MXPA96005829A - Inhibition of the union of h. influenzae to cellulashuma - Google Patents

Abstract

The present invention relates to the binding of H. influenzae to human cells, such as oropharyngeal cells, can be inhibited by human E-casein, a recombinant form of E. casein contained in human milk and hydrolysates of both . The human E-casein or the hydrolyzate may be contained in a liquid enteral nutrient product such as an infant formula. the enteral nutrient product can be used, for example for the prevention and treatment of otitis media in infants. The human E-casein or the hydrolysis can also be administered as a spray for the throat or nasally using drops or a spray.

Description

INHIBITION OF THE UNION OF H. INFLUENZAS TO HUMAN CELLS The present invention relates, in general terms, to inhibiting the binding of Haemophilus influenzae to human cells, and more specifically to the use of native or recombinant human β-Casein and hydrolysates thereof to inhibit the binding of Haemophilus influenza (H. influenzae ) to human cells. Haemophilus are small, gram-negative, non-motile bacilli that do not form spores with complex growth requirements. Diseases caused by H. influenzae usually begin as nasopharyngitis, possibly precipitated by viral infection of the upper respiratory tract. Morese, and collaborators "Haemophilus". MICROBIOLOGY FOURTH EDIT ON. published by J.B. Lipincott Company, pages 615-618 (1990). H. influenzae is transmitted from person to person by droplets of the respiratory air or by direct contact with secretions. To colonize H. influenzae, it must fight with the ciliary cleaning mechanisms of the nasopharyngeal mucosal surface and with the mucus barrier. Once the mucus barrier and the ciliary scaler pass, H. influenzae binds to the epithelial cells of the mucosa. The invasion of mucosal surfaces seems to be an important characteristic of pathogenic bacteria. Stephens, and collaborators.

"Pathogenic Events During Infection of the Human Nasopharynx Neieseria meningi tis and Haemophilus influenzae. "REVIEWS OF INFECTIOUS DISEASES. 13: 22-23 (1991). It has also been reported that H. influenzae housed in the nasopharynx are a key factor in the development of middle ear infections (otitis media), and that the nontypable H. influenzae adhere to the nasopharyngeal and nasal mucosa cells.

Harada et al .. "Adherence of Haemophilus influenzae to nasal, nasopharyngeal and oral epithelial cells from patients with otitis media" EUROPEAN ARCHIVES OF OTO-RHINO-LARINGOLOGY. 247: 122-124 (1990). Stenfors et al .. "Abundant Attachment of Bacteria to Nasopharyngeal Epithelium in Otitis-Prone Children". THE JOURNAL OF INFECTIOUS DISEASES. 165: 1148-1150 (1992). According to the present invention β-casein isolated from human milk or a recombinant thereof, or a hydrolyzate of one or the other is employed to inhibit the adhesion of H. influenzae to human cells. Breastfeeding exclusively for human infants during the first four months of life has been associated with a decrease in the incidence of both acute and recurrent otitis media. However, the question remains whether it is the breast milk itself or is the mechanism of breastfeeding that exerts the protective effect. Sheard, "Breast-Feeding Protects Against Otitis Media." NUTRITION REVIEWS. Vol. 51, No. 9, pages 275-227 (1993). In a study of 1013 infants followed during their first full year, 47 percent had at least one episode of otitis media and 17 percent had recurrent otitis media. Infants who were breastfed exclusively for four or more months had half the average number of episodes of acute otitis media than those who were never breastfed and 40 percent less than infants whose diets were supplemented with other foods before the four months. The rate of recurrent otitis media in breastfed infants exclusively for six months or more was 10 percent and was 20.5 percent in breastfed infants for less than four months. Those researchers presented speculation that IgA, or micronutrients or Prostaglandin E, in breast milk may be protective, but concluded that the mechanism for a protective effect of breastfeeding against otitis media was unclear. Duncan and collaborators .. "Exclusive Breast-Feeding for at Least 4 Months Protects Against Otitis Media". PEDIATRICS. Vol. 91, No. 5, pages 867-872 (1993). WO 91/06308 presented by Andersson et al. For "ANTIBACTERIAL COMPOSITION", and an article published by the same authors (Aniansson et al .. "Antiadhesive activity of human casein against Streptococcus pneumonia and Haemophilus influenzae." MICROBIAL PATHOGENESIS. 8: 315-323 (1990) describes the use of a milk fraction having a molecular weight of at least 5000 daltons for "therapeutic prophylactic and / or diagnostic use in infections caused by S. pneumonae and H. influenzae", but suggests in these publications that the beneficial effect is provided by kappa-casein. However, the present invention relates to the use of native or recombinant human ß-casein, hydrolysates of both to inhibit H. influenzae infections. The patent W093 / 04172 refers to the sequence of DNA encoding human ß-casein, but does not describe the ability of either native or recombinant human ß-casein to inhibit the binding of H. influenzae to human cells. The patent W091 / 08675 describes a infant formula that contains recombinant forms of both human alpha-lactalbumin and human β-casein. However, this publication only describes that these proteins in human milk "will provide a formula of simulated human breast milk that does not exhibit the allergenic properties associated with formulas based on cow proteins or other foreign proteins." (page 3, lines 20-22). The use of β-casein to inhibit the binding of H. influenzae to human cells is not discussed or suggested in that publication. The two assays (a radiolabelled assay and the ELISA assay) that were used to determine the bioactivity of β-casein are described below. These trials have not been published so far, although the ELISA trial was based on established methodology. MATERIALS USED IN BOTH TESTS Haemophilus influenzae The cultures of Haemophilus influenzae. { H. influenzae) (fimbriated, non-typable) that were implicated in otitis media were obtained from Dr. Lauren Bakeletz of Ohio State University, Columbus, Ohio, U.S.A. The use of these organisms in tests has been described in Bakaletz and collaborators .. "Frequeney of Fimbriation of Nontypable Haemophilus influenzae and its Ability to Adhere to Chinchilla and Human Respiratory Epithelium ". Infection and Immunity 56: 331-335 (1988). H. influenzae were smeared onto chocolate agar plates (BBL-Becton Dickinson &Co., Cockeysville, Maryland, U.S.A.) from frozen aliquots of a low number of passages and incubated at 37 ° C in an incubator with 5 percent C02 for approximately 18 hours to obtain logarithmically growing cultures. H. influenzae was used in both the Assay Immuno Sorbent Linked with Enzymes (ELISA) as in the radio-labeling assay described below. Native human ß-casein ß-casein isolated from human milk was purchased from Symbicom AB. P.O. Box 1451. S-901 24 Umea, Sweden.

Recombinant human ß-casein The applicants obtained the ß-casein cDNA and the Symbicom AB expression system. P.O. Box 1451. S-901 24 Umea, Sweden. The cDNA of the human ß-casein used had been previously cloned and sequenced by Lonnerdal et al. Cloning and sequencing of a cDNA encoding human milk ß-casein. (SEQ ID NO: 1) Federation of European Biochemical Societies Letters 269. 153-156 (1990). Recombinant human ß-casein was obtained from E. coli and purified according to the method of Hansson et al .. Expression of Human Milk ß-Casein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms. Protein Expression and Purifi cation 4, 373-381 (1993). To express human ß-casein in E. coli, the ß-casein cDNA was cloned under the control of a T7 promoter in two different expression vectors. A pS26 vector was designed for intracellular expression. The other vector, pS28, has a signal sequence for extracellular expression. The procedure followed was substantially that described by Hansson et al. The cDNA of human ß-casein was isolated by Hansson et al. As an EcoRI fragment of 1.1-kb from a human gt lambda mammary gland library, and subcloned in pUC19. which was designated pS21. The cDNA was modified by the introduction of synthetic oligonucleotides in the 5 'and 3' terms. To introduce a suitable cloning site at the 5 'end, Ndel, a translation start, was inserted in front of the sequence coding for mature human ß-casein. To adapt the initial part of the translated sequence to the codon usage of E. coli, six synthetic oligonucleotides were constructed and ligated. PstI and EcoRI sites were also inserted in front of the Ndel site. The sequence of the synthetic fragment was 5'-CTGCAGAATTCATATGCGTGAAACCATCGAATCCCTGAGCTCGAGCGAAGAATCGATCAC CGAATACAAAAAAGTTGAAAAAGTTAAACACGAGGACCAGGATCC -3 '. (SEQ ID NO: 2 :) The sequence encoding the sequence is underlined. The synthetic fragment was cloned into pUC19 digested with PstI / BamHI resulting in a pS24 plasmid. To insert the rest of the ß-casein coding sequence, it was isolated and an AccI / BglII fragment of 303 base pairs was cloned into a derivative of pUC18 and designated as plasmid pS22. Four synthetic oligonucleotides containing the sequence encoding the terminal carboxy terminus and the translation stop were constructed followed by the BamHl and EcoRI sites resulting in the sequence 5'-A GA TCTACCC TG T GA CT CAG C CA CTTGCCCC AGTTCATAACCCCATTAGTGTCTAATAAGGATCCGAATTC-3 '(SEQ ID NO: 3 :) where the sequence encoding the protein is underlined. The synthetic fragment was cloned into pS22 digested by BglII / EcoRI, resulting in plasmid pS23. To obtain the fragment encoding the recombinant modified ß-casein, three fragments were ligated: a PstI / AvalI fragment of 89 base pairs from pS24; an Avall / Accl fragment of 197 pairs of baees of pS21; and pS23 digested by 5 PstI / AccI. The resulting plasmid pS25 was digested with Ndel / BamHI and a 641 base pair fragment was isolated and cloned into the pET-3a vector. The resulting expression vector was designated pS26. In order to construct a vector that mediates the extracellular expression of lC-, the sequence of the E. coli signal of the STII gene of the enterotoxin was introduced in front of the sequence coding for β-casein. A sequence of modified STII with compatible Ncol and Ndel ends and an internal Clal site was obtained by using a synthetic oligonucleotide. 5' TGCATATG -31 (SEQ ID NO: 4 :). To insert the signal sequence in front of the ß-caffeine coding sequence, pS25 was digested with Aval / EcoRI and a fragment of 619 base pairs was elated. This fragment was ligated with a fragment of synthetic oligonucleotides: 5'- CATATGCACGTGAAA CCATCGAATCCCTGAGCTCGAG -3 '(SEQ ID NO: 5), and pUC19 digested by Ndel / EcoRI. The resulting plasmid was designated pS27. The final expression vector pS28 was constructed by ligating three fragments: a fragment of Ndel / HindIII β-casein of 700 5 base pairs of p527. the STII signal sequence, and a pACAT7 vector digested by NcoI / HindIII. The expression vectors pS26 and pS28 were used to transform E. coli strains BL2KDE3), BL21 (DE3) pLysS, and BL21 (DE3) pLysE. lae bacteria were cultured in Luria Broth culture medium containing 50 μg / milliliter of carbenicillin, and when BL21 (DE3) pLysS, and BL21 (DE3) pLysE were used, the medium was supplemented with 25 μg / milliliter of chloramphenicol. For the induction of expression the l? "Cultures were cultured at a density yielding an optical density (OD) of 0.6 at a wavelength of 600 nanometers (OD ^), then 0.4 mM of IPTG was added to induce the T7 system Cells were harvested approximately 90 minutes after induction.15 Recombinant β-casein was isolated using standard procedures The expression system based on inducible T7 resulted in a high level of expression of the recombinant β-casein. were harvested and the cells were granulated by centrifugation. The supernatant contained the periplasmic proteins and the granule of the cytoplasmic fraction. The recombinant proteins obtained were compared with the native ß-casein, which had been purified by standard methods including either chromatography by ion exchange followed by the reverse HPLC phase or gel filtration. The recombinant and native ß-casein was compared using standard biochemical techniques comprising SDA-PAGE, Western blotting, amino acid analysis, peptide mapping, foefate analysis, and maea eepectrometry. It was found that the recombinant ß-caffeine excreted in E. coli comigra with the native human ß-caffeine throughout its length, not phosphorylated, which is one of seven native isoforms. Recombinant human β-caffeine has also been expressed in S. cerevisiae using the pYES 2.0 vector (Invitrogen Corp .. San Diego, CA), but the level of expression was approximately 10 percent of that obtained in E. coli. However, Hanseon and collaborator found that S. cerivisiae appeared to excrete the ß-caffeine from the foeforized human milk. Β-caffeine hydrolieatoe Human ß-caffeine (both native and recombinant) was digested with the endoproteinase specific GLU-C (Sigma, degree of sequence) that catalyses the hydrolysis of peptide bonds at the C-terminal of the acid residue glutamic After monitoring the digestion using high pressure liquid chromatography, an enzyme to protein ratio of 1: 100 (weight / weight) was chosen for a 30 hour digestion at 37 ° C in 0.1 M NH 4 HCO 3, pH 7.8. These digests were dried and resuspended in appropriate regulators before the tests described below. RADIOETIOUTE TEST Detroit 562 Cells Detroit pharyngeal carcinoma cells were obtained 562 of the American Type Culture Collection (Rockville, Maryland, U.S.A.). The use of this type of cells in assays has been described in Takahaehi, and collaborated, "Phoeophorylation of a Surface Receptor Bound Urokinase-Type Plasminogen Activator in a Human Meta- Metatic Carcinomatoue Cell Line" BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. 182: 1466-72. Lae membranae Detroit ee cultivated in Dulbecco's Modified Eagle Medium (GIBCO, Grand Island, New York, U.S.A.) supplemented with 10 percent of fetal bovine serum (Hyclone, Logan, Utah, U.S.A.). Cells were routinely subcultured in 75 centimeter-square flasks (Costar Cambridge, Maseachusetts. U.S.A.) using Tripeyn-EDTA (0.25 percent tripeine, lmM EDTA (ethylenediaminetetraacetic acid).

(GIBCO) to separate cells. The subculture steps of cells 48-75 were used for adhesion studies. Lae cells were seeded in 96-well plates (Costar) at a density of 20,000 cells per well and were maintained at 37 ° C in an incubator with 5 percent C02 for 8-10 days providing a confluent monolayer for adhesion studies . Plates were washed three times with 200 μl of Hanke Balanced ealine (HBS) (GIBCO), supplemented with 30 mM of N-2-hydroxyethylpeperazine-N "-2-ethane sulfonic acid (HBS) (GIBCO) to remove protein lae. The bacteria harvested in phosphate-buffered saline supplemented 0.05 percent bovine serum albumin (PBS-BSA) were centrifuged and resuspended in an oven, before the addition of the bacteria in the adhesion assays. Serum volume regulated with bovine serum phosphate-albumin producing an optical density of 2.4 at a wavelength of 600 nanometers (OD, ^). nl-indium-oxine (! -In) (Amersham, Arlington Heights, Illinois, U.S.A.) used a high-energy, short-lived tracer to radiolabel the bacteria. (The, p-In penetrates the membrane of the cell, where once inside the cell it dissociates resulting in the binding of the ions of the p-indium with the cytoplasmic components.). The use of the p-in labeling in another essay has been written in Ardehali, et al. "p? -Indium Labeling of Microorganisms to Facilitate the Investigation of Bacterial Adhesion", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 27: 269-275 (1993). 50 μCi of a solution of p? -In was added to 2.5 milliliters of the bacterial suspension at 37 ° C and incubated for 20 minutes. The radiolabelled bacteria were then washed twice and resuspended in 5 milliliters of HBS supplemented with 30 mM of HEPES regulator. 25 μl of the bacterial suspeneion labeled with? -In with 25 μl of ß-casein in a 96 well plate of polypropylene for 15 minutes at 37 ° C to allow the binding of β-casein to the bacteria. Measurement of radiolabeled bacterial adhesion 25 μl of the pre-incubation mixture containing both radiolabelled bacteria and β-caffeine were pipetted into each well of the eneay plate containing Detroit 562 cells. The assay plate was incubated for 20 minutes at 37μC to allow the adhesion of bacteria to the cell monolayer. The plates were then washed three times with HBS to remove unattached bacteria from the Detroit 562 cells. The assay was terminated by the addition of 100 μl of 1 N of eodium hydroxide (NaOH) to disrupt the cell monolayer. The entire content of each well was placed in a Cobra polypropylene tube (12 millimeters in diameter and 75 millimeters in height) (Packard, Meriden, Connecticut, U.S.A.) and counted in a Cobra gamma counter (also from Packard). The results were calculated by averaging the results of four replicates after the subtraction of the background radiation. The results are presented as the percentage inhibition of bacterial binding in control and agent wells.

RESULTS OF THE RADIO-ETIOUTE TEST The solutions of human and bovine ß-caffeine were first prepared in 20 mM ethanolamine, 6 m urea, pH 9. 5 And then they were washed twice in phosphate-buffered serum by ultrafiltration using Centricon membrane filter.

(Amicon, MA) with a closure of 3,000 daltons. After returning to sleep in the appropriate regulator for the radiolabeling session described above, the tester is tested in the room. Experiments were carried out with dietintoe numbere deeignadoe in different days. As shown in Table 1, human ß-casein exhibited 50 percent or greater inhibition at concentrations of 0.75 milligrams / milliliter or greater. It should be noted that when referring to Table 1, a higher percentage of inhibition indicates a higher level of "AGENT" bioactivity, and a lower percentage of inhibition indicates a lower level of "AGENT" activity. Bovine ß-casein was not significantly active yet at 1.5 milligrams / milliliter.

These results indicated that ß-casein from human milk has a different bioactivity compared to ß-casein from bovine milk. The hydrolyzate of human β-casein obtained with the enzyme GLU-C (prepared as described above) was also active (> 50 percent inhibition) at concentrations of 0.75 milligrams / milliliter or greater. When the purified GLU-C hydrolyzate of the purified recombinant β-casein was tested at 3.0 milligrams / milliliter, it exhibited activity similar to that of the β-caffeine hydrolieate in human milk. Therefore, this recombinant protein ee could be produced at a large scale from bacteriae to provide an abundant supply of a protein that retains the anti-adhesion activity of ß-casein from native human milk against H. influenzae.

TABLE 1 PERCENTAGE OF INHIBITION OF THE UNION OF H. influenzae TO THE DETROIT 562 CELLS * NH = NOT MADE ELISA TEST Preparation of H. influenzae The H. influenzae harvested from the chocolate agar plates were suspended in 7 milliliters of bovine serum phosphate-buffered albumin and were bio-stained by incubation in 350 μl of N-hydroxyl euccinimide ether. bio-tin caproate (0.1 grams of biotestin / 1 milliliter of dimethyl eulfoxide): Sigma, St. Louis Missouri, USA) for 15 minutes at room temperature. The bioestañadae bacteria washed tree vecee at 2,700 rpm for 30 minutes each time to remove the excess of bioethanol. The labeled bacteria were then re-eepended to an optical deficiency of 0000 of 2.4 using serum-buffered bovine serum albumin. Oropharyngeal cells Oropharyngeal (OP) cells were harvested from donadoree and combined in ealine regulatory solution with foefate (serum regulated with foefate). Lae euspensionee de célèe ee washed once in phosphate-regulated serum were re-suspended and counted on a hemocytometer.

The cells were adjusted to a density of 2.5 x 10 5 cells per milliliter in phosphate-regulated serum. To promote the union of the cells orofaríngeae, lae placae of 96 pozoe (Linbro-ICN, Costa Mesa, California, U.S.A.) were coated with L-lysine followed by exposure to 1.25 percent glutaraldehyde (creates cross-linking). Plates were washed thoroughly to remove any residual glutaraldehyde. Each well of the 96-well plate was inoculated with 50 μl of the oropharyngeal cell preparation yielding a final concentration of 1.25 x 104 cells per well. The designated wells were incubated with phosphate-buffered serum only (no lae cell oropharyngeal) to serve as background control wells to measure non-specific bacterial binding to the plastic. The inoculated plates were centrifuged at 2,700-3,000 rpm for ten minutes to pellet the preparation of suependidae cells, aerated and incubated overnight at 37 ° C in a humid chamber. The next morning, the plaque was plated with oropharyngeal cells and treated with serum with phoephate containing 5 percent bovine serum albumin for four hours to avoid non-specific bacterial binding. The plates were washed three times with 200 μl serum-buffered bovine serum albumin before its use in adhesion eneayoe. Measurement of the adhesion Loe H. influenzae bio-tinged and diluted 1: 1 with ß-casein or control buffer (serum regulated with phosphate-albumin of bovine serum) and incubated at 37 ° C in a shaking water bath for 30 minutes . 50 μl of the pre-incubation mixture was placed into the appropriate well of the oropharyngeal cell assay plate and incubated for 60 minutes at 37 ° C. The assay was stopped by washing the test plate three times with serum buffered with bovine serum albumin and the heat of the plate fixed at 65 ° C for 10 minutes. After the plate was cooled to room temperature, 100 μl of the Extravidin-peroxidase conjugate (Sigma), diluted 100 vecee in serum-buffered bovine serum albumin, was added to each well and the plate was incubated for 40 minutes at 37 ° C. This conjugate binds to the bacteria labeled with bioethanol. The excess of the conjugate ein uniree was removed by washing the eneayo plate tree times with bovine serum phosphate-buffered albumin and 100 μl of 2.2'-azino-bie (3-ethylbenzothiazolin-6-eulphonic acid) of peroxidase eect. added to each well. The platelets were incubated for 10 minutes and subsequently monitored for color revelation on a Thermomax 96-well plate reader (Molecular Devices, Menlo Park, California, USA) until positive control wells containing oropharyngeal and bacterial cells were found. bioethanol (without ß-casein) reached OD ^ from 2.5 to 3.0. The results of the union were calculated by averaging the results of replica tree. RESULTS OF THE ELISA TEST Since the Detroit 562 ee cells were derived from a pharyngeal tumor, the proteins described in Table 1 were also tested in an anti-adhesion assay with normal human oropharyngeal cells of volunteers. The results of this ELISA assay are shown in Table 2. Once again, ß-casein from native human milk and recombinant human ß-casein was found to be active at 0.5 milligrams / milliliter while bovine ß-casein is active. was inactive in the experiments described in SERIE I. After the analysis of the reading were adjusted for the maximum sensitivity of the assay, the human ß-caffeine and its GLU-C hydrolieate were still active in the concentrations of 0.5 milligram / milliliter and more. (SERIE II). Therefore, these results indicated that recombinant human ß-casein, ß-casein from human milk and its hydrolyzate inhibits the binding of H. influenzae to normal human oropharyngeal cells as well as to human tumor cells.

TABLE 2 PERCENTAGE OF INHIBITION OF THE UNION OF H. influenzae TO THE HUMAN OROPHARING CELLS * NH = NOT MADE From the previous experiments it is concluded that ß-caffeine isolated from breast milk, a recombinant form of ß-casein contained in human milk, and the hydrolyzate of ambae, inhibit the binding of H. influenzae to the human cell 5humanae. In addition, where H influenzae has been identified in the literature as being associated with otitis media, it has been concluded that the identified forms of β-casein can be used in the prevention and treatment of $ 1. otitis media in humans, especially in infant and human. In view of the therapeutic effect of enterally ingested human milk, which contains β-caffeine on otitis media, it is concluded that the previously identified forms of human ß-casein have a therapeutic benefit when ingested enterally (orally). The therapeutic effects described in the preceding paragraph ee may be provided by a liquid enteral nutritional product, such as an infant formula, comprising one or more proteins not contained in human milk in combination with a therapeutically effective amount of At least one of the forms of β-casein described in the preceding paragraph. It is also concluded that the binding of H. influenzae to the human oropharyngeal celle ee can be inhibited by administering it via a naeal screen, or as a throat spray, a formulation containing an amount Therapeutic effect of when less than one of the forms of human '*' ß-casein identified in the preceding paragraph. A nasally administered formulation may be in the form of either droplets or a spray. Intravenous throat and nasal spray products and methods are believed to be effective in inhibiting the binding of H. influenzae to human cells because the interaction of ß-casein and H. influenzae is thought to occur in the nasopharynx via the direct contact after the digestion and ß-casein absorption. It is believed that the previously identified forms of β-casein can be incorporated into any specialized or standard enteral liquid nutrient product containing at least one protein not found in human milk, such as infant formulas based on bovine milk or baeadae in eoya and other drinks consumed by niñoe pequeñoe. In a preferred embodiment in the liquid enteral nutrient product there are no proteins or lactose hydrolysates of breast milk, other than β-casein. Such a product has utility in the treatment and prevention of otitis media in infant and human patients. Although preferred embodiments of the invention have been decribed, it will be apparent to those skilled in the art that changes and modifications can be made in the same divereoe without departing from the spirit or scope of this invention.

**** 'SEQUENCE OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: Abbott Laboratories (ii) TITLE OF THE INVENTION: Inhibition of the Union of H. Influenzae to Human Cells (iii) SEQUENCE NUMBER: 5 (iv) CORRESPONDENCE DIRECTION : (A) RECIPIENT: Lonnie R. Drayer ROSS Products Division Abbott Laboratories (B) STREET: 625 Cleveland Avenue (C) CITY: Columbus (D) STATE: Ohio (E) COUNTRY: United States of America (F) ZIP: 43215 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Diskette, 3.5 inches, 1.44? D memory (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) DATA OF THIS APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF SUBMISSION: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 249,556 ( B) DATE OF SUBMISSION: 26-MAY-1994 (A) NUMBER OF APPLICATION: US 08 / 249,584 (B) DATE OF SUBMISSION: 26-MAY-1994 (ix) ) TELECOMMUNICATIONS INFORMATION: (A) TELEPHONE: (614)624-3774 (B) TELEFAX: (614)624-3074 r, (C) TELEX: None (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCES. (A) LENGTH: 1065 base pairs (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Deconocidated (ii) TYPE OF MOLECULE: cloned cDNA that represents. product of a segment of human genomic DNA (A) DESCRIPTION: human milk ß-casein (iii) HYPOTHETICAL: (iv) UNSENSE: (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: Human (A) ORGANISM: Homo sapiens (B) CEPA: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STATUS: Adult (E) HAPLOTIPO: (F) TYPE OF TISSUE: Mammary gland (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ORGANEL: (vii) IMMEDIATE SOURCE: Human mammary gland (A) LIBRARY: (B) CLONE: (viii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: (B) POSITION ON THE MAP: (C) UNITS: ( ix) FEATURES: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and retry analysis. (D) OTHER INFORMATION: The encoded product of the nucleotide of SEQ ID NO: 1 is the human milk protein, β-caffeine. (x) PUBLICATION INFORMATION: (A) AUTHORS: B. Lonnerdal, and collaborator (B) TITLE: Cloning and eequencing of a cDNA encoding human milk beta-caeein.

'-' (C) REVISTA: Federation European Biochemical Society Letters (D) VOLUME: 269 (E) ISSUE: (F) PAGES: 153-156 (G) DATE: 1990 (H) DOCUMENT NUMBER: (I) DATE OF PRESENTATION: (J) DATE OF PUBLICATION: (K) RELEVANT RESIDUES: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 CGG ATG AAG GTC CTC ATC CTC GCC TGC CTG GTG GCT CTT GCT CTT GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT ACÁ GAA TAC AAG AAA GTT GAG AAG GTT AAA CAT GAG GAC CAG CAG 1 CAA GGA GAG GAT GAA CAC CAG GAT AAA ATC TAC CCC TCT TTC CAG 1 CCA CAG CCT CTG ATC TAT CCA 'TTC GTT GAA CCT ATC CCC TAT GGT 2 TTT CTT CCA CAA AAC ATT CTG CCT CTT GCT CAG CCT GCT GTG GTG 2 CTG CCT GTC 'CCT CAG CCT GAA ATA ATG GAA GTC CCT AAA GCT AAA 3 GAC ACT GTC TAC ACT AAG GGC AGA GTG ATG CCT GTC CTT AAA TCT 3 . CCA ACG ATA CCC TTT TTT GAC CCT CAA ATC CCA AAA CTC ACT GAT 4 CTT GAA AAT CTG CAT CTT CCT CTG CCT CTG CTC CAG CCC TTG ATG 4 CAG CAG GTC CCT CAG CCT ATT CCT CAG ACT CTT GCA CTT CCC CCT 4 CAG CCC CTG TGG TCT GTT CCT CAG CCC AAA GTC CTG CCT ATC CCC 5 CAG CAA GTG GTG CCC TAC 'CCT CAG AGA GCT GTG CCT GTT CAA GCC 5 CTT CTG CTC AAC CAA GAA CTT CTA CTT AAC CCC ACC CAC CAG ATC 6 TAC CCT GTG ACT CAG CCA CTT GCC CCA GTT CAT AAC CCC ATT AGT 6 GTC TAA GAA GAT TTC AAA GTT AAT TTT CCC TCC TTA TTT TTG AAT 7 TGA CTG AGA CTG GAA ATA TGA TGC CTT TTC CGT CTT TGT ATC ACG 7 TTA CCC CAA ATT AAG TAT GTT TGA ATG AGT TTA TAT GGA AAA AAT 8 GAA CTT TGT CCC TTT ATT TAT TTT ATA TAT TAT GTC ATT CAT TTA 8 ATT TGA AAT TTG ACT CAT GAA CTA TTT ACA TTT TCC AAA TCT TAA 9 TTC AAC TAG TAC CAC AGA AGT TCA ATA CTC AT "TGG AAA TGC TAC 9 AAA CAT ATC AAA CAT ATG TAT ACÁ AAT TGT TTC TGG AAT TGT GCT 9 TAT TTT TAT TTC TTT AAG AAT CTA TTT CCT TTC CAG TCA TTT CAA 10 TAA ATT ATT CTT AAG CAT AAA AAA AAA AAA 10) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCES. (A) LENGTH: 105 paree of baees (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Unknown (ii) TYPE OF MOLECULE: synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: ( iv) WITHOUT SENSE: (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: Synthetic oligonucleotide sequence (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STATE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ORGANELO: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN THE GENOMA: ( A) CHROMOSOME / SEGMENT: (B) POSITION ON THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: analysis of £? DNA restriction. (D) OTHER INFORMATION: The eynthetic oligonucleotide assigns the adaptation of the translation sequence to the codon usage of E. coli. (x) PUBLICATION INFORMATION: (A) AUTHORS: L. Haneeon, and collaborator (B) TITLE: Expreeeion of Human Milk ß-caeein in Escherichia coli: Comparison of Recombinant Protein with Native Ieoform. (C) MAGAZINE: Protein Expre- sentation and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373-381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) DATE OF SUBMISSION: (J) ) DATE OF PUBLICATION: (K) RELEVANT RESIDUES: (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2 CTG CAG AAT TCA TAT GCG TGA AAC CAT CGA ATC CCT GAG CTC GAG 45 CGA AGA ATC GAT CAC CGA ATA CAA AAA AGT TGA AAA AGT TAA ACA 90 CGA GGA CCA GGA TCC 105 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCES. (A) LENGTH: 71 base pairs (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Unknown (ii) TYPE OF MOLECULE: synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: ( iv) WITHOUT SENSE: (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: Synthetic oligonucleotide sequence (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STATE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ORGANEL: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN THE GENOME: (A) CHROMOSOME / SEGMENT: (B) POSITION ON THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: analysis of sequence and re-certification of DNA. (D) OTHER INFORMATION: (x) PUBLICATION INFORMATION: (A) AUTHORS: L. Haneeeon, and colaboradoree (B) TITLE: Expreeeion of Human Milk ß-caeein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms. (C) MAGAZINE: Protein Expreeeion and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373-381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) DATE OF PRESENTATION: (J) ) DATE OF PUBLICATION: (K) RELEVANT RESIDUES: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 YEAR A TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA 45 'rTA GTG TCT AAT AAG GAT CCG AAT TC TL (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF SEQUENCES. (A) LENGTH: 68 base pairs (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Unknown (ii) TYPE OF MOLECULE: synthetic oligonucleotide (A) DESCRIPTION: (Üi) HYPOTHETICAL: ( iv) WITHOUT SENSE: (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: Synthetic oligonucleotide sequence (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STATE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ORGANELO: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN THE GENOMA: ( A) CHROMOSOME / SEGMENT: (B) POSITION ON THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: sequencing and DNA restriction analysis . (D) OTHER INFORMATION: (x) PUBLICATION INFORMATION: (A) AUTHORS: L. Hansseon, and collaborator (B) TITLE: Expression of Human Milk ß-casein in Escherichia coli: Comparison of Recombinant Protein with tive Ieoforme. (C) MAGAZINE: Protein Expreeeion and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373-381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) 'DATE OF SUBMISSION: • (J) DATE OF PUBLICATION: ( K) RELEVANT RESIDUES: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 CAT GAA AAA GAA TAT CGC ATT TCT TCT TGC ATC GAT GTT CGT TTT 45 TTC TAT TGC TAC AAA TGC TTA TG ffi (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCES. (A) LENGTH: 37 base pairs (B) TYPE: Nucleic acid (C) TYPE OF CHAIN: Simple (D) TOPOLOGY: Unknown (ii) TYPE OF MOLECULE: synthetic oligonucleotide (A) DESCRIPTION: (Üi) HYPOTHETICAL: ( iv) WITHOUT SENSE: (v) TYPE OF FRAGMENT: (vi) ORIGINAL SOURCE: Synthetic oligonucleotide sequence (A) ORGANISM: (B) CEPA: (C) INDIVIDUAL ISOLATED: (D) DEVELOPMENT STATE: (E) HAPLOTIPO: (F) TYPE OF TISSUE: (G) TYPE OF CELL: (H) CELLULAR LINE: (I) ORGANELO: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN THE GENOMA: ( A) CHROMOSOME / SEGMENT: (B) POSITION ON THE MAP: (C) UNITS: (ix) CHARACTERISTICS: (A) NAME / KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: analysis of DNA sequencing and restriction . (D) OTHER INFORMATION: (x) PUBLICATION INFORMATION: (A) AUTHORS: L. Hanssson, and collaborator (B) TITLE: Expression of Human Milk ß-casein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms. (C) MAGAZINE: Protein Expreseion and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373-381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) DATE OF SUBMISSION: (J) DATE OF PUBLICATION: (K) RELEVANT RESIDUES: (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5 CAT ATG CAC GTG AAA CCA TCG AAT CCC TGA GCT CGA G 37

Claims (14)

1. CLAIMS 1. A liquid enteral nutrient product comprising at least one protein not contained in human milk in combination with at least one material selected from the group consisting of ß-caffeine isolated from human milk, a recombinant form of ß-casein contained in human milk and hydrolysates of both in a therapeutically effective amount that inhibits the binding of H. influenzae to human cells.
2. 2. A liquid enteral nutrient product according to claim 1, wherein the product is a formula for infants.
3. 3. A liquid enteral nutrient product according to claim 1, wherein the human cells are oropharyngeal cells.
4. 4. A liquid enteral nutrient product according to claim 2, wherein the human cells are oropharyngeal cells.
5. 5. A nutritious enteral liquid infant formula comprising at least one protein not containing human milk in combination with at least one material selected from the group consisting of β-casein isolated from human milk, a recombinant form of β-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the binding of H. infl uenzae 'human cells, said infant formula not containing other proteins found in human milk.
6. 6. A liquid enteral nutrient formula according to claim 5, wherein the human cells are 5célulae orofaríngeae.
7. 7. A naturally-administrable formulation comprising at least one material selected from the group which was isolated from ß-casein isolated from human milk, a ß-casein form of the ß-casein contained in human milk, and lOhydrolisate from both in an effective amount Therapeutically it inhibits the binding of H. influenzae to human oropharyngeal cells.
8. 8. A spray formulation comprising at least one material selected from the group that 15considered in ß-casein isolated from human milk, a recombinant form of ß-casein contained in human milk and hydrolysates of both in a therapeutically effective amount that inhibits the binding of H. influenzae to human oropharyngeal cells.
9. 9. A method for inhibiting the binding of H. influenzae to human cells by enterally ingesting a liquid nutritional product comprising at least one protein not contained in human milk in combination with an effective amount of at least one The material selected from the group consisting of ß-casein-slated from human milk, a recombinant form of β-caffeine contained in human milk and hydrolysates of both.
10. 10. A method to inhibit the binding of H. influenzae to human cells in a human infant, Feeding the human infant enterally to the human infant with an infant formula comprising at least one protein not contained in human milk in combination with an effective amount of at least one material selected from the group consisting of β-caffeine The honey of human milk, a recombinant form of ß-casein contained in human milk and hydrolyzate of ambae.
11. 11. A method to treat and prevent otitis media in a human by inhibiting the binding of H. influenzae to human cells by feeding the human with a nutritious product Which comprises at least one protein not contained in human milk in combination with an effective amount of when , menoe a material selected from the group that connects ß-casein isolated from human milk, a recombinant form of ß-casein contained in human milk and hydrolisatoe 20 both
12. 12. A method for treating and preventing otitis media in a human infant by inhibiting the binding of H. influenzae to human cells by feeding the human infant a nutritional product comprising at least one non-contained protein in human milk in combination with a human quantity * of at least one material selected from the group consisting of β-casein isolated from human milk, a recombinant form of ß-casein contained in human milk and hydrolysates of both.
13. 13. A method for inhibiting the binding of H. influenzae to human cells oropharynxes by administering via a nasal passage a formulation containing a therapeutically effective amount of at least one material selected from the group consisting of β-casein isolated from the milk. human, a recombinant form of ß-casein contained in human milk and hydrolysates of both.
14. 14. A method for inhibiting the binding of H. influenzae to human oropharyngeal cells by administering a spray formulation for the throat containing a Therapeutically effective amount of at least one material selected from the group consisting of β-casein isolated from human milk, a recombinant form of β-caffeine contained in human milk and hydrolysates of both.