability of a homologue to enable a cell to invade a host cell can be measured by anchoring the homologue in a lipid bilayer compound (i.e., liposome) , presenting the bound homologue to a host cell and determining if the lipid bilayer compound is phagocytozed by the host cell.

Intemalin protein ho ologues can result from natural allelic variation or natural mutation. Intemalin protein homologues can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. Isolated proteins of the present invention, including homologues, can be identified in a straight-forward manner by the ability of antibodies specific for intemalin to bind to the homologue, and/or testing such homologues to assess their ability to promote invasion of a host cell, and/or by their ability to bind to an intemalin receptor.

The minimum size of an isolated intemalin protein of the present invention is sufficient to form an epitope, a size that is typically at least from about 7 to about 9 amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope. In accordance with the present invention, a mimetope refers to any compound that is able to mimic the ability of an isolated intemalin protein of the present invention. A mimetope can be a peptide that has been modified to decrease its susceptibility to degradation but that still retains the ability to bind to an intemalin receptor and/or the ability to elicit an immune response against at least one epitope of intemalin. Other examples of mimetopes include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti- idiotypic antibodies and/or catalytic antibodies, or fragments thereof. A mimetope can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds that have desired identifying characteristics of intemalin and/or intemalin antibodies. A mimetope can also be obtained by, for example, rational drug design. In a rational drug design procedure, the three-dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography. The three- dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modelling. The predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source (e.g., plants, animals, bacteria and fungi). A preferred intemalin protein or mimetope has the characteristics of intemalin found on Listeria cells. A suitable Listeria of the present invention includes Listeria monocytogenes and Listeria ivanovii . An internalin protein of the present invention has the further characteristic of being encoded by a nucleic acid molecule that is capable of hybridizing, under stringent conditions, with a nucleic acid comprising at least a portion of the nucleic acid sequence encoding an intemalin protein, such as that disclosed in SEQ ID NO:l. As used herein, the phrase "at least a portion of" an entity refers to an amount of the entity that is at least sufficient to have the functional aspects of that entity. For example, at least a portion of a nucleic acid sequence, as used herein, is an amount of a nucleic acid sequence capable of forming a stable hybrid under stringent hybridization conditions. SEQ ID NO:l represents the deduced sequence of a DNA nucleic acid molecule of intemalin. (It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO:l, at best, represents an apparent nucleic acid sequence of the nucleic acid molecule encoding at least a portion of intemalin) . As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules (or sequences) are used to identify similar sequences. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Labs Press, 1989. One embodiment of the present invention is a fusion protein that includes an intemalin-containing domain attached to a fusion segment. Depending on the segment's characteristics, a fusion segment can function as a tool to simplify purification of an intemalin protein, such as to enable purification of the resultant fusion protein using affinity chromatography. Furthermore, a fusion segment can act as an immunopotentiator to enhance the immune response mounted by an animal immunized with an intemalin protein containing such a fusion segment. A suitable fusion segment can be a domain of any size that has desired function (e.g., purification tool, increased immunogenicity, and/or increased stability) . It is within the scope of the present invention to use one or more fusion segments. Fusion segments can be joined to amino and/or carboxyl termini of the internalin-domain of the protein. Linkages between fusion segments and intemalin-containing domains of fusion proteins can be susceptible to cleavage in order to enable straight-forward recovery of the intemalin-containing domains of such proteins. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an intemalin- containing domain.

A preferred fusion segment for use in the present invention includes a glutathione binding domain, such as Schistosoma japonicum glutathione-S-transferase (GST) or a portion thereof capable of binding to glutathione; a sugar binding domain such as a maltose binding domain from a maltose binding protein; a metal binding domain, such as a poly-histidine segment capable of binding to a divalent metal ion; an immunoglobulin binding domain, such as Protein A, Protein G, T cell, B cell, Fc receptor or complement protein antibody-binding domains; and/or a "tag" domain, for example, at least a portion of j3-galactosidase, other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies. A more preferred fusion segment includes a glutathione binding domain and a maltose binding domain. An example of a particularly preferred fusion protein of the present invention is encoded by the expression vector pGEX3X4.3.526, the production of which is disclosed herein. As used herein, the nucleic acid sequence contained in the expression vector pGEX3X4.3.526 encodes the intemalin fusion protein referred to herein as GST-INL. One aspect of the present invention is directed to the use of antibodies that are capable of binding to intemalin protein. Such uses include, an assay capable of detecting the presence of intemalin in samples, the detection of pathogenic microorganisms, methods to inhibit propagation of Listeria, ' and a method for identifying an intemalin receptor. The following detailed discussion relates to various embodiments of the present invention that involve an anti-intemalin antibody.

One embodiment of the present invention includes antibodies capable of selectively binding to an intemalin protein or mimetope thereof. Such an antibody is herein referred to as an anti-intemalin antibody. As used herein, the term "selectively binds to" refers to the ability of such an antibody to preferentially bind to intemalin (including homologues of intemalin) and mimetopes thereof. Antibodies of the present invention can be either polyclonal or monoclonal antibodies. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein or mimetope used to obtain the antibodies. Antibodies of the present invention can include chimeric antibodies in which at least a portion of the heavy chain and/or light chain of an antibody is replaced with a corresponding portion from a different antibody. For example, a chimeric antibody of the present invention can include an antibody having an altered heavy chain constant region (e.g., altered isotype) , an antibody having protein sequences derived from two or more different species of animal, and an antibody having altered heavy and/or light chain variable regions (e.g., altered affinity or specificity) . Preferred antibodies are raised in response to proteins, peptides or mimetopes thereof of intemalin. More preferred antibodies are raised by proteins, or mimetopes thereof, that are encoded, at least in part, by an intemalin nucleic acid molecule. Generally, in the production of an antibody, a suitable experimental animal, such as a rabbit, hamster, guinea pig or mouse, is exposed to an antigen against which an antibody is desired. Typically, an animal is immunized with an effective amount of antigen that is injected into the animal. An effective amount of antigen refers to an amount needed to induce antibody production by the animal. The animal's immune system is then allowed to respond over a pre-determined period of time. The immunization process can be repeated until the immune system is found to be producing antibodies to the antigen. In order to obtain polyclonal antibodies specific for the antigen, serum is collected from the animal that contains the desired antibodies. Such serum is useful as a reagent. Polyclonal antibodies can be further purified from the serum by, for example, treating the serum with ammonium sulfate. In order to obtain monoclonal antibodies, the immunized animal is sacrificed and B lymphocytes are recovered from the spleen. The B lymphocytes are then fused with myeloma cells to obtain a population of hybridoma cells capable of continual growth in suitable culture medium. Hybridomas producing a desired antibody are selected by testing the ability of an antibody produced by a hybridoma to bind to the antigen.

A preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of an intemalin protein or mimetope thereof to produce antibodies thereto and recovering such antibodies. Preferred intemalin protein for administration to an animal includes, but is not limited to, intemalin fusion protein, recombinant intemalin, natural intemalin, and/or mimetopes thereof. Preferred intemalin proteins for producing antibodies include an intemalin peptide, recombinant intemalin, and intemalin fusion protein. More preferred intemalin protein includes a peptide having the amino acid sequence CNNQIADITPLANLTNLT (SEQ ID NO:3), intemalin protein encoded by at least a portion of the nucleic acid sequence represented by SEQ ID NO:l, and GST- INL encoded by the expression vector pGEX3X4.3.526.

Preferred anti-intemalin antibody useful for inhibiting the invasion of Listeria cells includes antibodies capable of specifically binding to an intemalin peptide having the amino acid sequence SEQ ID NO:3; antibodies capable of specifically binding to the intemalin fusion protein GST-INL (described in detail in the Examples) ; or antibodies capable of specifically binding to an intemalin protein encoded by at least a portion of the nucleic acid sequence represented by SEQ ID NO:l. Preferred polyclonal anti-intemalin antibodies include rabbit anti-GST-INL antibodies, rabbit anti-ACrlNL antibodies and rabbit anti-KLH3054 antibodies. Preferred monoclonal anti-intemalin antibodies of the present invention have the isotype IgGl, IgG2a, IgG2b, IgA or IgM. More preferred monoclonal anti-intemalin antibodies have substantially the same binding characteristics as the monoclonal antibody produced by the hybridoma cell lines 292; 1847; 1339; 804; 1630; 1360; 1835; 1042; 469; and 678.

One embodiment of the present invention relates to the use of intemalin antibodies (both monoclonal and polyclonal) to form immunocomplexes, and the use of such immunocomplexes to inhibit invasion of internalin-bearing cells into host cells. An immunocomplex refers to a complex comprising an antibody and its ligand (i.e., antigen) . Such an immunocomplex formation substantially inhibits the ability of intemalin to bind to a host cell. According to the present invention, inhibition of invasion refers to the ability of the antibody to prevent preferably at least about 50% more preferably at least about 70%, and even more preferably at least about 90% of intemalin- bearing cells from invading a host cell. In particular, an anti-intemalin antibody of the present invention is capable of substantially inhibiting the ability of a Listeria cell to invade a host cell. Preferred anti- intemalin antibodies useful for inhibiting the invasion of Listeria cells include the anti-intemalin antibodies disclosed herein.

A monoclonal antibody of the present invention is capable of specifically binding to intemalin protein, thereby forming an immunocomplex therewith. A preferred monoclonal antibody of the present invention has the isotype IgGl, IgG2a, IgG2b, IgA or IgM. A preferred monoclonal antibody has substantially the same binding characteristics as the monoclonal antibody produced by the hybridoma cell lines 292; 1847; 1339; 804; 1630; 1360; 1835; 1042; 469; and 678.

Listeria is the causative agent for the disease listeriosis in humans and other animals. Direct transmission of Listeria can occur between farm animals and humans. Clinical symptoms of listeriosis can include central nervous system infections, primary bacteremia, endocarditis, gastroenteritis and focal listeriosis. Presently, effective treatment requires diagnosis of the infective agent such that the appropriate antibiotic therapy can be implemented.

Listeria infection, in both sporadic and epidemic listeriosis, is often transmitted by ingestion of food and food products. The present invention includes the use of a novel screening method capable of detecting pathogenic Listeria in contaminated samples, particularly clinical samples and food samples. In addition, because intemalin is more abundant on the surface of Listeria monocytogenes during log phase of growth and less during stationary phase of growth, the screening method of the present invention is capable of detecting an expanding population of Listeria cells in a contaminated sample. Moreover, because intemalin may be secreted from Listeria cells and because intemalin is present on dead or dying Listeria cells, the screening method is capable of detecting Listeria that cannot be cultured, and this permits one to distinguish between pathogenic microorganisms and those microorganisms that are not pathogenic despite ostensive indications to the contrary. One embodiment of the present invention relates to a method to screen samples for intemalin and includes the steps of: (a) immunoreacting a sample with an antibody capable of selectively binding to intemalin to form an immunocomplex; and (b) determining the presence of the immunocomplex. A novel and unexpected aspect of the screening method of the present invention is the ability of an anti-intemalin antibody to detect intemalin in a sample having other components which can disrupt the screening method. For example, both food samples and clinical samples can contain substantial amounts of lipids which can cause anti-intemalin antibody to bind non- specifically to a substrate to which a sample is attached. In addition, proteins present in samples can hinder the ability of an anti-intemalin antibody to bind to intemalin. In addition, a protein in a food sample could cross-react with an anti-intemalin antibody thereby preventing binding of the antibody to intemalin. Thus, to be useful in a screening method, an anti-internalin antibody must have sufficient specificity, affinity and/or avidity to overcome any inhibition of binding due to the presence of undesired proteins and/or lipids. Preferred antibodies for use with a screening method of the present invention include anti-internalin antibodies of the present invention as disclosed herein.

Suitable samples for use with the screening method of the present invention include any sample suspected of having intemalin, in particular samples suspected of being contaminated with Listeria . Preferred samples for use in conjunction with the present invention include, but are not limited to, foods, clinical specimens, and environmental samples. Food samples can include cooked and raw food. Particularly preferred food samples include poultry, cattle, pig, goat, sheep, lamb, fish, seafood, dairy samples, fruit, vegetables and grains. A preferred poultry sample includes, but is not limited to chicken, turkey and eggs. A preferred pig sample includes, but is not limited to minced pork, bacon, ham, salami, and sausage. Mixed meat product samples can include hot dogs, pate, and meat extracts such as broth. A preferred dairy sample includes, but is not limited to, fermented and non-fermented dairy products. More preferred dairy products include milk, cheese, whey, butter, baby formula, ice cream, and yogurt.

Particularly preferred cheese samples include whey cheese, hard cheese, and soft cheese. Raw fish, fermented fish, shrimp and crab are particularly preferred seafood samples.

Clinical samples suitable for screening using an assay of the present invention include animal fluid, cellular, tissue and excrement, such as feces and urine specimens. Particularly preferred clinical fluid samples include blood, plasma, serum, saliva, cerebo-spinal fluid, exudates such as pus, amniotic fluid, interstitial fluid, synovial fluid and autopsy specimens. Particularly preferred cellular samples include macrophages, endothelial cells and epithelial cells. Particularly preferred clinical tissue samples include brain, lung, liver, spleen, lymphnodes, bone, muscle, placental tissue and abotrtuses. Environmental samples suitable for screening using an assay of the present invention include surface water, plant, soil, silage, slaughterhouse waste and sewage samples. A screening assay of the present invention is particularly useful for screening samples removed from food storage and/or processing facilities, and food transportation vehicles.

The screening method of the present invention can further comprise comparing the formation of particular immunocomplexes obtained in the testing of samples with immunocomplexes formed using a control solution. A control solution can include a negative control solution and/or a positive control solution. A positive control solution of the present invention contains an effective amount of at least one compound known to bind to an anti-internalin antibody of the present invention. As such, immunocomplexes form using positive control solutions. Preferred compounds for use in a positive control solution of the present invention include, but are not limited to, a natural intemalin protein, a recombinant intemalin protein, an intemalin fusion protein, an intemalin peptide, Listeria monocytogenes cells, bacterial or eukaryotic cells transfected with an expressable form of intemalin nucleic acid sequence, and inert particles coated with intemalin. More preferred positive control compounds include recombinant intemalin protein encoded by at least a portion of the nucleic acid sequence represented by SEQ ID NO:l, fusion intemalin protein encoded by the expression vector pGEX3X4.3.526, and an intemalin peptide having the amino acid sequence represented by SEQ ID NO:3.

A negative control solution of the present invention can comprise a solution that is known not to bind to an anti-intemalin antibody of the present invention. As such, immunocomplexes do not form using negative control solutions. A negative control solution can comprise a solution having compounds essentially incapable of binding to an anti-intemalin antibody of the present invention, such as Listeria cells having no intemalin, or a solution having no compounds contained therein (e.g., saline + bovine serum albumin only) . Preferred negative control solutions include, but are not limited to phosphate buffered saline solutions with or without irrelevant proteins (i.e., non-internalin protein), tris buffered saline with or without irrelevant proteins, and parallel samples known not to contain intemalin. A negative control solution of the present invention can also include a solution having antibodies essentially incapable of binding to intemalin, or a solution having substantially no antibodies contained therein. Particularly preferred negative control solutions include solutions containing antibodies is the pre-immune serum taken from the animal from which a particular anti-internalin antibody was derived, which represents the antibody repertoire present in the animal prior to immunization and solutions containing antibodies having the same isotype as the anti- internalin antibody used in a screening assay but which does not bind to intemalin.

The conditions under which an anti-internalin antibody of the present invention is immunoreacted with intemalin in a sample are conditions in which the antibody and the intemalin can associate in a specific manner to form an immunocomplex. Such conditions include, for example, an effective incubation temperature to encourage association of the proteins but that does not result in degradation of the proteins, an effective incubation time to allow optimal formation of immunocomplexes, and an effective incubation buffer in which the structural integrity of the proteins is maintained. Each aforementioned condition can vary significantly depending upon various well known parameters such as the concentration, isotype and specificity of the antibody used, and the type of sample being tested. A preferred incubation temperature for performing an immunoreaction of the present invention ranges from about 1°C to about 45^βC, more preferably from about 3°C to about 40^βC, and even more preferably from about 4°C to about 37°C. Suitable incubation times can vary with the temperature at which the immunoreaction is performed. For example, the incubation time can be decreased as the incubation temperature is raised. A preferred incubation time for an immunoreaction of the present invention ranges from about 2 hours to about 24 hours if the incubation is performed at a temperature ranging from about 4°C to about 20°C (i.e., room temperature), and from about 1 hour to 12 hours if the incubation is performed at a temperature ranging from about 25°C to about 40°C. A particularly preferred incubation time is about 1-3 hours if the incubation is performed at about 37°C. An effective incubation buffer is an aqueous solution containing buffering compounds which adjust the solution to a suitable ionic strength to encourage an immunoreaction. Examples of incubation buffers of the present invention include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, Hank's solution and other aqueous physiologically balanced solutions. Particularly preferred buffers of the present invention include phosphate buffered saline solutions with or without irrelevant proteins (i.e., non-intemalin protein) and tris buffered saline with or without irrelevant proteins.

In one embodiment, a screening method of the present invention is capable of detecting pathogenic microorganisms that are dead or dying. As such, a screening method of the present invention does not include the step of expanding a population of microorganisms contained in the sample.

In one embodiment, a screening method of the present invention comprises a first step of coating one or more portions of a solid substrate with a suitable amount of a first layer comprising an anti-internalin antibody of the present invention or a sample to be tested using the present invention, and of coating one or more other portions of the solid substrate with a suitable amount of a positive and/or a negative control solution. Suitable substrates of the present invention include, but are not limited to, plastic, nitrocellulose, filters, glass, latex, paper, sepharose, agarose and liposomes. A preferred substrate can include an ELISA plate, a radioimmunoassay plate, a dipstick, agarose beads, plastic beads, and/or fliters. A more preferred solid substrate includes an ELISA plate, a dipstick and a radioimmunoassay plate, with an ELISA plate being even more preferred.

The conditions under which a first layer and a control solution of the present invention are immobilized on a substrate are conditions in which the optimal amount of protein can irreversibly associate with the substrate. Such conditions include, for example, an effective temperature at which a protein can bind to the substrate without being degraded, an effective incubation time to optimize immobilization of a protein to a substrate, and an effective buffer which maintains the structurally integrity of a protein. A preferred temperature for immobilizing a first layer and a control solution of the present invention to a substrate ranges from about 4°C to about 45°C, more preferred from about 15°C to about 40°C, and even more preferred from about 20°C to about 37^βC. Incubation times can vary significantly depending on the temperature used. A preferred incubation time ranges from about 3 hours to about 24 hours if the incubation is performed at a temperature ranging from about 4°C to about 20^βC (i.e., room temperature) , and from about 1 hour to 12 hours if the incubation is performed at a temperature ranging from about 30°C to about 40°C. A particularly preferred incubation time is about 1-3 hours if the incubation is performed at about 37°C. An effective buffer for immobilizing a first layer and a control solution of the present invention to a substrate is an aqueous solution containing buffering compounds which adjust the solution to a suitable ionic strength to maintain the structural integrity of the protein but not containing any additional proteins which could interfere with the binding to the substrate of the antibody, sample or control solution. Examples of such buffers include, but are not limited to water, phosphate buffered saline, Ringer's solution, dextrose solution, Hank's solution and other aqueous physiologically balanced solutions. Particularly preferred buffers of the present invention include phosphate buffered saline with or without irrelevant proteins and tris buffered saline with or without irrelevant proteins.

Following the foregoing immobilization step, excess amounts of the first layer and control solution are washed from the substrate. A second step is then performed comprising contacting the substrate with a blocking solution having compounds incapable of binding to a compound used to detect the presence of immunocomplexes (described in detail below) . Suitable compounds of a blocking solution include, but are not limited to bovine serum albumin, dissolved powdered milk and detergents such as Tween 20 (Sigma, St. Louis, MO) . Following the foregoing blocking step, excess amounts of the blocking solution are washed from the substrate. A third step is then performed comprising the immunoreaction step disclosed below.

The screening method of the present invention may further comprise determining the formation of an immunocomplex of the present invention using a compound capable of detecting immunocomplexes between an anti- internalin antibody and intemalin. A detection compound of the present invention can be any compound capable of being attached to a protein and capable of being detected. A detection compound presents or displays a suitable detectable tag which can include, for example, a radioisotope, an enzyme, a chromophore, a fluorescent compound and a chemiluminescent compound. Preferred detectable tags of the present invention include enzymes, with alkaline phosphatase and peroxidase being more preferred. Detection of the tag can be accomplished using a variety of well-known techniques, depending on the method. For example, an enzymatic assay, (e.g., use of alkaline phosphatase or horseradish peroxidase) often yields a colorimetric or fluorescent product that can be detected visually or by an instrument such as a densitometer, ellipsometer or a spectrophotometer. A detection compound may also include a carrier to which a detectable tag is attached. Such carriers can include, for example, an anti-internalin antibody; an antibody capable of binding to an anti-internalin antibody; a bacterial surface protein that binds to antibodies, such as Protein A or Protein G; a biotin-streptavidin or biotin-avidin coupled detection system; a cell that interacts with antibodies, such as a T cell or B cell or macrophage; an eukaryotic cell surface protein that binds to antibodies, such as an FC receptor; and a complement protein. A preferred carrier of the present invention includes, but is not limited to, an anti-internalin antibody; an antibody capable of binding to an anti-internalin antibody; a bacterial surface protein that binds to antibodies, such as Protein A or Protein G; and/or a biotin-streptavidin or biotin-avidin coupled detection system. A more preferred carrier includes an antibody capable of binding to an anti- internalin antibody; a bacterial surface protein that binds to antibodies, such as Protein A or Protein G; and/or a biotin-streptavidin or biotin-avidin coupled detection system. An even more preferred carrier includes an antibody capable of binding to an anti-internalin antibody. It is within the scope of the present invention that the amount of immunocomplex formation can be determined using one or more layers of detection compounds. For example, an untagged antibody can be bound to an anti-internalin antibody and the untagged antibody can then be bound by a tagged antibody.

The concentration of a detection compound used to detect an immunoreaction can vary significantly depending upon the efficiency with which the carrier associates during an immunoreaction and the presence of non- specifically associated carriers. A preferred concentration of tagged anti-internalin antibody useful for detecting immunocomplexes can range from about 0.1 micrograms (μg) of antibody per milliliter (ml) of buffer to about 1000 μg of antibody per ml of buffer, a more preferred concentration can range from about 1 μg of antibody per ml of buffer to about 10 μg of antibody per ml of buffer, and an even more preferred concentration can be about 1 μg of antibody per ml of buffer to about 5 μg of antibody per ml of buffer.

A preferred concentration of a tagged antibody capable of binding to an anti-internalin antibody can range from about 0.001 μg of antibody per ml of buffer to about 100 μg of antibody per ml of buffer, a more preferred concentration can range from about 0.005 μg of antibody per ml of buffer to about 50 μg of antibody per ml of buffer, and an even more preferred concentration can range from about 0.01 μg of antibody per ml of buffer to about 5 μg of antibody per ml of buffer.

A preferred concentration of Protein A or Protein G can range from about 0.1 μg of Protein A or Protein G per ml of buffer to about 100 μg of Protein A or Protein G per ml of buffer, a more preferred concentration can range from about 1 μg of Protein A or Protein G per ml of buffer to about 10 μg of Protein A or Protein G per ml of buffer, and an even more preferred concentration can be about 2 μg of Protein A or Protein G per ml of buffer. A preferred concentration of avidin added to a solution having biotinylated antibody can range from about 0.01 μg of avidin per ml of buffer to about 100 μg of avidin per ml of buffer, a more preferred concentration can range from about 0.1 μg of avidin per ml of buffer to about 10 μg of avidin per ml of buffer, and an even more preferred concentration can be about 1 μg of avidin per ml of buffer. Preferred methods to determine if a sample contains intemalin by measuring bound immunocomplexes, include immunoblot assays (e.g., Western blot) and plate immunoassays (e.g., ELISA and radioimmunoassays) . Preferred methods to determine if a sample contains intemalin by measuring unbound immunocomplexes, include immunofluorescent antibody assays (e.g., fluorescence activated cell sort (FACS) analysis) and immunoprecipitation assays (e.g., agglutination assays). A contaminated sample is identified by comparing the level or degree of immunocomplex formation when anti- internalin antibodies and intemalin contained in test samples are reacted, with the level of immunocomplex formation observed in control samples. As such, if a test sample results in immunocomplex formation greater than or equal to immunocomplex formation using a positive control sample, then the sample contains intemalin. Conversely, if a sample results in immunocomplex formation similar to immunocomplex formation using a negative control sample, then the sample does not contain intemalin. This testing method is useful in determining the presence of pathogenic Listeria in a sample since only pathogenic Listeria expresses intemalin on its surface.

It is within the scope of the present invention that a number of parameters can be optimized to increase the immunocomplex formation between an anti-internalin antibody of the present invention and intemalin, and the detection of such immunocomplexes. Changes in various parameters can increase the sensitivity and specificity of the screening assay. For example, the ratio of the antibody and the sample used in the immunoreaction step of the present invention can be varied, and different combinations of anti-internalin antibodies can be used for detection of intemalin. In addition to such modifications, the incubation conditions, buffers (ionic strength, pH) , de- lipidizing a sample and other assay parameters can be manipulated to improve the assay. All of these modifications to the embodiments described herein are deemed to be within the purview of the present invention.

A screening method of the present invention is capable of detecting intemalin on at least about 1 x IO⁶ Listeria cells per ml, preferably at least about 1 x IO⁴ Listeria per ml, and more preferably at least about 100 Listeria cells per ml.

A screening assay of the present invention is also capable of detecting soluble intemalin in a sample. The presence of soluble intemalin in a sample indicates that intemalin-bearing cells are or at least were present in the sample being tested.

One embodiment of the present invention is a kit useful for screening samples for intemalin. A kit of the present invention comprises an anti-internalin antibody of the present invention and a means for determining an immunoreaction between the antibody and intemalin. A compound for determining an immunoreaction between the antibody and intemalin can include a detection compound of the present invention as described in detail above. A kit of the present invention further comprises at least one control solution such as those disclosed herein. It is within the skill of one in the art to modify a kit of the present invention for detecting intemalin by, for example, adding to the kit means for collecting samples to be tested with a kit, such as cotton swabs, syringes or spatulas. A test kit of the present invention preferably contains anti- internalin antibody which is in lyophilized form or in solution.

In one embodiment, a kit of the present invention comprises: (1) a first anti-internalin antibody capable of binding to a first epitope on intemalin; (2) a second anti-internalin antibody capable of binding to a second epitope on intemalin that has an enzyme tag associated therewith; (3) a means for reacting the enzyme bound to the second anti-internalin antibody such that the enzyme produces an indicator (e.g., color) that is detectable by eye or by instrumentation. Preferably, the first anti- intemalin antibody is immobilized on a solid substrate, such as an ELISA plate, filter or dipstick and the second anti-internalin antibody is provided separate from the first anti-internalin antibody. Preferred anti-internalin antibodies for use with a kit of the present invention include the anti-internalin antibodies disclosed herein. Preferred enzyme tags for the present kit include, but are not limited to alkaline phosphatase, peroxidase and other enzymes capable of producing a color reaction. Suitable means for reacting with an enzyme include an enzyme substrate capable of being altered by the enzyme attached to the second anti-internalin antibody. A kit of the present invention can also include aqueous solutions necessary for inducing the enzyme reaction.

Another aspect of the present ivention is a wearable badge useful for detecting the presence of intemalin- bearing cells in the environment. A badge of the present invention comprises a solid substrate coated with an anti- intemalin antibody, which upon binding of intemalin, changes in a detectable form (e.g., visual). For example, elipsometric detection methods can be used in which an antigen binding to an antibody bound to a solid substrate causes the substrate to change color. Such techniques are well known by those of skill in the art and are incorparated by reference herein. Preferred anti-internalin antibodies for use with a badge of the present invention include the anti-internalin antibodies disclosed herein.

Another aspect of the present invention involves a method to remove an intemalin-bearing cell from a contaminated food product by contacting the food product with an antibody capable of binding to intemalin to form an immunocomplex and separating the immunocomplex from the food product. Preferred anti-internalin antibodies for use with a removal method of the present invention include the anti-internalin antibodies disclosed herein. Preferably, an anti-internalin antibody of the present invention is securely attached to a solid substrate. In one embodiment of a removal method of the present invention, a solid substrate over which aqueous food products can be passed is coated with an anti-internalin antibody of the present invention. Suitable solid substrates for purification of aqueous food products include, for example, mesh material, nylon filters, agarose beads, and plastic beads. Preferred substrates include mesh material and filters. The rate at which an aqueous food product is passed over a solid substrate depends upon the thickness of the food product and the type of solid substrate used. Such rates can be determined using methods known to those of skill in the art.

In another embodiment of a removal method of the present invention, a solid substrate which can be passed over a solid food product is coated with an anti-internalin antibody of the present invention. Suitable solid substrates for use with a removal purification of aqueous food products include, for example, strips of plastic arranged in a broom-like fashion, plastic film and paper. Another aspect of the present invention involves a method to inhibit the propagation of Listeria having intemalin associated therewith, by specifically regulating the invasion ability of Listeria into a host cell. The method comprises administering a therapeutic composition capable of inhibiting the uptake of intemalin-bearing cells by a host cell. As used herein, uptake refers to the active entry of an object into a host cell via receptor mediated endocytosis. As such, uptake of a cell having intemalin refers to the cellular driven endocytosis by a host cell of an intemalin-bearing cell due to the interaction of intemalin with an intemalin receptor on the host cell. A therapeutic composition of the present invention is particularly useful for preventing the infection of an animal by intemalin-bearing cells, preventing the propagation of an intemalin-bearing cell in an animal and treating an animal having a disease caused by infection of an intemalin-bearing cell. The invasion regulation method of the present invention is particularly useful for inhibiting the propagation of Listeria cells having intemalin. As such, a therapeutic composition is particularly useful for preventing the infection of an animal by Listeria, preventing the propagation of Listeria in an animal and treating an animal having listeriosis.

A variety of therapeutic compositions can be used to perform the invasion regulation method of the present invention. Such therapeutic compositions include, but are not limited to, at least a portion of an intemalin protein, an anti-intemalin antibody and an inhibitory compound of the present invention (described in detail below) . Preferred intemalin protein for use in a therapeutic composition includes intemalin protein encoded by at least a portion of the nucleic acid sequence represented by SEQ ID NO:l, GST-INL fusion intemalin protein encoded by the expression vector pGEX3X4.3.526, and the intemalin peptide having the amino acid sequence represented by SEQ ID NO:3. Preferred anti-internalin antibodies for use with a purification method of the present invention include the anti-internalin antibodies of the present invention disclosed herein. Preferably, the ability of intemalin and/or an anti-internalin antibody of the present invention to inhibit the binding of intemalin to the is measured by the ability of such therapeutic compositions to prevent preferably at least about 50%, more preferably at least about 75%, and even more preferably at least about 90% of intemalin protein from binding to cells having intemalin receptor.

An inhibitory compound capable of inhibiting the binding of intemalin to an intemalin receptor can be identified by a method including the steps of: (a) contacting an intemalin protein with a putative inhibitory compound to form a reaction mixture; (2) combining the reaction mixture with a cell having an intemalin receptor; and (3) determining if the putative compound inhibits binding of the intemalin protein to the cell. Preferably, the ability of the putative compound to inhibit the binding of intemalin to the cell is determined by measuring the amount of intemalin that binds to the cell. For example, inhibition of intemalin binding to intemalin receptor can be determined by measuring the ability of an inhibitory compound to prevent preferably at least about 50%, more preferably at least about 70%, and even more preferably at least about 90% of intemalin from binding to cells having intemalin receptor. The amount of intemalin bound to a cell can be determined by contacting the cell with an anti- internalin antibody of the present invention and using a detection compound of the present invention described herein. Preferred intemalin for use with a method to identify an inhibitory compound includes at least a portion of an intemalin that is capable of binding to an intemalin receptor, more preferred is a full-length intemalin protein, and even more preferred is an intemalin protein encoded by at least a portion of the nucleic acid sequence represented by SEQ ID NO:l. A preferred cell for use with a method to identify an inhibitory compound includes a cell capable of specifically binding to intemalin (i.e., having surface-bound intemalin receptor) . A more preferred cell includes, but is not limited to, J774 cell, a CaCo-2 cell or a human umbilical vein cells (HUVEC) .

Putative inhibitory compounds can include compounds which mimic the structure of at least the portion of intemalin capable of binding to an intemalin receptor, or conversely that mimic or conform to, at least the portion of an intemalin receptor capable of binding to intemalin. Such compounds can include, but are not limited to, protein-based compounds, carbohydrate-based compounds, natural organic compounds, synthetically derived organic compounds, and anti-idiotypic antibodies, or fragments thereof.

A therapeutic composition of the present invention can be administered to any animal, preferably to avians or mammals, and more preferably to chickens, turkeys, humans, horses, cattle, goat, sheep and other economic food animals.

A therapeutic composition of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions, and animal feed. Non-aqueous carriers, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc. , to which sterile water or saline can be added prior to administration. In order to inhibit the propagation of intemalin- bearing cells in an animal, a therapeutic composition of the present invention is administered to the animal in an effective manner such that the composition is capable of inhibiting the invasion of the intemalin-bearing cells into host cells. For example, an anti-internalin antibody or mimetope thereof, when administered to an animal in an effective manner, is able to bind to intemalin-bearing cells, thereby preventing the association of such intemalin with intemalin receptors on the surface of host cells. Similarly, an intemalin protein of the present invention, when administered to an animal in an effective manner, is able to bind to intemalin receptors on the surface of host cells, thereby preventing the association of the intemalin receptors with intemalin on the surface of the intemalin-bearing cells.

Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art depending upon a variety of variables, including the animal to be treated and the stage of infection. A suitable single dose is a dose that is capable of substantially inhibiting the propagation of intemalin-bearing cells in an animal when administered one or more times over a suitable time period. A preferred single dose of an intemalin protein ranges from about 0.1 μg to about 100 milligrams (mg) of a therapeutic composition per individual, more preferred ranges being from about 0.5 μg to about 50 mg of a therapeutic composition per individual, and even more preferred ranges being from about 1 μg to about 10 mg of a therapeutic composition per individual. A preferred single dose of an anti-internalin antibody therapeutic composition ranges from about 1 μg to about 100 mg of the therapeutic composition per kilogram body weight of the animal, more preferred ranges being from about 0.5 μg to about 50 mg of a therapeutic composition per kilogram body weight of the animal, and even more preferred ranges being from about 1 μg to about 10 mg per kilogram body weight of the animal. A preferred administration schedule is one in which the preferred amount the therapeutic composition is administered over a time period of from about 1 hour to about biweekly for 3 weeks. Modes of administration can include parenteral, topical, oral or local administration, such as intradermally or by aerosol. A further aspect of the present invention includes a method for delivering a reagent to a desired site in an animal using a target vehicle comprising an intemalin protein joined to at least one heterologous compound. A target vehicle of the present invention is capable of delivering a desired compound in a site specific manner, in particular, to a cell bearing an intemalin receptor. A heterologous compound of the present invention comprises an intemalin protein of the present invention joined to a heterologous compound to be delivered to form a chimeric molecule. A preferred heterologous compound of the present invention includes, but is not limited to, an antigen; a drug, such as antibiotics, anti-neoplastic drugs; a marker; a hormone; an antibody; a cytokine; and/or a growth factor. A more preferred heterologous compound includes a protein, a peptide, a toxin, a dye, a microbial agent, and/or an inert particle. An even more preferred heterologous compound includes antibiotics, anti-neoplasties, hormones, cytokines and/or growth factors.

A target vehicle of the present invention can be produced by direct association of a heterologous compound to an intemalin protein. An intemalin protein can be non-covalently associated with a heterologous compound by, for example, mixing the two components, with the any portion of the intemalin protein that does not interfere with the ability of the intemalin protein to bind to an intemalin receptor. An intemalin protein can also be covalently associated covalently to a heterologous compound by several methods including, for example, glutaraldehyde linkage, photoaffinity labelling, treatment with carbodiimides, treatment with chemicals capable of linking di-sulfide bonds, and treatment with other cross-linking chemicals standard in the art. An intracellular target vehicle of the present invention can also be produced by ligating a nucleic acid sequence encoding a heterologous compound to a nucleic acid sequence encoding at least a portion of an intemalin protein using, for example, recombinant DNA techniques that are standard in the art. An oligonucleotide which encodes a desired peptide is synthesized using known codons for the amino acid sequence, preferably those codons which have preferred utilization in the organism used for expression. Such a nucleic acid molecule comprising two or more nucleic acid domains are joined together in such a manner that the resulting nucleic acid molecule is expressed as a chimeric compound containing at least a portion of an intemalin protein, preferably one that has a strong affinity for an intemalin receptor and a compound to be delivered. Examples of an intracellular target vehicle nucleic acid molecule include, but are not limited to, a nucleic acid sequence encoding a full-length intemalin protein ligated to: a nucleic acid sequence encoding an antigenic peptide capable of binding to a major histocompatibility complex (MHC) molecule; and a nucleic acid sequence encoding a peptide capable of regulating normal cellular function, such as protein secretion, cellular signal transduction or RNA transcription.

For a target vehicle of the present invention, the dose will vary according to, for example, the particular complex, the manner of administration, the particular purpose for the delivery (e.g., treatment of disease or delivery of an imaging reagent) , the overall health and condition of the recipient and the judgement of the physician or technician administering the target vehicle. Acceptable protocols to administer target vehicles in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. Preferred dosages for target vehicles of the present invention range fro about 1 nanogram (ng) to about 100 mg of protein per individual, more preferred dosages range from about 5 ng to about 10 mg of protein per individual, and even more preferred dosages range from about 10 ng to about 1 mg of protein per individual.

A target vehicle of the present invention can be administered to an animal using a variety of methods. Such delivery methods can include parenteral, topical, oral or local administration, such as intradermally or by aerosol. A target vehicle can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration to the intestinal region of an animal include powder, tablets, pills and capsules. Preferred delivery methods for a target vehicle of the present invention include intravenous administration, local administration by, for example, injection, intradermal injection, intramuscular injection and inhalation. For particular modes of delivery, a target vehicle of the present invention can be formulated in an excipient of the present invention. A target vehicle of the present invention can be administered to any animal, preferably to mammals, and more preferably to humans.

Another aspect of the present invention is directed to a vaccine capable of specifically inducing a Class I restricted immune response in an animal. According to the present invention, a Class I restricted immune response refers to an immune response initiated by the interaction of a Class I MHC protein bound to an antigenic peptide with a T cell receptor. As used herein, an antigen refers to a foreign compound capable of inducing an immune response. Antigenic peptides are produced by proteolysis of antigens prior to binding of the peptide to an MHC protein. Class I MHC proteins typically bind to antigenic peptides derived from proteins actively synthesized by the same cell expressing the Class I protein. In contrast. Class II MHC proteins typically bind to antigenic peptides derived from exogenous protein that enter a cell's endocytic pathway. Intracellular trafficking permits an antigenic peptide to become associated with an MHC protein. The resulting MHC- peptide complex then travels to the surface of the cell where it is available for interaction with a T Cell receptor (TCR) .

It is known that Class I proteins bind to antigenic peptides in the endoplasmic reticulum and that Class II proteins bind to antigenic peptides in an endocytic vesicle. Intracellularly-derived proteins destined for binding to Class I proteins are proteolytically cleaved into antigenic peptides in the cytoplasm of the cell. The antigenic peptide is then bound by a proteinaceous transporter complex which transports the peptide into the endoplasmic reticulum. The antigenic peptide is then available to bind to a Class I protein.

Typically, exogenous antigens enter the endocytic pathway of a cell and remain in a vesicular compartment in which the antigens are cleaved into antigenic peptides. Class II proteins bind to the antigenic peptide when the vesicle containing the peptide fuses with the vesicle containing the Class II proteins. As such, exogenous antigens typically do not enter the cellular compartment containing Class I proteins and, therefore, do not bind to Class I proteins. Thus, in order to target an exogenous antigen to a Class I pathway the antigen must be able to enter a cell, escape the endocytic pathway and enter the endoplasmic reticulum of the cell. The present invention provides for a vaccine capable of entering a host cell and escaping from a membrane-bound cytoplasmic vesicle within the cell cytoplasm. In one embodiment, a vaccine of the present invention comprises a carrier cell expressing intemalin, listeriolysin O and a heterologous antigen. Suitable carrier cells of the present invention comprise non-pathogenic cells (naturally occurring and/or rendered non-pathogenic) . As such, Listeria monocytogenes is not a preferred carrier cell of the present invention. Moreover, Listeria monocytogenes has been shown to be a primary pathogen in mice when about 2 x IO⁴ cells were administered intravenously. Carrier cells of the present invention include, but are not limited to non-pathogenic bacteria, fungi, insect, plant and mammalian cells. Suitable carrier cells include bacterial cells that are part of the natural microflora of an animal to which the vaccine is to be administered. Preferred carrier cells of the present invention include attenuated pathogenic bacteria, E . coli , various species of Bacillus , various species of Pseudomonas , various species of Salmonella , various species of Mycobacterium , vaccinia virus, adenovirus and adenoassociated virus.

According to the present invention, a carrier cell is transformed with at least one nucleic acid molecule encoding intemalin protein and a heterologous antigen. A preferred intemalin nucleic acid molecule comprises at least a portion of the nucleic acid sequence represented by SEQ ID NO:l. A heterologous antigen of the present invention refers to an antigen that is not naturally expressed by the carrier cell expressing the antigen. A heterologous antigen can be a protein capable of being secreted from a carrier cell (i.e., having secretory signals) or capable of being surface-bound. Preferred heterologous antigens of the present invention are antigens capable of being secreted from the carrier cell. More preferred heterologous antigens are chimeric proteins comprising an antigenic peptide epitope joined to a protein that is known to be secreted by a cell. For example, an antigenic peptide epitope can be joined to the listeriolysin O which is known to be secreted from Listeria monocytogenes cells. A heterologous antigen can include any antigen which is capable of producing peptides capable of binding to an MHC Class I molecule. Preferred heterologous antigens include antigens found on infectious pathogens such as bacteria, virus, parasites and fungi.

Construction of desired expression vectors containing the nucleic acid molecules of the present invention can be performed by methods known to those skilled in the art for expression in a carrier cell. If the carrier cell is a prokaryotic cell, plasmids are used that contain replication sites and control sequences derived from a species compatible with the carrier cell. Control sequences can include, but are not limited to promoters, operators, enhancers, ribosome binding sites, and Shine-Dalgamo sequences. If the carrier cell is a eukaryotic cell, plasmids are used that contain promoters derived from appropriate eukaryotic genes. Useful mammalian promoters include early and late promoters from SV40 or other viral promoters such as those derived from baculovirus, polyoma virus, adenovirus, bovine papilloma virus or avian sarcoma virus. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in a carrier cell of the present invention including bacterial, yeast, other fungal, and mammalian cells.

An expression system can be constructed from any of the foregoing control elements operatively linked to the nucleic acid sequences of the present invention using methods known to those of skilled in the art. (see, for example, Sambrook et al., ibid . )

Another embodiment of a vaccine of the present invention comprises a liposome or micelle having intemalin, hemolysin and an antigen. Liposomes and micelles can be prepared using methods standard in the art. For example, multilamellar vesicles can be produced by dissolving lipids in a suitable organic solvent and drying the lipids under vacuum to form a thin lipid film. The film can be covered with an aqueous solution containing intemalin, he-colysin and an antigen, and allowed to hydrate. Immunization protocols using a vaccine of the present invention can vary according to the antigenic complex used and the mode of administration used. Preferred modes of administration include intravenous administration, local administration, oral and aerosol. Doses will vary according to the size of the recipient of the vaccine and/or the disease being treated.

The following experimental results are provided for purposes of illustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1

The following example describes the cloning of the intemalin gene from Listeria monocytogenes . 1. The isolation of Listeria monocytogenes genomic DNA.

Listeria monocytogenes genomic DNA was prepared by the following method. Listeria monocytogenes bacteria were incubated overnight in a 37°C shaking water bath in 100 ml of tryptose-peptone broth (BRL, Gaithersburg, MD) and harvested by centrifugation at 11,000 X g for 10 min at

4^βC. The pellet was washed three times by centrifugation in

5 ml of 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0.

The washed pellet was resuspended in 10 ml of 0.01 M sodium phosphate buffer, pH 7.0, plus 20% (W/V) sucrose and 2.5 mg/ml of lysozyme (Sigma, St. Louis, MO) and incubated in a 37°C shaking water bath for 1 h. Following incubation, 10 ml of 10 mM tris-HCl, pH 8.0, plus 1 mM EDTA, 1% SDS and 1 mg of proteinase K/ml was added to the mixture and incubated for 2 h in a 55°C water bath. The enzyme digest was transferred to two polypropylene screw cap tubes, an equal volume of 1:1 phenol:CHC1₃ was added, mixed by vortexing and centrifuged at 5,000 X g for 10 min at room temperature. The phenol:chloroform extraction was repeated two times followed by two extractions with 100% chloroform. The aqueous phase was brought to 0.3 M sodium acetate and the DNA precipitated with 2.5 volumes of ice cold 100% ethanol followed by reprecipitation with 70% ethanol. The DNA pellet was resuspended in 0.5 ml of 10 mM tris, pH 8.0, plus 1 mM EDTA (TE) , 1% SDS and l mg/ml of proteinase K, and the proteinase K digestion performed as above. The final DNA pellet was resuspended in TE plus 1 μg/ml RNAase. 2. Cloning of the intemalin gene by the polymerase chain reaction.

The intemalin gene was cloned from the genomic DNA using the polymerase chain reaction (PCR) . Oligonucleotide probes for PCR were synthesized using an Applied Biosystems model 392 DNA synthesizer and standard methods. One oligonucleotide was homologous to the amino terminus of intemalin and incorporated a recognition site for BamHI (5ΑTGGATCCTGAAGACGGTCTTAGGAAA3' ; SEQ ID NO:4). The other oligonucleotide was complimentary to the carboxyl terminus and termination codon of intemalin (5ΑTGAATTCCTATTTACTA3'; SEQ ID NO:5). PCR was performed using a Perkin-Elmer DNA Thermal Cycler set for 2 min at 94°C to denature, 2 min at 50°C to anneal and 2 min at 72°C for extension for 30 cycles and using TAQ DNA polymerase (Perkin-Elmer, Norwalk, CT) in 15 mM MgCl₂. The PCR product was brought to 0.3 M sodium acetate and extracted with 1:1 phenol:chloroform, precipitated with ethanol and the precipitate washed once with 70% ethanol to remove the sodium acetate. The PCR amplified intemalin gene was resuspended in TE buffer.

3. Cloning of the intemalin gene into the pCRII vector.

The intemalin gene was ligated directly into the cloning plasmid vector pCRII (Invitrogen, San Diego, CA) which has over-hanging thymidine residues at either 3' end of the open cloning site within its polylinker. Ligation was performed overnight in a 15°C water bath using T4 DNA ligase (Perkin-Elmer, Norwalk, CT) . Controls included PCR product or the cloning vector alone incubated in the presence or absence of ligase, incubated with DH5α. Five μl of ligation mixture was incubated for 1 h on ice with 200 μl of competent E . coli DH5α, heat shocked for 1 min in a 42°C water bath and 800 ul of LB broth was added followed by incubation for 1 h in a 37°C shaking incubator. Various dilutions of the cultures were plated on LB agar plus 50 μg/ml of ampicillin, 1 mM IPTG and 10 mM X-gal (5 Prime-3 Prime, Boulder, CO) and incubated overnight at 37°C. Positive colonies, resistant to ampicillin were selected for on the basis of alpha complementation of the lac gene, transferred to 5 ml of LB-ampicillin broth and incubated overnight in a 37°C shaking incubator. DNA plasmid mini- preps were screened for insertion of and the appropriate orientation of the intemalin gene in pCRII by restriction mapping using Bam HI, Eco RI and Hind III and the restriction digests were viewed on 0.7% agarose-ethidium bromide gels. A positive clone designated pCRII4.3 was found to contain the intemalin gene inserted in the 3' to 5' direction. The clone was cultivated overnight in 1 liter of TB-amp and DNA plasmid maxi-preps were made. pCRII4.3 DNA was digested with Bam-HI and the intemalin gene was separated by electrophoresis on 0.7% agarose- ethidium bromide, and isolation of the band containing the intemalin gene was removed from the gel and electroeluted at 100 V for 1 h in 0.5 trisborate buffer, pH 7.4. Eluted DNA was precipitated with ethanol as described above and resuspended in TE-RNAase. 4. Cloning the Bam-HI digested intemalin gene into the pGEX3X expression vector.

The Bam-HI DNA fragment encoding intemalin was ligated into the expression vector pGEX3X (Pharmacia, Stockholm, Sweden) using the ligation techniques described above. The pGEX3X expression system permits cloning of a desired DNA immediately downstream of a recognition site for factor Xa protease which is located within the gene encoding glutathione S-transferase (GST) . Thus, the GST- rlNL fusion polypeptide may be immobilized on sepahrose- glutathione (Pharmacia, Stockholm, Sweden) and the intemalin protein cut from the matrix with the proteolytic enzyme factor Xa (Boehringer-Mannheim, Germany) . Competent DH5α were transfected with the ligation mixtures and positive colonies selected for on LB-amp after incubation overnight at 37°C. Plasmid DNA mini-preps were screened by restriction enzyme digestion for the presence and orientation of the intemalin gene. One clone, pGEX3X4.3.526, was selected which contained the intemalin gene having the correct orientation and the correct nucleotide sequence as determined by standard nucleotide sequencing methods using the pGEX3X sequencing primer 5'GCATGGCCTTTGCAGGG (SEQ ID NO:6). The intemalin gene was found to be in-frame with GST and the factor Xa restriction site.

Example 2

The following example describes the production of intemalin fusion protein and recombinant intemalin. Intemalin fusion protein was produced by the following method. DH5α transformed with pGEX3X4.3.526 were incubated in 100 ml of TB-amp overnight in a 37°C shaking incubator. The overnight culture was transferred to 400 ml of TB-amp in a 2 liter Erlenmeyer flask and incubated for 2 h in a 37°C shaking incubator. Sterile 1 mM IPTG (5 Prime-3 Prime, Boulder, CO) was added to the culture and incubated 16-18 h in a 37°C shaking incubator. Bacteria were centrifuged at 3,000 X g and the pellets from 250 ml of culture were resuspended in 10 ml of lysis buffer (TNE with 10 mg/ml lysozyme) and incubated in a shaking water bath at 37°C for 1 h. To this mixture, 20 mg of sodium deoxycholate was added followed by incubation in a 37°C shaking water bath for 1 h. One hundred μl of 1 mg/ml DNAase I was added to the lysate for an additional 30 min of incubation at room temperature. The lysate was centrifuged at 3,000 X g for 30 min at room temperature and the supernatant discarded. The pellet was resuspended in 10 ml/250 ml of pelted culture of sterile 0.1 M tris, pH 8.4 plus 1% Triton X 100 and 6 M urea. The pellets were extracted for 2 h at 4°C on a rocking platform. The extract was centrifuged at 3,000 X g for 30 min at room temperature and the pellet discarded. The intemalin fusion protein containing supernatant was dialyzed overnight at 4°C against double distilled water which resulted in a cloudy precipitate in the dialysate. The dialysate was centrifuged at 3,000 X g for 30 min at room temperature and the pellet discarded. Recombinant intemalin protein was produced by the following method. Ten mg of sephadexglutathione (Sigma, St. Louis, MO) was washed three times with sterile PBS by centrifugation for 5 min at 10,000 X g and added to 1 ml of the 6 M urea dialysate plus 1% Triton X 100. This was incubated overnight at 4°C on a rocking platform. The affinity matrix was microcentrifuged for 5 min at 10,000 X g, the supernatant was removed and frozen at -20°C. The affinity matrix was washed three times in sterile PBS and resuspended in 1 ml of PBS plus 1% Triton X 100, 1 mM CaCl₂ and 5 μg of factor Xa. The enzyme digest was incubated overnight at 4°C on a rocking platform. The digest was microcentrifuged for 5 min at 10,000 X g and the supernatant transferred to a new eppindorf tube. Ten μl of enzyme inhibitor cocktail (1 mg/ml each of: α-1 anti trypsin; leupeptin and aprotinin) was added per ml of supernatant and stored at -20°C. Recombinant intemalin protein (rINL) was separated on 10% SDS-PAGE mini-gels and visualized by staining either with Coomassie blue dye or by silver staining (Bio Rad, CA) .

Recombinant intemalin was identified by ELISA and Western immunoblot analysis using rabbit polyclonal antibodies developed by immunizing rabbits with an 18 mer peptide CNNQIADITPLANLTNLT which was synthesized and coupled to KLH by the malamide reaction. The peptide is referred to herein as KLH3054.

Example 3 The following example describes the production of antibodies capable of binding to recombinant intemalin protein, intemalin fusion protein and intemalin peptide KLH3054.

One mg of recombinant intemalin (rINL) was cut from a 10% SDS-PAGE mini-gel and emulsified with a Dounce homogenizer in 1 ml of sterile PBS. KLH3054 peptide or acrylamide-recombinant intemalin (herein referred to as ACrlNL) were separately emulsified in an equivalent volume of Freund's complete adjuvant (FCA) . Rabbits were immunized by subcutaneous injection of FCA and 250 μg of antigen. This was followed with bi-weekly injections of 250 μg of corresponding antigen emulsified in an equivalent amount of Incomplete Freund's adjuvant. Following immunization, rabbits were bled and their serum tested for the presence of anti-internalin fusion protein, anti-ACrlNL antibodies and anti-internalin peptide antibodies by ELISA and by Western immunoblot analysis. Such polyclonal antibodies are referred to herein as anti-GST-INL antibodies, anti- ACrlNL antibodies and anti-KLH3054 antibodies, respectively.

ELISA assays were performed by incubating 50 μl of 1 mg/ml of either rINL or intemalin fusion protein in flat bottomed 96 well microtiter plates overnight at 4°C. KLH3054 peptide was coupled to the plastic dish by incubating 50 μl of 1 mg/ml of KLH3054 with 50 μl of 1.5% glutaraldehyde for 1 h at room temperature. All ELISA plates were washed one time with sterile PBS-tween 20 (PBST) and incubated overnight with 100 μl of PBS plus 1% BSA (block) . Plates were washed 3 times with PBST, 100 μl of rabbit anti-serum added to the first well at a 1:100 dilution in block and diluted two-fold serially to well number 8. Wells 9 and 10 served as secondary antibody only controls and wells 11 and 12 served as block only controls. Anti-KLH3054 or anti-ACrlNL antibody were added to the plates and incubated for 45 min at 37°C and then 1 h at 4°C. The plates were washed 3 times with PBST and the secondary antibody, goat anti-rabbit IgG coupled to HRP at a 1:3,000 dilution in block, was added to wells 1 through 10 with block only in wells 11-12. The plates were incubated for 30 min at 37°C and then 30 min at 4°C. The plates were washed 3 times with PBST, developed using a BioRad HRP kit (BioRad, CA) , the reaction stopped with 2% oxalic acid and the plates read at 402 nm. Table 2 shows a representative titration of antiserum from the rabbits immunized either with acrylamide recombinant intemalin (ACrlNL) or KLH3054, and bled after the fourth immunization. Serum from rabbits injected with ACrlNL had anti-KLH3054 antibodies which could be detected at a titer in excess of 100,000. IgG from this antiserum was affinity purified using Affigel coupled to Protein A (Sigma, St. Louis, MO) according to the manufacturers specifications and designated anti-ACrlNL antibodies hereafter. ELISA of anti-ACrlNL antibodies for both intemalin fusion protein and recombinant intemalin (rINL) were equally positive demonstrating that the rabbit had responded specifically to recombinant intemalin immunization.

Table 2. Titration of rabbit anti-ACrlNL and anti-KLH3054 antisera against peptide KLH3054 by ELISA.

EXPERIMENTAL TITER Rabbit pre-immune serum, full strength 0

Secondary antiserum only 0

Block only 0

Anti-KLH3054, bleed #4 160,000

Anti-ACrlNL, bleed #4 160,000 Anti-ACrlNL, affinity purified IgGl 100,000

For Western immunoblot analysis, intemalin fusion protein, rINL or KLH3054 peptide were separated on 10% SDS- PAGE mini-gels and electroblotted to nitrocellulose for 1 h at 80 V. For preliminary studies these proteins and the KLH3054 peptide were electrophoresed as a single large band down the entire SDS-PAGE gel, transferred together to the nitrocellulose paper and the paper cut into strips. Thus, individual strip blots could be tested using small volumes of antiserum in order to conserve reagents. Blots were washed once in PBST and incubated overnight at 4°C on a Nutator in plastic bags containing 20 ml of PBST plus 2% non-fat dry milk block. Blots were washed 3 times in PBST and transferred to a new plastic bag containing 20 ml of milk block plus anti-ACrlNL antisera to be tested. The optimal titer for the antiserum in Western blot analysis was found to be 1:200-1:10,000. Blots plus anti-ACrlNL antibodies were incubated at 4°C on a rocking platform for 2 h, washed 3 times in PBST and then incubated at room temperature in 20 ml of secondary antibody, goat anti- rabbit-HRP antibody diluted 1:300 in block, washed and developed using a BioRad HRP kit. Strip blots probed with the rabbit anti-ACrlNL IgGl demonstrated a specific reaction for rINL, intemalin fusion protein and for the KLH3054 consensus sequence peptide. The results indicate that the immunization of rabbits with ACrlNL resulted in the production of specific anti-rlNL antibodies which bound both the rINL and intemalin fusion proteins and the consensus repeat sequence of intemalin (represented by KLH3054) . Based on this result, the ELISA and Western immunoblot analysis could be used to screen the immune responses of mice immunized with ACrlNL and following immunization these tests could be used to screen the production anti-internalin monoclonal antibodies by clones isolated from immunized mice.

Anti-internalin monoclonal antibodies (INL mAbs) were prepared as follows. Male BALB/c mice, 6-8 weeks old were injected subcutaneously with 100 μg of ACrlNL in complete Freund's adjuvant. Four weeks later the mice were boosted by intraperitoneal injection of 100 μg of ACrlNL in incomplete Freund's adjuvant. Booster injections were repeated biweekly and beginning on the second biweekly injection mice were bled from the tail vein and their antiserum tested by ELISA and Western im unoblots for the presence of anti-ACrlNL antibodies. At approximately ten weeks after the primary immunization, mice displayed serum ELISA titers to recombinant intemalin at approximately 1:3000 and were positive for binding to recombinant intemalin and the 3054 peptide on Western immunoblots. Mice were sacrificed, their spleens removed and splenocytes were fused with myeloma fusion partners, selected based upon standard methods and sub cloned. Following sub cloning, the supematants from clones which grew well were tested for the presence of anti-rlNL mAbs by ELISA and Western immunoblot analysis. Positive clones were subcloned to single cells, grown to the appropriate number and the supematants from wells containing these clones were re-tested.

Multiple hybridomas producing antibodies capable of binding to KLH3054 peptide, rINL and intemalin fusion protein were identified and are referred to as hybridomas 292; 1847; 1339; 804; 1630; 1360; 1835; 1042; 469; and 678. These 10 positive clones which reacted with the KLH3054 peptide, intemalin fusion protein and rINL by ELISA and by Western immunoblot analysis were incubated in RPMI plus 15% fetal bovine serum. Cells from cultivation were preserved on liquid nitrogen in fetal calf serum containing 10% DMSO and antibiotics. Cells removed from storage could be batch cultivated for the production of mAbs from culture fluid and for the isolation of hybridoma cells for ascites production. Ascitic fluid was prepared by injecting hybridomas intraperitoneally into BALB/c nu+/nu+ Mice (Jackson Laboratories, Bar Harbor, ME) and harvested several weeks after injection. The immunoglobulin G fractions were isolated from the ascitic fluid by affinity chromatography on Affigel-protein A (Sigma, St. Louis, MO) . Hybridoma clones 292, 804, 1847, 1360 and 1339 were IgGl mAbs whereas clone 1630 was an IgG2a subclass Ab.

Example 4

The following example describes detection of intemalin on Listeria cells using the antibodies described in Example 3.

1. Staining of Listeria with anti-internalin antibodies. The objective of these experiments was to determine whether rabbit anti-internalin antibodies or mouse anti- internalin mAbs was able to detect intemalin on the surface of various Listeria . Two studies were performed, one comparing and contrasting the reactivity of anti- internalin antibody with various mutant strains of Listeria and a second study evaluating the expression of intemalin on various Listeria species. Stained cells were evaluated by visual fluorescence microscopic examination and by flow cytometry. In all instances negative staining controls included primary antibody staining with either rabbit gamma globulin as a control for anti-ACrlNL polyclonal antibodies or two isotype specific mAbs as a control for the anti- intemalin mAbs. The isotype controls were mAbs B344.1 which is a mouse IgGl against Staphylococcus enterotoxin B and MR12-5 which is a mouse IgGl against the Vβl region of the T cell receptor. a. Fluorescence staining of mutant Listeria by anti- internalin antibodies. The following strains of Listeria were used in this experiment: Wild type Listeria monocytogenes designated MP- 10; wild type L . innocua which does not express intemalin and is non-invasive for CaCo-2 cells; Bug 8, a transposon mutant of wild type Listeria monocytogenes which fails to express intemalin and fails to invade CaCo-2 cells; and L . innocua +, designated Li+, which is an intemalin negative saprophytic species of Listeria transformed with a plasmid encoding the intemalin gene which enables the expression of intemalin on the surface and which is invasive for CaCo-2 cells.

Bacteria were cultivated for 3 h at 37°C in a shaking water bath in the appropriate medium, harvested by centrifugation at 10,000 X g for 3 min in a microcentrifuge, washed 3 times by centrifugation with sterile PBS and resuspended in 1 ml of sterile PBS. Ten microliters of the appropriate culture was transferred to a new tube, and pelleted by centrifugation. The cell pellet was resuspended in ten ul of the appropriate antibody preparation or control antibody and incubated for

1 h at 37°C. Five hundred ul of 4% paraformaldehyde in PBS was added to each tube and incubated for 10 min at room temperature. The cells were spun at 10,000 X g for 3 min and washed once in sterile PBS. The cells were then resuspended in 100 μl of secondary antibody, CY3 conjugated donkey anti-mouse immunoglobulin or anti-rabbit immunoglobulin diluted 1:1000 in PBS, and the tubes incubated for 1 h at 37°C. The cells were washed 3 times with sterile PBS by centrifugation, resuspended in 20 μl of PBS and 10 μl was mounted on a glass slide and viewed using a Nikon fluorescence microscope. The remaining 10 μl of stained cells were transferred to 1 ml of PBS and analyzed using a Coulter model 751 flow cytometer at 514 nm. Table 3 shows that rabbit anti-ACrlNL antibody was able to detect intemalin on the surface of MP-10 and Li+ but failed to detect intemalin on the surface of the intemalin negative Bug 8 or on wild type L . innocua .

Table 3. Fluorescence staining of various Listeria species with anti-internalin antibodies.

MICROORGANISM STAINING REACTION

Anti-ACrlNL Rabbit IgG

MP-10 4+ negative

Bug 8 negative negative

L . innocua negative negative

Li+ 4+ negative

No fluorescence was detected when the various bacterial strains were stained CY3 conjugated donkey anti-rabbit immunoglobulin alone. Figure 1 shows a shift in the mean fluorescence intensity for MP-10 stained with anti-ACrlNL in comparison to its negative control with a mean intensity of 18, demonstrating a two-fold increase in the mean fluorescence intensity, to 34, for the stained wild type MP-10. Table 4 shows that the anti-internalin mAbs could also be detected on the surface of MP-10 and Li+ after being incubated in ascitic fluid for 1 h at 37°C. The various anti-internalin mAbs failed to react with either Bug 8, the intemalin knockout mutant, or with wild type L . innocua .

Table 4. Fluorescence staining of various Listeria species with anti intemalin mAbs.

MICROORGANISM STAINING REACTION TO mAb

292 804 1630

MP-10 4+ 3+ 4+

BUG 8 negative negative negative

L . innocua negative negative negative

Li+ 4+ 1+ 2+

In all instances primary staining with the isotype control mAbs were negative. Anti-internalin mAbs 804, 1339 and 1847 have not yet been evaluated. Figure 2 shows the shift in a mean fluorescence intensity of 20 for MP-10 stained with secondary antibody alone to a mean fluorescence intensity of 48 for MP-10 stained either with anti-internalin mAb 292 or 1630.

Together, these studies show that both the rabbit anti-ACrlNL as well as the anti-internalin mAbs specifically bound to wild type Listeria monocytogenes MP- 10 and the transformed Li+ which expresses intemalin but failed to bind to either wild type L. innocua or to the intemalin knockout mutant Bug 8. This study also demonstrates that the anti-internalin mAbs were able to detect the presence of intemalin on wild type MP-10 in a pattern similar to that seen for staining with polyclonal rabbit anti-ACrlNL antibodies. b. Fluorescence staining of various Listeria species with anti-internalin antibodies.

The objective of this study was to determine whether rabbit anti-ACrlNL antibodies or anti-internalin mAbs were able to bind to pathogenic versus non-invasive, non- pathogenic species of Listeria . Wild type Listeria monocytogenes MP-10, L . innocua (ATCC 33090), L . ivanovii (ATCC 19119), L . seeligerii (ATCC 35967), L . welshimeri (ATCC 35897) and L . urrayi (ATCC 25401) were cultivated for 3 h in TPB at 37°C in a shaking water bath and stained as described above using anti-ACrlNL. Table 5 shows that the rabbit anti-ACrlNL antiserum as well as several of the anti-internalin mAbs were able to differentiate the invasive MP-10 wild type Listeria monocytogenes as well as the invasive species L . ivanovii . By comparison, non- invasive species failed to bind the anti-ACrlNL antibody suggesting that invasive Listeria species may express surface intemalin or an epitope which reacts with the anti-internalin antibody. Both specificity of staining and its absence were confirmed by flow cytometry. Table 5. Fluorescence staining of invasive and non- invasive Listeria species with rabbit anti-ACrlNL or with anti-internalin mAb.

MICROORGANISM STAINING REACTION

ANTI-ACrlNL 292 1630 SECONDARY ALONE

MP-10 4+ 3+ 3+ NEG.

L . innocua NEG. NEG. NEG. NEG.

L. ivanovii 4+ 3+ 3+ NEG.

L. seeligerii NEG. NEG. NEG. NEG.

L. welshii NEG. NEG. NEG. NEG.

L . murrayi NEG. NEG. NEG. NEG.

Figure 3 shows the shift in fluorescence intensity for L . ivanovii stained either with secondary antibody alone (mean intensity of 20) or with anti-internalin 292 (mean intensity of 52) or anti-internalin 1630 mAb (mean intensity of 48) .

Together these data show that both the rabbit anti-

ACrlNL antibodies and the anti-internalin mAbs were able to distinguish between invasive, pathogenic Listeria monocytogenes MP-10 and L . ivanovii and the non-invasive, non-pathogenic, saprophytic Listeria species. c. The kinetics of intemalin expression on Listeria monocytogenes MP-10. The overall objective of this study was to use the rabbit anti-ACrlNL antibodies to follow the expression of intemalin on the surface of wild type Listeria monocytogenes MP-10 during growth of the bacteria in vitro.

MP-10 was cultivated in 100 ml of TPB and at various intervals an aliquot was removed and stained as above for the presence of intemalin using rabbit anti-ACrlNL antibodies. Table 6 shows that intemalin was expressed on the surface of MP-10 beginning at about mid-log of the growth curve and was uniformly expressed on the surface of MP-10 at the stationary phase of growth.

Table 6. The kinetics of intemalin expression on Listeria monocytogenes MP-10 as detected by anti-ACrlNL.

HOURS OF GROWTH ANTI-ACrlNL REACTION 0 negative 2 1+ 4 3+ 8 4+ 16 4+

d. Fluorescence staining of Listeria during invasion of CaCo-2 enterocytes and J774 macrophages.

The objective of this study was to determine whether intemalin is expressed on the surface of wild type MP-10 during infection of either CaCo-2 cells (ATCC CTB 37) or the macrophage like murine cell line J774 (ATCC TIB 67) . CaCo-2 cells were cultivated as described by Gailliard et al. (iJid.) whereas J774 cells were cultivated in RPMI 1640 supplemented with 10% fetal calf serum, penicillin, streptomycin and fungizone. Five hundred thousand cells were cultivated at 37°C in an humidified atmosphere containing 5% C0₂ as monolayers on sterile 35 mm coverslips. Listeria monocytogenes MP-10 was incubated overnight at 37°C in 5 ml of TPB in a shaking water bath and washed 3 times with sterile PBS by centrifugation. Cell monolayers were washed three times with sterile, serum free, RPMI 1640 then infected with MP-10 at a 10:1 multiplicity of infection. The infected monolayers were incubated at 37°C in an humidified atmosphere containing 5% C0₂. At various intervals after infection the monolayers plus bacteria were fixed with 4% paraformaldehyde in PBS, washed with RPMI 1640, stained with rabbit anti-ACrlNL antibodies and, as a secondary antibody, CY3 conjugated donkey anti-rabbit immunoglobulin. In both CaCo-2 and J774 cell lines the presence of intemalin positive MP-10 was detected throughout the cycle of infection. In both instances, MP- 10 was able to escape restriction within the cell phagosome where it entered the cytoplasm and polymerized actin as demonstrated by staining for actin on the surface of the bacteria. In parallel experiments, cell monolayers were also infected either with Bug 8 or Li+. The anti-ACrlNL antibody failed to detect Bug 8 within cells although intraphagosomal Bug 8 could be demonstrated in sparse numbers in some macrophages, possibly phagocytosed through a lectin-receptor mechanism. By comparison, anti-ACrlNL antibodies was able to detect intemalin positive Li+ within the phagosomes of both cell types. Thus, during the infection of permissive cells, intemalin was found on the surface of MP-10 throughout it's intracellular life cycle. Taken together, these data show that polyclonal rabbit anti-ACrlNL IgG antibodies and urine anti-internalin mAbs were able to detect the presence of intemalin on pathogenic, invasive Listeria monocytogenes . Intemalin was also detected on the surface of invasive L . ivanovii , however, intemalin was not detected on the surface of saprophytic and non-invasive Listeria species. Intemalin was expressed on the surface of Listeria monocytogenes beginning at approximately mid-log phase of in vitro growth and was found on bacteria in stationary growth phase. Intemalin could also be demonstrated on the surface of MP- 10 throughout its life cycle in both CaCo-2 and J774 cells. These experiments demonstrate the applicability of anti- internalin antibodies in the detection of pathogenic Listeria monocytogenes . Example 5

This example demonstrates that anti-internalin antibodies were capable of blocking the entry of Listeria monocytogenes into permissive cell lines.

CaCo-2 and J774 cells were cultivated as described in Example 4. Human umbilical vein cells (HUVEC, ATCC CRL 1730) were cultivated at 37°C in an humidified atmosphere containing 5% C0₂ in F-12K medium supplemented with 10% fetal bovine serum and growth factors (Sigma, St. Louis, MO) according to ATCC specifications. For all experiments, five hundred thousand cells were incubated in 24 well flat bottom culture dishes and washed 3 times in serum free balanced salts solution. Cells were resuspended in serum free culture medium and Listeria monocytogenes added to the cultures at an effector:target ratio (E:T) of 1:10. The cultures containing bacteria were then incubated at various intervals in the absence or presence of various concentrations of anti-intemalin antibody and lysed with sterile distilled water plus 10% saponin. Lysates were diluted serially ten-fold and plated on tryptic soy agar. Plates were incubated overnight at 37°C and the number of colony forming units (CFU) from anti-internalin antibody treated cells compared to that for untreated cells. Figure 4 shows the log CFU of MP-10 recovered after 2 h of incubation on CaCo-2 monolayers in media alone or in the presence of 10% normal mouse serum. Parallel cultures were treated with dilutions of either rabbit anti-ACrlNL antibodies or rabbit anti-KLH3054 antibodies. Treatment with either antibody resulted in significantly fewer numbers of MP-10 recovered from cultures suggesting that the antibodies inhibited the association of MP-10 with CaCo-2 cell monolayers.

Figure 5a shows the results of a similar experiment performed using J774 monolayers. Because the magnitude of CFUs recovered in the presence of anti-internalin antibodies was on the order of 2 or more logarithms less, the data is presented as a percent of a normalized (100%) control. The results show that incubation of MP-10 in the presence of either anti-ACrlNL or anti-KLH3054 antibodies significantly inhibited the association of MP-10 with J774 macrophages. Fig. 5b shows the results of a similar experiment to that shown in Fig. 5a except that the IgG fraction purified from the antisera was used to inhibit the association of MP-10 and Li⁺ cells with J774 cells. Figure 6 shows the results of an experiment performed using HUVEC monolayers incubated with MP-10 and Li+ in the absence and presence of affinity purified IgG fractions of anti-internalin mAbs. The presence of anti-internalin mAbs significantly inhibited the association of both intemalin bearing bacteria with HUVECs.

Taken together, the data suggests that anti-internalin antibody blocks the uptake of Listeria monocytogenes by several disparate cell lines including epithelial, endothelial and macrophage-like cells. Further, anti- internalin antibody inhibits uptake of the transformed intemalin expressing mutant Li+. It is reasonable to conclude that anti-internalin antibody combines with intemalin on the surface of Listeria monocytogenes thereby preventing it from binding the putative intemalin receptor on the host cell. Example 6

The following example demonstrates the presence of an intemalin receptor on CaCo-2 and J774 cells. The objective of this study was to determine whether recombinant intemalin could bind to the surface of CaCo-2, HUVEC and J774 cells in a specific manner. Cell lines were incubated in a 35 mm culture well on sterile glass slides, as above, and fixed with 4% paraformaldehyde and/or 100% methanol. Recombinant intemalin was then added to the cultures at various concentrations. The cells were incubated for 1 h at 37°C in an humidified atmosphere containing 5% C0₂, washed three times and then incubated for 1 h, with rabbit anti-ACrlNL antibodies. The cells were washed three times and incubated for 1 h, with CY3 conjugated donkey anti-rabbit immunoglobulin. Washed cells were then viewed using a Leitz fluorescence microscope. In a parallel set of experiments, cells were also stained with anti-internalin mAb 292, 804 or 1630. Controls for this study included staining of fixed and unfixed cells with normal mouse serum or pre-immune rabbit IgG antibody followed by the appropriate secondary antibody and staining with the secondary antibody alone. The staining results indicated that recombinant intemalin bound to the surface of both CaCo-2, HUVEC and J774 cells.

To further demonstrate a possible intemalin receptor on the surface of these cells, membrane preparations of lysed CaCo-2 and J774 cells, prepared by ultracentrifugation were extracted in 20 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40 and 1 mg/ml of anti-trypsin, leupeptin and aprotinin. Cell membrane detergent lysates were electroblotted to nitrocellulose papers from 10% SDS- PAGE gels and blocked overnight at 4°C in PBST plus 2% non¬ fat dry milk. Western blots were then incubated for 1 h at 4°C in block containing approximately 10 ug of recombinant intemalin, washed 3 times with PBST, incubated 1 h at 4°C with either polyclonal anti-ACrlNL antibodies or anti- intemalin 292 mAb, washed and then stained with the appropriate secondary antibody coupled to horseradish peroxidase (HRP) . Developed western immunoblots demonstrated the presence of recombinant intemalin bound to a 180,000 MWr dalton polypeptide in the membrane detergent extracts from both CaCo-2 and J774 cells. Example 7

The following example illustrates the imporatnce of intemalin protein in the escape of Listeria cells from a phagosome of a host cell.

Mutant Bug 8 cells (intemalin negative) were cultured using the method described in Example 4. Host J774 cells were cultures using the method described in Example 5. About 10* Bug 8 cells and about 10⁶ J774 cells were co- cultured for 0.5 hours at 37°C. The cells were then thoroughly washed and incubated for 4 hours at 37°C. Samples of bacteria were removed at 0 time (at washing stage) , at 2 hours and at 4 hours and the number of Bug 8 cells in the culture were determined by plating serial dilutions of each sample in triplicate on soy agar plates. The number of Bug 8 cells was shown not to increase during the incubation period. Internalization of Bug 8 cells into J774 cells was confirmed by permeabilizing the membrane of the J774 cells and treating the cells with an anti-Listeria antibody specific for an eptiope different from intemalin and tagged with a fluorescent label. The labelled cells were then visualized by fluorescence microscopy. In addition, Bug 8 cells were visualized in phagosomes using Lucifer Yellow. J774 cells were incubated with 350 μg/ l lucifer yellow for 72 hours prior to co-incubation with Bug 8 cells. Uptake of Bug 8 cells into J774 cells was detected by visually obeserving the fusion between Bug 8 containing phagosomes and lucifer yellow loaded lysosomes under a fluorescent microscope. Bug 8 cells contained within fused phago-lysosomes were clearly outlined by the lucifer yellow. Thus, the intemalin negative mutant Bug 8 were phagocytized by the J774 cells but were not able escape the phagosome. The results indicate that intemalin is needed for the escape of Listeria cells from a phagosome.

A second study was performed that determined if the mutant Bug 8 cells tested above were able to produce listeriolysin O which may effect the ability of a Bug 8 cell to escape a phagasome. Bug 8 cells were plated on blood agar plates and cultured overnight. The production of 0-hemolysin by the Bug 8 cells was measured by observing the extent of blood cell lysis surrounding each colony of cells. Blood cells were lysed greater than 5 mm around each Bug 8 colony indicating that the Bug 8 cells were producing substantial amounts of 0-hemolysin.

Taken together, the data indicate that the interaction of intemalin with its host cell receptor followed by phagocytosis of bacteria within a phagosome can alter the phagosomal membrane in such a way as to be susceptible to the lytic action of listeriolysin O, thereby promoting the escape of bacteria from the phagosome. Example 8

The following example describes enzyme linked immunoassay experiments to detect intemalin on the surface of Listeria monocytogenes cells using rabbit anti- internalin antibodies.

A series of enzyme linked immunoassay (ELISA) experiments were performed to determine the number of Listeria monocytogenes cells that could be detected using the rabbit anti-ACrlNL antibody described above in Example

3.

Fifty μl of fetal calf serum diluted 1:100 in phosphate buffered saline (PBS) + azide was added to each well of a 96 well Immulon ELISA plate. Fifty μl of rabbit gamma globulin (25 μg/ml in PBS-azide) was added to 3 wells as a control sample. The plate was allowed to incubate for 1.5 hours at room temperature. Following the incubation, excess liquid was removed and 50 μl of Listeria monocytogenes cells at the appropriate dilution in PBS- azide was added to each well. Appropriate dilutions were determined based on the total number of Listeria monocytogenes cells to be added to each well (see Figs. 7-9 for numbers of Listeria monocytogenes cells used in different wells) . The plate was centrifuged for 10 min. at 1500 rpm (400 X G) at room temperature and the supernatant was removed and 50 μl of rabbit anti-ACrlNL antibodies was added to the wells at the appropriate dilution in blocking buffer (PBS + 0.05% Tween 20 + 1% bovine serum albumin). The appropriate dilutions of the rabbit anti-ACrlNL antibodies are indicated in Figs. 7-9. The rabbit anti- ACrlNL antibodies were incubated overnight at 4°C. Following the incubation, the plate was centrifuged for 10 in. at 1500 rpm (400 X G) and the supernatant was removed. The plate was then washed two times by filling each well with washing buffer (PBS + 0.05% Tween 20), spinning the plate for 10 min. at 1500 rpm and removing the supernatant. Fifty μl of a 1:3000 dilution in blocking buffer of goat anti-rabbit-horse radish peroxidase conjugated antibody was added to each well. The plate was incubated overnight at 4°C and washed two times using the method stated immediately above. One hundred μl of horse radish peroxidase substrate (2,2'-azino-di-[3- ethylbenzthioazoline-6-sulfonic acid + hydrogen peroxide) was added to each well and incubated for 30 min. at room temperature. One hundred μl of 2% oxalic acid was then added to each well to stop the peroxidase reaction. The extent of enzyme reactivity was determined by measuring the color change in each well at 405 nm using a standard ELISA reader. The optical density measurements were then corrected by subtracting the optical density measurements of the samples which did not have Listeria monocytogenes added to the well from the measurements of the Listeria monocytogenes containing samples. The values were then plotted and are shown in Figs. 7-9.

The results shown in Fig. 7 indicate that the optical density measurements using a 1:50 dilution of the rabbit anti-ACrlNL antibodies are capable of detecting at least 10 Listeria monocytogenes cells per well. The results shown in Fig. 8 indicate that the number of Listeria monocytogenes cells detected using a 1:50 dilution of the rabbit anti- ACrlNL antibodies is proportional to the number of Listeria monocytogenes cells detected using a 1:100 dilution of the rabbit anti-ACrlNL antibodies. The optical density measurements, however, do not necessarily coincide with the number of number of Listeria monocytogenes cells per well. Such variability may be due to a technical error in the dilution of Listeria monocytogenes cells. In addition, the variability may be due to the loss of Listeria monocytogenes cells during the washing steps since the assay is not a capture assay. Fig. 9 is a schematic representation of the optical density measurements derived from 3 separate ELISA plates using a 1:50 dilution of the rabbit anti-ACrlNL antibodies. The results indicate that the at least 10 Listeria monocytogenes cells can be detected using a 1:50 dilution of the rabbit anti-ACrlNL antibodies. The results also illustrate the variability of optical density measurements between different ELISA plates. Such variability may be due to a technical error in the dilution of Listeria monocytogenes cells. The variability in efficiency of binding that can be seen between the different ELISA tests represented by Figs. 7-9. Such variability can arise due to technician error and variations in the condition of the Listeria monocytogenes cells on the different days of each test. Overall, the results indicate that the ELISA assay was able to detect at least 10 Listeria monocytogenes cells per well using a 1:50 dilution of the rabbit anti-ACrlNL antibodies. Example 9

The following example describes studies testing the ability of anti-internalin antibodies of the present invention to detect Listeria monocytogenes contamination in food samples.

1. Preparation of Food Samples.

In a first sample, 900 μl of tryptose phosphate broth was mixed with 100 μl of juice removed from a series of packages of hot dogs (different brands) and incubated in a 4 ml snap-cap tube for 3 days at room temperature. In a second sample, 900 μl of tryptose phosphate broth was mixed with a 0.3 cm³ mashed piece of hot dog meat from each package in a 4 ml snap-cap tube for 3 days at room temperature. Prior to treatment of the hot dog meat sample with anti-internalin antibody, the sample was filtered using a Whatman #1 filter to separate the mashed meat from the culture medium. Samples were prepared in two separate experiments (Experiment 1 and Experiment 2) . Both hot dog juice and hot dog meat were tested in Experiment 1 and hot dog juice alone was tested in Experiment 2.

2. ELISA Assays.

In a 96 well Immulon 3 plate, 50 μl of fetal calf serum diluted 1:100 in phosphate buffered saline (PBS) + azide was added to the plate. Fifty μl of rabbit gamma globulin (25 μg/ml in PBS-azide) was added to wells 10-12 in row B. PBS-azide buffer alone was added to wells 7-9 in row B. The plate was allowed to incubate for 1.5 hours at room temperature. Following the incubation, excess liquid was removed and the wells were filled with the blocking buffer described above in Example 8 and allowed to incubate for 1 hour at room temperature. Excess liquid was then removed. Fifty μl of each food sample (undiluted or 1:10 dilution) were added to 4 wells per sample. The plate was centrifuged for 10 min. at 1500 rpm at room temperature and the supernatant was removed. Fifty μl of a 1:50 dilution of rabbit anti-ACrlNL antibodies in PBS-azide was added to at least one well for each sample and a 1:50 dilution of pre-immune serum taken from the same rabbit as the anti- ACrlNL antibodies was added to the a different well containing the same sample. Samples tested in Experiment 1 were performed in duplicate and samples tested in Experiment 2 were tested in triplicate. The plate was incubated overnight at 4°C. Following the incubation, the plate was centrifuged for 10 min. at 1500 rpm and the supernatant was removed. The plate was then washed two times by filling each well with washing buffer (PBS + 0.05% Tween 20) , spinning the plate for 10 min. at 1500 rpm and removing the supernatant. Fifty μl of a 1:3000 dilution in blocking buffer of goat anti-rabbit-horse radish peroxidase conjugated antibody was added to each well. The plate was incubated overnight at 4°C and washed two times using the method stated immediately above. One hundred μl of horse radish peroxidase substrate was added to each well and incubated for 30 min. at room temperature. One hundred μl of 2% oxalic acid was then added to each well to stop the peroxidase reaction. The extent of enzyme reactivity was determined by measuring the color change in each well at 405 nm using a standard ELISA reader. 4. Results.

The results of the two experiments are shown in Figs. 10 and 11. The optical density measurements were corrected by subtracting the optical density measurements of the control samples from the measurements of the food samples. Referring to Figs. 10 and 11, the sample numbers correspond to the following commercial hot dog products: Sample 1 Sigman Top Dog Jumbos

Sample 2 John Morrel German Grand Weiners

Sample 3 Hygrades

Sample 4 Oscar Meyer

Sample 5 Wilson Jumbos Sample 6 Bar S Big Beef Franks

Sample 7 King Soopers Turkey Franks

Sample 8 Sinai 48 Kosher Franks

Sample 9 Ball Park

Sample 10 Healthy Choice Jumbo Franks Sample 11 Hormel 97% Fat Free

Sample 12 King Soopers Jumbo Franks

Different packages of hot dogs were used for Experiment 1 and Experiment 2. The results shown in Fig. 10 indicate that intemalin was detected in the hot dog juice Samples 7, 9 and 10. Intemalin was well detected in the hot dog meat Samples 7,

8 and 12 and less well detected in Samples 2, 3, 4, 5, 6,

9 and 10. The results shown in Fig. 11 indicate that intemalin was well detected in the hot dog juice Samples

8 and 9 and less well detected in Samples 3, 4, 5, 6 and 11. Taken together, the data indicates that intemalin can be detected in both hot dog juice and hot dog meat samples using a 1:50 dilution of rabbit anti-ACrlNL antibodies. The presence of internalin in the hot dog samples indicates the presence of pathogenic Listeria in the samples.

Referring to Fig. 10, in certain hot dog meat samples (see Samples 2, 3, 4, 5, 9 , and 12), the undiluted sample and the diluted sample differed in whether the sample was positive or negative (i.e., contaminated or not contaminated) . The variation can be due to differing amounts of lipid in each sample due to the effectiveness of the filtration step. Such lipid would cause the anti-ACrlNL antibodies added to the sample to bind non-specifically to the well of the ELISA plate. In addition, hot dog meat protein aggregates can escape filtration and disrupt the binding of the anti-ACrlNL antibodies to internalin in the sample. Together, the results of Experiment 1 and Experiment 2 indicate that internalin protein can be detected in hot dog juice and hot dog meat samples. Thus, an antibody- based screening assay using anti-internalin antibodies is an effective method for screening food samples for pathogenic Listeria .

SEQUENCE LISTING The following Sequence Listing is submitted pursuant to 37 CFR SI.821. A copy in computer readable form is also submitted herewith. Applicants assert pursuant to 37 CFR S1.821(f) that the content of the paper and computer readable copies of SEQ ID Nθ:l through SEQ ID NO:6 submitted herewith are identical.

(1) GENERAL INFORMATION:

(i) APPLICANT: Campbell, Priscilla Potter, Terry Sawyer, Richard

Drevets, Douglas Freed, John

(ii) TITLE OF INVENTION: INTERNALIN PRODUCTS AND PROCESSES

(iii) NUMBER OF SEQUENCES: 6

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Sheridan Ross & Mclntosh

(B) STREET: 1700 Lincoln Street, 35th Floor

(C) CITY: Denver (D) STATE: Colorado

(E) COUNTRY: U.S.A.

(F) ZIP: 80203

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US NOT YET ASSIGNED (B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Kovari , Joseph E.

(B) REGISTRATION NUMBER: 33,005 (C) REFERENCE/DOCKET NUMBER: 2879-11

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 303/863-9700

(B) TELEFAX: 303/863-0223 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2241 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..2241

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

GGG ATC CTG AAG ACG GTC TTA GGA AAA ACG AAT GTA ACA GAC ACG GTC 48 Gly He Leu Lys Thr Val Leu Gly Lys Thr Asn Val Thr Asp Thr Val 1 5 10 15 TCG CAA ACA GAT CTA GAC CAA GTT ACA ACG CTT CAG GCG GAT AGA TTA 96 Ser Gin Thr Asp Leu Asp Gin Val Thr Thr Leu Gin Ala Asp Arg Leu 20 25 30

GGG ATA AAA TCT ATC GAT GGA TTG GAA TAC TTG AAC AAT TTA ACA CAA 144 Gly He Lys Ser He Asp Gly Leu Glu Tyr Leu Asn Asn Leu Thr Gin 35 40 45

ATA AAT TTC AGC AAT AAT CAA CTT ACG GAT ATA ACG CCA CTT AAA GAT 192 He Asn Phe Ser Asn Asn Gin Leu Thr Asp He Thr Pro Leu Lys Asp 50 55 60

TTA ACT AAG TTA GTT GAT ATT TTG ATG AAT AAT AAT CAA ATA GCA GAT 240 Leu Thr Lys Leu Val Asp He Leu Met Asn Asn Asn Gin He Ala Asp 65 70 75 80

ATA ACT CCG CTA GCT AAT TTG ACG AAT CTA ACT GGT TTG ACT TTG TTC 288 He Thr Pro Leu Ala Asn Leu Thr Asn Leu Thr Gly Leu Thr Leu Phe 85 90 95 AAC AAT CAG ATA ACA GAT ATA GAC CCG CTT AAA AAT CTA ACA AAT TTA 336 Asn Asn Gin He Thr Asp He Asp Pro Leu Lys Asn Leu Thr Asn Leu 100 105 110

AAT CGG CTA GAA CTA TCT AGT AAC ACG ATT AGT GAT ATT AGT GCG CTT 384 Asn Arg Leu Glu Leu Ser Ser Asn Thr He Ser Asp He Ser Ala Leu 115 120 125

TCA GGT TTA ACT AAT CTA CAG CAA TTA TCT TTT GGT AAT CAA GTG ACA 432 Ser Gly Leu Thr Asn Leu Gin Gin Leu Ser Phe Gly Asn Gin Val Thr 130 135 140

GAT TTA AAA CCA TTA GCT AAT TTA ACA ACA CTA GAA CGA CTA GAT ATT 480 Asp Leu Lys Pro Leu Ala Asn Leu Thr Thr Leu Glu Arg Leu Asp He 145 150 155 160

TCA AGT AAT AAG GTG TCA GAT ATT AGT GTT CTG GCT AAA TTA ACC AAT 528 Ser Ser Asn Lys Val Ser Asp He Ser Val Leu Ala Lys Leu Thr Asn 165 170 175 TTA GAA AGT CTT ATC GCT ACT AAC AAC CAA ATA AGT GAT ATA ACT CCA 576 Leu Glu Ser Leu He Ala Thr Asn Asn Gin He Ser Asp He Thr Pro 180 185 190 CTT GGG ATT TTA ACA AAT TTG GAC GAA TTA TCC TTA AAT GGT AAC CAG 624 Leu Gly He Leu Thr Asn Leu Asp Glu Leu Ser Leu Asn Gly Asn Gin 195 200 205

TTA AAA GAT ATA GGC ACA TTG GCG AGT TTA ACA AAC CTT ACA GAT TTA 672 Leu Lys Asp He Gly Thr Leu Ala Ser Leu Thr Asn Leu Thr Asp Leu 210 215 220

GAT TTA GCA AAT AAC CAA ATT AGT AAT CTA GCA CCA CTG TCG GGT CTA 720 Asp Leu Ala Asn Asn Gin He Ser Asn Leu Ala Pro Leu Ser Gly Leu 225 230 235 240 ACA AAA CTA ACT GAG TTA AAA CTT GGA GCT AAC CAA ATA AGT AAC ATC 768 Thr Lys Leu Thr Glu Leu Lys Leu Gly Ala Asn Gin He Ser Asn He 245 250 255

AGT CCC CTA GCA GGT TTA ACC GCA CTC ACT AAC TTA GAG CTT AAT GAA 816 Ser Pro Leu Ala Gly Leu Thr Ala Leu Thr Asn Leu Glu Leu Asn Glu 260 265 270

AAT CAG CTG GAA GAT ATT AGC CCA ATT TCT AAC CTG AAA AAT CTC ACA 864 Asn Gin Leu Glu Asp He Ser Pro He Ser Asn Leu Lys Asn Leu Thr 275 280 285

TAT TTA ACG TTG TAC TTT AAT AAT ATA AGT GAT ATA AGC CCA GTT TCT 912 Tyr Leu Thr Leu Tyr Phe Asn Asn He Ser Asp He Ser Pro Val Ser 290 295 300

AGT TTA ACA AAG CTT CAA AGA TTA TTT TTC TAT AAT AAC AAG GTA AGT 960 Ser Leu Thr Lys Leu Gin Arg Leu Phe Phe Tyr Asn Asn Lys Val Ser 305 310 315 320 GAC GTA AGC TCA CTT GCG AAC TTA ACC AAT ATT AAT TGG CTT TCA GCT 1008 Asp Val Ser Ser Leu Ala Asn Leu Thr Asn He Asn Trp Leu Ser Ala 325 330 335

GGG CAT AAC CAA ATT AGC GAT CTT ACA CCA TTG GCT AAT TTA ACA AGA 1056 Gly His Asn Gin He Ser Asp Leu Thr Pro Leu Ala Asn Leu Thr Arg 340 345 350

ATC ACC CAA CTA GGG TTG AAT GAT CAA GCA TGG ACA AAT GCA CCA GTA 1104 He Thr Gin Leu Gly Leu Asn Asp Gin Ala Trp Thr Asn Ala Pro Val 355 360 365

AAC TAC AAA GCA AAT GTA TCC ATT CCA AAC ACG GTG AAA AAT GTG ACT 1152 Asn Tyr Lys Ala Asn Val Ser He Pro Asn Thr Val Lys Asn Val Thr 370 375 380

GGC GCT TTG ATT GCA CCT GCT ACT ATT AGC GAT GGC GGT AGT TAC GCA 1200 Gly Ala Leu He Ala Pro Ala Thr He Ser Asp Gly Gly Ser Tyr Ala 385 390 395 400 GAA CCG GAT ATA ACA TGG AAC TTA CCT AGT TAT ACA AAT GAA GTA AGC 1248 Glu Pro Asp He Thr Trp Asn Leu Pro Ser Tyr Thr Asn Glu Val Ser 405 410 415

TAT ACC TTT AGC CAA CCT GTC ACT ATT GGA AAA GGA ACG ACA ACA TTT 1296 Tyr Thr Phe Ser Gin Pro Val Thr He Gly Lys Gly Thr Thr Thr Phe 420 425 430

AGT GGA ACC GTG ACG CAG CCA CTT AAG GCA ATT TTT AAT GCT AAG TTT 1344 Ser Gly Thr Val Thr Gin Pro Leu Lys Ala He Phe Asn Ala Lys Phe 435 440 445

CAT GTG GAC GGC AAA GAA ACA ACC AAA GAA GTG GAA GCT GGG AAT TTA 1392 His Val Asp Gly Lys Glu Thr Thr Lys Glu Val Glu Ala Gly Asn Leu 450 455 460 TTG ACT GAA CCA GCT AAG CCC GTA AAA GAA GGT CAC ACA TTT GTT GGT 1440 Leu Thr Glu Pro Ala Lys Pro Val Lys Glu Gly His Thr Phe Val Gly 465 470 475 480

TGG TTT GAT GCC CAA ACA GGC GGA ACT AAA TGG AAT TTC AGT ACG GAT 1488 Trp Phe Asp Ala Gin Thr Gly Gly Thr Lys Trp Asn Phe Ser Thr Asp

485 490 495

AAA ATG CCG ACA AAT GAC ATC AAT TTA TAT GCA CAA TTT AGT ATT AAC 1536 Lys Met Pro Thr Asn Asp He Asn Leu Tyr Ala Gin Phe Ser He Asn 500 505 510 AGC TAC ACA GCA ACC TTT GAG AAT GAC GGT GTA ACA ACA TCT CAA ACA 1584 Ser Tyr Thr Ala Thr Phe Glu Asn Asp Gly Val Thr Thr Ser Gin Thr 515 520 525

GTA GAT TAT CAA GGC TTG TTA CAA GAA CCT ACA CCA CCA ACA AAA GAA 1632 Val Asp Tyr Gin Gly Leu Leu Gin Glu Pro Thr Pro Pro Thr Lys Glu 530 535 540

GGT TAT ACT TTC AAA GGC TGG TAT GAC GCA AAA ACT GGT GGT GAC AAG 1680 Gly Tyr Thr Phe Lys Gly Trp Tyr Asp Ala Lys Thr Gly Gly Asp Lys 545 550 555 560

TGG GAT TTC GCA ACT AGC AAA ATG CCT GCT AAA AAC ATC ACC TTA TAT 1728 Trp Asp Phe Ala Thr Ser Lys Met Pro Ala Lys Asn He Thr Leu Tyr

565 570 575

GCC CAA TAT AGC GCC AAT AGC TAT ACA GCA ACG TTT GAT GTT GAT GGA 1776 Ala Gin Tyr Ser Ala Asn Ser Tyr Thr Ala Thr Phe Asp Val Asp Gly 580 585 590 AAA TCA ACG ACT CAA GCA GTA GAC TAT CAA GGA CTT CTA AAA GAA CCA 1824 Lys Ser Thr Thr Gin Ala Val Asp Tyr Gin Gly Leu Leu Lys Glu Pro 595 600 605

AAG GCA CCA ACG AAA GCC GGA TAT ACT TTC AAA GGC TGG TAT GAC GAA 1872 Lys Ala Pro Thr Lys Ala Gly Tyr Thr Phe Lys Gly Trp Tyr Asp Glu 610 615 620

AAA ACA GAT GGG AAA AAA TGG GAT TTT GCG ACG GAT AAA ATG CCA GCA 1920 Lys Thr Asp Gly Lys Lys Trp Asp Phe Ala Thr Asp Lys Met Pro Ala 625 630 635 640

AAT GAC ATT ACG CTG TAC GCT CAA TTT ACG AAA AAT CCT GTG GCA CCA 1968 Asn Asp He Thr Leu Tyr Ala Gin Phe Thr Lys Asn Pro Val Ala Pro

645 650 655

CCA ACA ACT GGA GGG AAC ACA CCG CCT ACA ACA AAT AAC GGC GGG AAT 2016 Pro Thr Thr Gly Gly Asn Thr Pro Pro Thr Thr Asn Asn Gly Gly Asn 660 665 670 ACT ACA CCA CCT TCC GCA AAT ATA CCT GGA AGC GAC ACA TCT AAC ACA 2064 Thr Thr Pro Pro Ser Ala Asn He Pro Gly Ser Asp Thr Ser Asn Thr 675 680 685

TCA ACT GGG AAT TCA GCC AGC ACA ACA AGT ACA ATG AAC GCT TAT GAC 2112 Ser Thr Gly Asn Ser Ala Ser Thr Thr Ser Thr Met Asn Ala Tyr Asp 690 695 700

CCT TAT AAT TCA AAA GAA GCT TCA CTC CCT ACA ACT GGC GAT AGC GAT 2160 Pro Tyr Asn Ser Lys Glu Ala Ser Leu Pro Thr Thr Gly Asp Ser Asp 705 710 715 720 AAT GCG CTC TAC CTT TTG TTA GGG TTA TTA GCA GTA GGA ACT GCA ATG 2208 Asn Ala Leu Tyr Leu Leu Leu Gly Leu Leu Ala Val Gly Thr Ala Met 725 730 735

GCT CTT ACT AAA AAA GCA CGT GCT AGT AAA TAG 2241 Ala Leu Thr Lys Lys Ala Arg Ala Ser Lys 740 745

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 746 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Gly He Leu Lys Thr Val Leu Gly Lys Thr Asn Val Thr Asp Thr Val 1 5 10 15

Ser Gin Thr Asp Leu Asp Gin Val Thr Thr Leu Gin Ala Asp Arg Leu 20 25 30

Gly He Lys Ser He Asp Gly Leu Glu Tyr Leu Asn Asn Leu Thr Gin 35 40 45 He Asn Phe Ser Asn Asn Gin Leu Thr Asp He Thr Pro Leu Lys Asp 50 55 60

Leu Thr Lys Leu Val Asp He Leu Met Asn Asn Asn Gin He Ala Asp 65 70 75 80

He Thr Pro Leu Ala Asn Leu Thr Asn Leu Thr Gly Leu Thr Leu Phe 85 90 95

Asn Asn Gin He Thr Asp He Asp Pro Leu Lys Asn Leu Thr Asn Leu 100 105 110

Asn Arg Leu Glu Leu Ser Ser Asn Thr He Ser Asp He Ser Ala Leu 115 120 125

Ser Gly Leu Thr Asn Leu Gin Gin Leu Ser Phe Gly Aβn Gin Val Thr 130 135 140

Asp Leu Lys Pro Leu Ala Asn Leu Thr Thr Leu Glu Arg Leu Asp He 145 150 155 160

Ser Ser Asn Lys Val Ser Asp He Ser Val Leu Ala Lys Leu Thr Aβn 165 170 175

Leu Glu Ser Leu He Ala Thr Asn Asn Gin He Ser Asp He Thr Pro 180 185 190

Leu Gly He Leu Thr Asn Leu Asp Glu Leu Ser Leu Asn Gly Asn Gin 195 200 205 Leu Lye Asp He Gly Thr Leu Ala Ser Leu Thr Asn Leu Thr Asp Leu 210 215 220

Asp Leu Ala Asn Asn Gin He Ser Asn Leu Ala Pro Leu Ser Gly Leu 225 230 235 240 Thr Lys Leu Thr Glu Leu Lys Leu Gly Ala Asn Gin He Ser Asn He 245 250 255

Ser Pro Leu Ala Gly Leu Thr Ala Leu Thr Asn Leu Glu Leu Asn Glu 260 265 270 Aβn Gin Leu Glu Asp He Ser Pro He Ser Asn Leu Lys Asn Leu Thr 275 280 285

Tyr Leu Thr Leu Tyr Phe Asn Asn He Ser Asp He Ser Pro Val Ser 290 295 300

Ser Leu Thr Lys Leu Gin Arg Leu Phe Phe Tyr Asn Asn Lys Val Ser 305 310 315 320

Asp Val Ser Ser Leu Ala Asn Leu Thr Asn He Asn Trp Leu Ser Ala 325 330 335

Gly His Asn Gin He Ser Asp Leu Thr Pro Leu Ala Aβn Leu Thr Arg 340 345 350 He Thr Gin Leu Gly Leu Asn Asp Gin Ala Trp Thr Asn Ala Pro Val 355 360 365

Asn Tyr Lye Ala Aβn Val Ser He Pro Asn Thr Val Lys Asn Val Thr 370 375 380

Gly Ala Leu He Ala Pro Ala Thr He Ser Asp Gly Gly Ser Tyr Ala 385 390 395 400

Glu Pro Asp He Thr Trp Asn Leu Pro Ser Tyr Thr Asn Glu Val Ser 405 410 415

Tyr Thr Phe Ser Gin Pro Val Thr He Gly Lys Gly Thr Thr Thr Phe 420 425 430 Ser Gly Thr Val Thr Gin Pro Leu Lys Ala He Phe Asn Ala Lys Phe 435 440 445

His Val Asp Gly Lye Glu Thr Thr Lys Glu Val Glu Ala Gly Asn Leu 450 455 460

Leu Thr Glu Pro Ala Lys Pro Val Lys Glu Gly His Thr Phe Val Gly 465 470 475 480

Trp Phe Asp Ala Gin Thr Gly Gly Thr Lys Trp Asn Phe Ser Thr Asp 485 490 495

Lys Met Pro Thr Asn Asp He Asn Leu Tyr Ala Gin Phe Ser He Asn 500 505 510 Ser Tyr Thr Ala Thr Phe Glu Asn Asp Gly Val Thr Thr Ser Gin Thr 515 520 525

Val Asp Tyr Gin Gly Leu Leu Gin Glu Pro Thr Pro Pro Thr Lye Glu 530 535 540

Gly Tyr Thr Phe Lys Gly Trp Tyr Asp Ala Lye Thr Gly Gly Aβp Lys 545 550 555 560

Trp Asp Phe Ala Thr Ser Lys Met Pro Ala Lys Asn He Thr Leu Tyr 565 570 575

Ala Gin Tyr Ser Ala Asn Ser Tyr Thr Ala Thr Phe Asp Val Asp Gly 580 585 590 Lys Ser Thr Thr Gin Ala Val Asp Tyr Gin Gly Leu Leu Lys Glu Pro 595 600 605

Lys Ala Pro Thr Lys Ala Gly Tyr Thr Phe Lye Gly Trp Tyr Asp Glu 610 615 620 Lye Thr Asp Gly Lys Lys Trp Asp Phe Ala Thr Asp Lys Met Pro Ala 625 630 635 640

Aβn Aβp He Thr Leu Tyr Ala Gin Phe Thr Lye Aβn Pro Val Ala Pro 645 650 655

Pro Thr Thr Gly Gly Asn Thr Pro Pro Thr Thr Asn Asn Gly Gly Asn 660 665 670

Thr Thr Pro Pro Ser Ala Asn He Pro Gly Ser Asp Thr Ser Asn Thr 675 680 685

Ser Thr Gly Asn Ser Ala Ser Thr Thr Ser Thr Met Asn Ala Tyr Asp 690 695 700 Pro Tyr Asn Ser Lys Glu Ala Ser Leu Pro Thr Thr Gly Asp Ser Aβp 705 710 715 720

Aβn Ala Leu Tyr Leu Leu Leu Gly Leu Leu Ala Val Gly Thr Ala Met 725 730 735

Ala Leu Thr Lye Lye Ala Arg Ala Ser Lye 740 745

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Cys Aβn Aβn Gin He Ala Aβp He Thr Pro Leu Ala Asn Leu Thr Asn 1 5 10 15 Leu Thr

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: ATGGATCCTG AAGACGGTCT TAGGAAA 27

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: ATGAATTCCT ATTTACTA 18

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

GCATGGCCTT TGCAGGG 17

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims:

Claims

What is claimed:

1. A method to screen a sample for internalin, comprising immunoreacting a sample with an antibody capable of selectively binding to internalin, and determining the presence of said immunoreaction.

2. A method to screen a sample for Listeria, comprising immunoreacting a sample with an antibody capable of selectively binding to internalin, and determining the presence of said immunoreaction.

3. A test kit for detecting internalin in a sample, said kit comprising:

(a) an antibody capable of selectively binding to internalin; and

(b) a means for determining an immunoreaction between said antibody and said internalin.

4. The method of Claim 1, Claim 2 or Claim 3, wherein said sample is selected from the group consisting of poultry, cattle, pig, goat, sheep, lamb, fish, seafood, dairy products, fruit, vegetables grains, milk, cheese, whey, butter, baby formula, ice cream, yogurt, blood, plasma, serum, tissue, cells, feces, cerebo-spinal fluid, saliva, exudates, amniotic fluid, interstitial fluid, synovial fluid, autopsy specimens, brain, lung, liver, spleen, lymph nodes, urine, bone, muscle, placental tissue and abortuses.

5. The method of Claim 1, Claim 2 or Claim 3, wherein said step of determining comprises measuring the presence of a detectable signal associated with said immunoreaction said signal selected from the group consisting of radioactivity, fluorescence, enzyme activity, and chromophoric activity.

6. The method of Claim 1, Claim 2 or Claim 3, wherein said antibody is capable of forming an immunocomplex that is capable of substantially inhibiting the ability of said internalin to bind to an internalin receptor.

7. An isolated antibody capable of selectively binding internalin found on Listeria monocytogenes , said antibody identified by its ability to inhibit the uptake of Listeria monocytogenes by a host cell selected from the group consisting of CaCo-2 cells and J774 cells when said host cell is co-incubated with said Listeria monocytogenes .

8. The antibody of Claim 7, wherein said antibody has substantially the same binding characteristics as the polyclonal antibody selected from the group consisting of anti-GST-INL antibodies, anti-ACrlNL antibodies and anti- KLH3054 antibodies.

9. A hybridoma that produces an antibody of Claim 7.

10. The hybridoma of Claim 9, wherein said hybridoma is selected from the group consisting of 292, 1847, 1339, 804, 1630, 1360, 1835, 1042, 469, and 678.