NZ283281A - Liquid Nutritional Product or throat spray containing beta-casein from Human milk for inhibiting attachment of Haemophilus influenzae to Human cells - Google Patents

Abstract

This use of b-casein contained in human milk, a recombinant form of the b-casein contained in human milk and hydrolysates of both in combination with at least one protein not contained in human milk, is in liquid nutritional products (i.e. milk formula) for inhibiting attachment of H. influenzae to human cells/

Description

% New Zealand No. International No. 283281 NEW ZEALAND PATENTS ACT 1 953 complete specification Title of Invention: Inhibition of attachment of H.Influenzae to human cells Name, address and nationality of applicant(s) as in international application form: ABBOTT LABORATORIES, No organisation found! ofDept. 377/AP6D, 100 Abbott Park Rad, Abbott Park, Illinois 60064-3537, United States of America m 283281 INHIBITION OF ATTACHMENT OF H. INFLUENZAE TQ HUMAN CELLS The present invention relates to the use of native or recombinant human ^■casein and hydrolysates thereof in the preparation of medicaments for inhibiting the attachment of Haemophilium influenzae (H. influenzae) to human cells.

Haemophilus are small. gram-negative, non-molotile, non-spore forming bacilli with complex growth requirements. Diseases caused by H. influenzae usually begin as a nasopharyngitis, possibly precipitated by a viral infection of the upper respiratory tract. Morse, et al. "Haemophilus". MICROBIOLOGY. FOURTH EDITION, published by J.B. Lipincott Company, pages 615-618 (1990).

H. influenzae are spread from person to person by airborne respiratory droplets or direct contact with secretions. To colonize, H. influenzae must contend with ciliary clearance mechanisms of the nasopharyngeal mucosal surface and the mucous barrier. Once past the mucous barrier and the ciliary escalator. H. influenzae attach to mucosal epithelial cells. Invasion of mucosal surfaces appears to be an important characteristic of pathogenic bacteria. Stephens, et al., "Pathogenic Events During Infection of the Human Nasopharynx with Neisseria meningitis and Haemophilus influenzaeREVIEWS OF INFECTIOUS DISEASES. 13:22-23 (1991). It has further been reported that H. influenzae harbored in the nasopharynx are a key factor in the development of middle ear infections (otitis media), and that nor-typable H. influenzae adhere to nasopharyngeal and nasal mucosal cells. Harada et al.. "Adherence of Haemophilus influenzae to nasal, nasopharyngeal and bucal epithelial cells from patients with otitis media" EUROPEAN ARCHIVES OF OTO-RHINO-LARYNGOLOGY. 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). In accordance with the present invention ^-casein isolated from human milk or a recombinant form thereof, or a hydrolysate of either is employed to inhibit the adhesion of H. influenzae to human cells.

Exclusive breast-feeding of human infants for the first four months of life has been associated with a decrease in the incidence of both acute | l^'lcLLECfUAL™pViL.^£HfroVf:iut OF hi. * - g jun 2000 DECEIVED 2 and recurrent otitis media. However, the question remained as to whether it is breast milk per se or the mechanics of breast feeding that exert the protective effect. Sheard. "Breast-Feeding Protects Against Otitis Media". NUTRITION REVIEWS, Vol. 51, No. 9, pages 275-277 (1993). In one study of 1,013 infants followed for their entire first year, All had at least one episode of otitis media and 17* had recurrent otitis media. Infants exclusively breast fed for four or more months had half the mean number of acute otitis media episodes as those not breast fed at all and 402 less than those infants whose diets were supplemented with other foods prior to four months. The recurrent otitis media rate in infants exclusively breast-fed for six months or more was 10£ and was 20.5? in those infants breast-fed for less than four months. Those investigators presented speculations that IgA, or micronutrients or Prostaglandin E, in breast milk may be protective, but concluded that the mechanism for a protective effect of breast-feeding against otitis media was not clear. Duncan et al.. "Exclusive Breast-Feeding for at Least 4 Months Protects Against Otitis Media". PEDIATRICS. Vol. 91. No. 5, pages 867-872 (1993).

WO 91/06308 filed by Andersson et al. for "ANTIBACTERIAL COMPOSITION", and a published article by the same authors (Aniansson et al., "Anti-adhesive activity of human casein against Streptococcus pneumonia and Haemophilus influenzae", MICROBIAL PATHOGENESIS. 8:315-323 (1990) disclose the use of a milk fraction having a molecular weight of at least 5,000 daltons for "therapeutic prophylactic, and/or diagnostic use in infections caused by 5. pneumonae and/or H. influenzae", but it is suggested 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 ^-casein and hydrolysates of both to inhibit H. influenzae infections.

W093/04172 relates to a DNA sequence encoding human /?-casein, but does not disclose the capacity of either native or recombinant human ^-casein to inhibit the attachment of H. influenzae to human cells.

W091/08675 discloses an infant formula which contains recombinant forms of both human alpha-lactalbumin and human /2-casein. However, this publication discloses only that these human milk proteins will "give a simulated human mother's milk formula that does not exhibit the allergenic properties associated with formulas based on cow or other foreign protein." (page 3, lines 20-22). The use of human /?-casein to inhibit the attachment 3 283281 of H. influenzae to human cells is not taught or suggested in said publication.

In a first aspect, the present invention provides the use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both, in the preparation of a liquid nutritional product also comprising at least one protein not containedin human milk, for inhibiting the attachment of H. influenzae to human cells.

In a further aspect, the present invention provides the use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both, in the preparation of an infant formula also- comprising at least one protein not in a human infant.

In yet a further aspect, the present invention provides the use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both, in the preparation of an enteral nutritional product also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human by inhibiting the attachment of H. influenzae to human cells.

In a further aspect, the present invention provides the use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of botn, in the preparation of an enteral formula also comprising at least one protein not inhibiting the attachment of H. influenzae to human cells.

In a still further aspect, the present invention provides the use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both, in the preparation of a formulation for inhibiting the attachment of H. influenzae to human oropharyngeal cells.

SUMMARY OF THE INVENTION for inhibiting the attachment of H. influenzae to human cells for treating and preventing otitis media in a human infant by INTELLECTUAL PROPEHTY QFRcEl OF N.2. ( ( allowed by page 3a) - 6 jun 2cu0 received 283281 In a yet further aspect, the present invention provides a use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for inhibiting the attachment of H. influenzae to human cells.

In a further aspect, the present invention provides a use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for inhibiting the attachment of H. influenzae to human cells in a human infant.

In a still further aspect, the present invention provides a use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the 3-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human by inhibiting the attachment of H. influenzae to human cells.

The present invention further provides a use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human infant by inhibiting the attachment of H, influenzae to human cells. 283281 The two assays Ca radiolabeled assay and an ELISA assay) which were used for determining the bioactivity of 0-casein are described below. These assays have not been published heretofore, although the ELISA assay was based upon established methodology.

MATERIALS USED IN BOTH ASSAYS Haemoohi lus influenzae Haemophilus influenzae {H. influenzae) cultures (fimbriated, nontypable) which have been implicated in otitis media were obtained from Dr. Lauren Bakeletz of The Ohio State University. Columbus, Ohio, U.S.A. The use of these organisms in assays has been described in Bakaletz. et al.. "Frequency of Fimbnation of Nontypable Haemophilus influenzae and its Ability to Adhere to Chinchilla and Human Respiratory Epithelium", Infection and Immunity. 56: 331-335 (1988). The H.influenzae were streaked onto Chocolate agar plates (BBL-Becton Dickinson & Co., Cockeysville. Maryland, U.S.A.) from frozen aliquots of a low passage number and incubated at 37°C in a 52 C02 incubator for about 18 hours to obtain logrithmically growing cultures. The H.influenzae was used in both the Enzyme Linked Imniuno Sorbent Assay (ELISA) assay and the radiolabeling assay as described below.

Native Human ff-Casein ^-casein isolated from human milk was purchased from Symbicom AB. P.O. Box 1451, S-901 24 Umea. Sweden.

Recombinant Human ff-Casein Applicants obtained ^-casein cDNA and the expression system from Symbicom AB. P.O. Box 1451, S-901 24 Ume3, Sweden. The human 0-casein cDNA used had been previously cloned and sequenced by Lonnerdal et al., Cloning and sequencing of a cDNA encoding human milk /?-casein. (SEQ 10 NO: I:) Federation of European Biochemical Societies Letters 269. 153-156 (1990). The recombinant human /?-casein was obtained from £". coli and purified according to the method of Hansson et al..Expression of Human Milk ^-Casein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms.

JNTEOECrjAT™p£KfTGffL^ U of N2. j (followed by page 4) - 6 jun 2000 received 4 Protein Expression and Purification 4, 373-381 (1993). To express human /?-casein in E.coli . /3-casein cDNA was cloned under control of a T7 promoter in wo different expression vectors. One vector, pS26, was designed for intracellular expression. The other vector, pS28, has a signal sequence for extracellular expression. The procedure followed was substantially that described cy Hansson et al.

Human ^-casein cDNA was isolated by Hansson et al. as a 1.1-kb fcoRI fragment from a human lambda gt mammary gland library, and was subcloned into pUC19. which was designated pS21. The cDNA was modified by introduction of synthetic oligonucleotides in the 5' and 3' termini. To introduce a suitable cloning site in the 5' end, Nde\. a translational start, was inserted in front of the sequence encoding mature human /?-casein. To adapt the initial part of the translated sequence to E.coli codon usage, six synthetic oligonucleotides were constructed and ligated. Also, PstI and EcoRl sites were inserted in front of the Ndel site. The sequence of the synthetic fragment was 5' - CTGCAGAATTCATATGCGT GAAACCATCGAATCCCTGAGCTCGAGCGAAGAATCGATCACCGAATACAAAAAAGTTGAAAAAGTTAAACACG AGGACCAGGATCC-3'. (SEQ ID NO: 2:) The protein encoding sequence is underlined. The synthetic fragment was cloned into Pstl/BamHI-digested pUC19 resulting in plasmid pS24. To insert the rest of the ^-casein encoding sequence, a 303-bp Accl/Bglll fragment was isolated and cloned into a pUC18 derivative and designated plasmid pS22. Four synthetic oligonucleotides containing the sequence encoding the carboxy-terminal end and translation stop followed by ffamHI and fcoRI sites were constructed resulting in the sequence 5' AGATCTACCCTGTG A CTCAGC CACTTGCCCCAGTTCATAACCC CATTAGTGTCTAATAAGGATCCGAATTC-3', (SEQ ID NO: 3:) where the protein encoding sequence is underlined, The synthetic fragment was cloned into Bglll/EcoRl digested pS22, resulting in plasmid pS23. To obtain the recombinant modified ^-casein encoding fragment, three fragments were ligated: an 89-bp Pstl/Avall fragment from pS24; a 197 - bp AvalHAccl fragment from pS21; and PstllAccl digested pS23. The resulting plasmid pS25 was digested with Ndel/Bamtil and a 641-bp fragment was isolated and cloned into the vector pET-3a. The resulting expression vector was designated pS26.

In order to construct a vector mediating extracellular expression, the E. coli signal sequence of the enterotoxin STII gene was introduced in front - . 5 of the /?-casein encoding sequence. A modified STII sequence with Ncol- and Ndel-compatible ends and an internal Clal site was obtained by using a synthetic oligonucleotide. 5'-CATGAAAAAGAATATCGCATTTCTTCTTGCATCGATGTTCGTTT TTTCTATTGCTACAAATGCATATG-3' (SEQ ID NO: 4:). To insert the signal sequence in front of the ^-casein encoding sequence, pS25 was digested with Aval/EcoRl and a 619-bp fragment was isolated. This fragment was ligated with a synthetic oligonucleotide fragment, 5'CATATGCACGTGAAACCATCGAATCCCTGAGCTCGAG-3' (SEQ ID NO: 5:), and Ndel/EcoRl-digested pUC19. The resulting plasmid was designated pS27. The final expression vector.pS28, was constructed by ligating three fragments: a 700-bp Ndel/Hindlll /?-casein fragment isolated from pS27, the STII signal sequence, and a Ncol/Hindlll-digested pACAT7 vector.

The expression vectors pS26 and pS28 were used to transform E.coli strains BL2KDE3), BL21(DE3)pLysS, and BL21(DE3)pLysE. The bacteria were grown in Luria Broth medium containing 50 /vg/ml carbenicillin. and when B121 (DE3)plysS and BL21(DE3)pLysE were used the medium was supplemented with 25 pg/ml chloramphenicol.

For induction of expression the cultures were grown to a density yielding an optical density (OD) of 0.6 at a wavelength of 600 nanometers (OD600). then 0.4 mM IPTG was added to induce the T7 system, The cells were harvested about 90 minutes after induction.

Recombinant /?-casein was isolated using standard procedures. The inducible T7-based expression system resulted in high-level expression of recombinant /?-casein. Bacteria were harvested and the cells pel letted by centrifugation. The supernatant contained the periplastic proteins and the pellet the cytoplasmic fraction. The recombinant proteins obtained were compared with native ^-casein, which had been purified by standard methods including either ion-exchange chromatography followed by reversed-phase HPLC or gel filtration. Recombinant and native ^-casein were compared by standard biochemical techniques comprising SDS-PAGE, Western blotting, amino acid analysis.peptide mapping, phosphate analysis, and mass spectrometry. Recombinant /?-casein expressed in E.coli was found to comigrate with full-length, nonphosphorylated native human /3-casein, which is one of seven native isoforms.

Recombinant human ^-casein has also been expressed in S.cerevisiae using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA), but the 6 expression level was approximately 102 of that obtained in E.coli. However, Hansson et al. found that S. cerevisiae appeared to express phosphorylated human milk ^-casein. g-Casein Hvdrolvsates The human ^-casein (both native and recombinant) was digested using the specific endoproteinase GLU-C (Sigma, sequencing grade) which catalyzes the hydrolysis of peptide bonds at the C-terminal of glutamic acid residue. After monitoring the digest 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 NH4HC03, pH 7.8. These digests were dried and resuspended in appropriate buffers prior to use in the assays discussed above. 7 RADIOLABELED ASSAY Detroit 562 cells Detroit 562 pharyngeal carcinoma cells were obtained from the American Type Culture Collection (Rockville, Maryland, U.S.A.). The use of this type of cell in assays has been described in Takahashi , et al. "Phosphorylation Of A Surface Receptor Bound Urokmase-Type Plasminogen Activator In a Human Metastatic Carcinomatous Cell Line", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 182:1466-72. The Detroit 562 cells were cultured in Dulbecco's Modified Eagle Medium (GIBCO, Grand Island, New York, U.S.A.) supplemented with 10£ Fetal Bovine Serum (Hyclone. Logan. Utah. U.S.A.) Cells were routinely subcultured in 75 cm2 flasks (Costar, Cambridge, Massachusetts, U.S.A.) using Trypsin-EDTA (0.25£ trypsin, ImM EDTA (Ethylenediaminetetraacetic acid), (GIBCO) to detach cells. Cell subculture passages 48-75 were utilized for adhesion studies. Cells were seeded into 96 well plates (Costar) at a density of 20,000 ce'. is per well and maintained at 37°C in a 5£ C02 incubator for 8-10 days providing a confluent monolayer for adhesion studies. Plates were washed three times with 200 jul Hanks Balanced salt solution (HBS) (GIBCO), supplemented with 30 mM of N-2-Hydroxyethylpeperazine-N'-2-Ethane Sulfonic Acid (HBS) (GIBCO) to remove serum proteins, before addition of bacteria in adhesion assays.

Radiolabeled of bacterium Harvested bacteria in phosphate buffered saline supplemented with 0,052 Bovine serum albumin (PBS-BSA) were centrifuged and resuspended in a volume of PBS-BSA yielding an optical density (OD) of 2.4 at a wave length of 600 nanometers (OD600). m-IndiuiThoxine (ni-In) (Amersham, Arlington Heights. Illinois, U.S.A.) a high energy, short lived tracer, was utilized to radiolabel the bacteria. (The m-In penetrates the cell membrane, where once inside the cell it dissociates resulting in the binding of U1-Indium ions with cytoplasmic components.) The use of 11L-In labeling in other assays has been described in Ardehali, et al. Mlil-Indium Labeling of Microorganisms to Facilitate the Investigation of Bacterial Adhesion". JOURNAL OF BIOMEDICAL MATERIALS RESEARCH. 27: 269-275 (1993). 50 pCi of an m-In solution was added to 2.5 ml of the bacterial suspension at 37°C and 8 incubated for 20 minutes. The radiolabeled bacteria were then washed two times and resuspended in 5 ml HBS supplemented with 30 mM HEPES buffer. 25 jul of the m-In labeled bacterial suspension were preincubated with 25 //I of ^-casein in a polypropylene 95 well plate for 15 minutes at 37°C to allow binding of /?-casein to bacteria.

Radiolabeled bacterial adhesion measurement /;1 of the preincubation mixture containing both radiolabeled bacteria and ^-casein was pipetted into each well of the assay plate containing Detroit 562 cells. The assay plate was incubated for 20 minutes at 37°C to allow adhesion of the bacterium to the cell monolayer. The plates were then washed three times with HBS to remove nonadhering bacteria from the Detroit 562 cells. The assay was terminated by the addition of 100 p~\ of 1 N sodium hydroxide (NaOH) to disrupt the cell monolayer. The entire contents of each well was placed in a Cobra polypropylene tube (12 mm in diameter and 75 mm in height) (Packard, Meriden, Connecticut, U.S.A.) and counted on a Cobra gamma counter (also from Packard). Results were calculated by averaging the results of four replicates after the subtraction of background radiation. Results are presented as percent inhibition of bacterial attachment in no agent control wells.

PCT/US9S/03789 9 RESULTS FROM RADIOLABELED ASSAY The human and bovine /?-casein solutions were prepared first in 20 mM ethanolamine, 6 M urea. pH 9.5 and then washed twice in PBS by ultrafiltration using Centricon membrane filters (Amicon. MA) with a cut-off of 3,000 daltons. After resuspending in appropriate buffer for the radiolabeled assay described above, these samples were tested in the assay. Experiments with different designated numbers were performed in different days. As shown in Table 1. human ^-casein exhibited an inhibition of 50X or more at concentrations of 0.75 mg/ml or greater. It should be noted than when referring to Table 1, a higher percent inhibition indicates a higher level of bioactivity of the "AGENT", and a lower percent inhibition indicates a lower level of activity of the "AGENT". Bovine /?-casein was not significantly active even at 1.5 mg/ml. These results indicated that /?-casein from human milk has different bioactivity compared to the bovine milk /3-casein.

Hydrolysate of human ^-casein obtained with GLU-C enzyme (prepared as described above) was also active (>50% inhibition) at concentrations of 0.75 mg/ml or higher. When the GLU-C hydrolysate of purified recombinant /?-casein was tested at 3.0 mg/ml, it exhibited activity similar to that of the human milk ^-casein hydrolysate. Hence, this recombinant protein could be produced in large-scale from bacteria to provide an abundant supply of a protein which retains the anti-adhesion activity of native human milk p-casein against H. influenzae. ' 3 o LA OJ K» TABLE 1 B PERCENT INHIBITION OF H. influenzae ATTACHMENT TO DETROIT 562 CELLS AGENT CONCENTRATION MG/ML EXP 1 EXP 2 EXP 3 AVERAGE /?-Casein Isolated from Human Milk 3 67 ND* ND* 67 1.5 71 34 45 50 0.75 60 ND* ND* 60 0.5 ND* 27 28 28 Hydrolysate of ^-Casein Isolated from Human Milk 3 1.5 0.75 60 51 52 Recombinant Human ^-Casein Hydrolysate 3.0 71 Bovine ^-Casein 1.5 0.5 31 24 4 ND = NOT DONE I 11 ELISA ASSAY H. influer)7ae Preparation Harvested H. influenzae from chocolate agar plates were suspended in 7 ml PBS-BSA and biotinylated by incubation with 350 pi of biotin-caproate N-hydroxysuccinimide ester (0.Q1 g biotin/lml dimethyl sulfoxide; Sigma, St. Louis. Missouri, U.S.A.) for 15 minutes at room temperature. The biotinylated bacteria were washed three times at 2,700 rpm for 30 minutes each to remove excess biotin. The labeled bacteria were then resuspended at an OD600 of 2.4 using PBS-BSA.

Oropharyngeal cells Oropharyngeal (OP) cells were collected from donors and pooled in phosphate buffered saline (PBS). Cell suspensions washed one time in PBS were resuspended and counted using a hemocytometer. Cells were adjusted to a density of 2.5 x 105 cells per ml in PBS. To promote OP cell attachment, 96-well plates (Linbro-ICN, Costa Mesa. California, U.S.A.) were coated with L-lysine followed by exposure to 1.25% glutaraldehyde (creates cross-linking). Plates were thoroughly washed to remove any residual glutaraldehyde. Each well of the 96-well plate was inoculated with 50 pi of the OP cell preparation yielding a final concentration of 1.25 x 104 cells per well. Designated wells were incubated with PBS only (no OP cells) to serve as background control wells to measure non specific bacterial binding to plastic. Inoculated plates were centrifuged at 2,700-3,000 rpm for ten minutes to sediment the suspended cell preparation, aspirated and incubated overnight at 37°C in a moist chamber. The next morning plates with OP cells were treated with PBS containing 5% BSA for four hours to prevent non-specific bacterial attachment. The plates were washed three times with 200 pi PBS-BSA before use in adhesion assays.

Adhesion Measurement The biotinylated H. Influenzae were diluted 1:1 with /?-casein or control buffer (PBS-BSA) and incubated at 37°C in a shaking water bath for 30 minutes. 50 pi of the preincubation mixture was placed into the appropriate well of the OP cell assay plate and incubated for 60 minutes at 12 37°C. The assay was halted by washing the assay plates three times with PBS-BSA and the plate heat fixed at 65°C for 10 minutes. After the plate cooled to room temperature, 100 /j! of Extravidin-peroxidase conjugate (Sigma), diluted 100-fold in PBS-BSA, was added to each well and the plate incubated for 40 minutes at 37°C. This conjugate binds to the biotin-labeled bacteria. Excess unbound conjugate was removed by washing the assay plate three times with PBS-BSA and 100 y 1 of peroxidate substrate 2,2'-Azino-bis(3-£thylbenzthiazoline-6-Sulfonic Acid) was added to each well. Plates were incubated for 10 minutes and subsequently monitored for color development on a Thermomax 96 well plate reader (Molecular Devices, Menlo Park, California, U.S.A.) until the positive control wells containing OP cells and biotinylated bacteria (no /?-casein) reached an 0D6M of 2.5 to 3.0, Binding results were calculated by averaging the results of three replicates. 13 RESULTS FROM ELISA AS5AY Since the Detroit 562 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 from volunteers. Results from this ELISA assay are shown in Table 2. Once again, native human milk ^-casein and recombinant human /?-casein were found to be active at 0.5 mg/ml while bovine ^-casein was inactive in the experiments described in SET I. After the analysis of the readings was adjusted for maximum sensitivity of the assay, human ^-casein and its GLU-C hydrolysate were still active at the concentrations of 0.5 mg/ml and above (SET II). Hence these results indicated that the recombinant human ^-casein, human milk /3-casein and its hydrolysate inhibit attachment of H. influenzae to normal human oropharyngeal cells as well as human tumor cells. tn TABLE 2 PERCENT INHIBITION OF H. influenzae ATTACHMENT TO HUMAN OROPHARYNGEAL CELLS tvj -J to SET I AGENT /3-Casein Isolated from Human Mi Ik Recombinant Human ^-Casein Bovine ^-Casein CONCENTRATION MG/ML 0.5 0.5 0.5 EXP 1 EXP 2 45 49 0 58 0 EXP 3 56 53 0 EXP 4 EXP 5 AVERAGE 45 31 37 40 50 0 0 0 SET II AGENT /?-Casein Isolated from Human Milk Hydrolysate of ^-Casein Isolated from Human Milk ND = Not Done CONCENTRATION MG/ML 1 0.5 0.25 1 0.5 EXP 1 EXP 2 88 53 12 20 70 ND* ND* 48 73 EXP 3 68 82 ND* AVERAGE 70 47 20 •A n H W rjt S w 00 PCTAJS95/03789 It has been concluded from the foregoing experiments that ^-casein isolated from human milk, a recombinant form of the ^-casein contained in human milk, and hydrolysates of both, inhibits the attachment of H. influenzae to human cells. Furthermore, inasmuch as H. influenzae have been identified in the literature as being associated with otitis media, it has been concluded that the above identified forms of human /?-casein may be employed in the prevention and treatment of otitis media in humans, especially in human infants. In view of the therapeutic effect of enterally ingested human milk, containing human /?-casein upon otitis media, it is concluded that the above identified forms of human jS-casein have a therapeutic benefit when enterally (orally) ingested.

The therapeutic effects described in the preceding paragraph may be provided by an enteral liquid nutritional product, such as 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 human /?-casein described in the preceding paragraph. It is further concluded that the attachment of H. influenzae to human oropharyngeal cells may be inhibited by administering via a nasal passageway, or as a throat spray, a formulation containing a therapeutically effective amount of at least one of the forms of human /J-casein identified in the preceding paragraph. Such a nasally administered formulation may be in the form of either drops or a spray.

The enteral, throat spray and nasal products and methods are believed to be effective in inhibiting the attachment of H. influenzae to human cells because the interaction of the human jS-casein and H. influenzae is believed to occur in the nasopharynx via direct contact rather than following digestion and absorption of the /?-casein.

It is believed that the above identified forms of human ^-casein may be incorporated into any standard or specialized enteral liquid nutritional product containing at least one protein not found in human milk, such as bovine milk based or soy based infant formulas, and other beverages consumed by young children. In a preferred embodiment no proteins or hydrolysates thereof found in human milk, other than ^-casein, are contained in the liquid enteral nutritional product. Such a product has utility in the treatment and prevention of otitis media in human infants.

While preferred embodiments of the invention have been disclosed, it 16 will be apparent to those skilled in the art that various changes and modifications may be made therein without deviating from the spirit or scope of this invention.

% WO 95/32728 PCT/US95/03789 17 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Abbott Laboratories (ii) TITLE OF INVENTION: Inhibition of Attachment ofH. Influenzae to Human Cells (iii) NUMBER OF SEQUENCES: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: 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 READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: WO 95/32728 PCTAJS95/03789 « 18 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/249,556 (B) FILING DATE: 26-MAY-1994 (A) APPLICATION NUMBER: US 08/249,584 (B) FILING DATE: 26-MAY-1994 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (614) 624-3774 (B) TELEFAX: (614) 624-3074 (C) TELEX: None (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1065 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Cloned cDNA representing the product of a human genomic DNA segment (A) DESCRIPTION: Human milk (Jeta-casein (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Human (A) ORGANISM: Homo sapiens (B) STRAIN: PCT7US95/03789 19 (C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE: Adult (E) HAPLOTYPE: (F) TISSUE TYPE: Mammary gland (G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: Human Mammary Gland (A) LIBRARY: (B) CLONE: (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT.

(B) MAP POSITION: (C) UNITS: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID NO: 1 is the human milk protein, p-casein. (x) PUBUBLICATION INFORMATION: (A) AUTHORS: B. LonnerdaL, et aL (B) TITLE: Cloning and sequencing of a cDNA encoding human milk beta-casein.

(C) JOURNAL: Federation European Biochemical Society Letters % PCT/U S95/03789 (D) VOLUME. 269 (E) ISSUE: (F) PAGES: 153-156 (G) DATE: 1990 (H) DOCUMENT NUMBER: (I) FILING DATE: (J) PUBLICATION DATE: (K) RELEVANT RESIDUES: <xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 CGG ATG AAG GTC CTC ATC CTC GCC TGC CTG GTG GCT CTT GCT CTT 4 5 GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT 90 ACA GAA TAC AAG AAA GTT GAG AAG GTT AAA CAT GAG GAC CAG CAG 13 5 CAA GGA GAG GAT GAA CAC CAG GAT AAA ATC TAC CCC TCT TTC CAG 180 CCA CAG CCT CTG ATC TAT CCA TTC GTT GAA CCT ATC CCC TAT GGT 225 TTT CTT CCA CAA AAC ATT CTG CCT CTT GCT CAG CCT GCT GTG GTG 2 70 CTG CCT GTC CCT CAG CCT GAA ATA ATG GAA GTC CCT AAA GCT AAA 315 GAC ACT GTC TAC ACT AAG GGC AGA GTG ATG CCT GTC CTT AAA TCT 36 0 CCA ACG ATA CCC TTT TTT GAC CCT CAA ATC CCA AAA CTC ACT GAT 40 5 CTT GAA AAT CTG CAT CTT CCT CTG CCT CTG CTC CAG CCC TTG ATG 45 0 CAG CAG GTC CCT CAG CCT ATT CCT CAG ACT CTT GCA CTT CCC CCT 495 CAG CCC CTG TGG TCT GTT CCT CAG CCC AAA GTC CTG CCT ATC CCC 54 0 CAG CAA GTG GTG CCC TAC CCT CAG AGA GCT GTG CCT GTT CAA GCC 5 85 CTT CTG CTC AAC CAA GAA CTT CTA CTT AAC CCC ACC CAC CAG ATC 63 0 TAC CCT GTG ACT CAG CCA CTT GCC CCA GTT CAT AAC CCC ATT AGT 67 5 GTC TAA GAA GAT TTC AAA GTT AAT TTT CCC TCC TTA TTT TTG AAT 72 0 TGA CTG AGA CTG GAA ATA TGA TGC CTT TTC CGT CTT TGT ATC ACG 765 TTA CCC CAA ATT AAG TAT GTT TGA ATG AGT TTA TAT GGA AAA AAT B10 GAA CTT TGT CCC TTT ATT TAT TTT ATA TAT TAT GTC ATT CAT TTA 855 ATT TGA AAT TTG ACT CAT GAA CTA TTT ACA TTT TCC AAA TCT TAA 90 0 TTC AAC TAG TAC CAC AGA AGT TCA ATA CTC ATT TGG AAA TGC TAC 94 5 AAA CAT ATC AAA CAT ATG TAT ACA AAT TGT TTC TGG AAT TGT GCT 99 0 TAT TTT TAT TTC TTT AAG AAT CTA TTT CCT TTC CAG TCA TTT CAA 103 5 TAA ATT ATT CTT AAG CAT AAA AAA AAA AAA 1055 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS: 21 (A) LENGTH: 105 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Synthetic Ohgonucleotide Sequence (A) ORGANISM: (B) STRAIN; (C) INDIVIDUAL ISOLATE; (D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE: (F) TISSUE TYPE: (G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: WO 95/32728 PCT/US95/03789 22 (B) MAP POSITION: (C) UNITS: FEATURE: (A) NAME/KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The synthetic ohgonucleotide assures adaptation of translation sequence to E. coh codon usage.

PUBUBLICATION INFORMATION: (A) AUTHORS: L. Hansson, et al.

(B) TITLE: Expression of Human Milk p-casein in Escherichia coh: Comparison of Recombinant Protein with Native Isoforms.

(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373 - 381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) FILING DATE: (J) PUBLICATION DATE: (K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: 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 (ix) (x) (2) INFORMATION FOR SEQ ID NO: 3 23 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 71 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Synthetic Oligonucleotide Sequence (A) ORGANISM: (B) STRAIN: (C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE: (F) TISSUE TYPE: (G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: (viii) POSITION IN GENOME: WO 95/32728 PCT/US95/03789 24 (A) CHROMOSOME/SEGMENT: (B) MAP POSITION: (C) UNITS: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The synthetic oligonucleotide encodes the carboxy-terminal end and tranlation stop. (x) PUBUBLICATION INFORMATION: (A) AUTHORS: L. Hansson, et al.

(B) TITLE: Expression of Human Milk p-casein in Escherichia coli: Comparison of Recombinant Proteb with Native Isofonns.

(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373 - 381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) FILING DATE: (J) PUBLICATION DATE: (K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ED NO: 3 AGA TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA 45 TTA GTG TCT AAT AAG GAT CCG AAT TC 71 INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 68 base pairs (B) TYPE: Nucleic acid (C) STRAKDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: (iv) ANTI- SENSE: (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Synthetic Ohgonucleotide Sequence (A) ORGANISM: (B) STRAIN: (C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE: (F) TISSUE TYPE: (G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: t WO 95/32728 PCT/US95/03789 26 (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT.

(B) MAP POSITION: (C) UNITS: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: Modified enterotoxin STII signal, sequence. (x) PUBUBLICATION INFORMATION: (A) AUTHORS: L. Hansson, et al.

(B) TITLE: Expression of Human Milk p-casein in Escherichia coh: Comparison of Recombinant Protein with Native Isoforms.

(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373 - 381 (G) DATE. 1993 (H) DOCUMENT NUMBER: (I) FILING DATE: (J) PUBLICATION DATE: (K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 CAT GAA AAA GAA TAT CGC ATT TCT TCT TGC ATC GAT GTT CGT TTT 4 5 TTC TAT TGC TAC AAA TGC ATA TG gg WO 95/32728 PCTAJS95/03789 * ) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 37 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY. Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide (A) DESCRIPTION: (iii) HYPOTHETICAL: (iv) ANTI-SENSE: (v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Synthetic Ohgonucleotide Sequence (A) ORGANISM: (B) STRAIN: (C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE: (E) HAPLOTYPE: (F) TISSUE TYPE: (G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE: (vii) IMMEDIATE SOURCE: (A) LIBRARY: (B) CLONE: t WO 95/32728 FCT/US95/03789 28 (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: (B) MAP POSITION: (C) UNITS: (ix) FEATURE: (A) NAME/KEY: (B) LOCATION: (C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: (x) PUBUBLICATION INFORMATION: (A) AUTHORS: L. Hansson, et aL (B) TITLE: Expression of Human Milk p-casein in Escherichia coh: Comparison Recombinant Protein with Native Isoforms.

(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE: (F) PAGES: 373 - 381 (G) DATE: 1993 (H) DOCUMENT NUMBER: (I) FILING DATE: (J) PUBLICATION DATE: (K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 CAT ATG CAC GTG AAA CCA TCG AAT CCC TGA GCT CGA G 3 29

Claims (12)

WHAT WE CLAIM IS: 28 32

1. Use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both, in the preparation of a liquid nutritional product also comprising at least one protein not contained in human milk, for inhibiting the attachment of H. influenzae to human cells.

2. Use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both, in the preparation of an infant formula also comprising at least one protein not contained in human milk, for inhibiting the attachment of H. influenzae to human cells in a human infant.

3. Use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both, in the preparation of an enteral nutritional product also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human by inhibiting the attachment of H. influenzae to human cells.

4. Use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both, in the preparation of an enteral formula also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human infant by inhibiting the attachment of H. influenzae to human cells.

5. Use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for inhibiting the attachment of H. influenzae to human cells.

6: Use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for inhltt^5(fhftSi^|3^RP^p^f|H. influenzae to human cells in a human infant. ' 0F ~ 6 jun 2000

7. Use of at least one material selected from the ^oup .consisting ofJ3-casfein 30 28 3 28 1 contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both in the preparation of a medicament also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human by inhibiting the attachment of H. influenzae to human cells.

8. Use of at least one material selected from the group consisting of P-casein contained in human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in the preparation of a medicament, also comprising at least one protein not contained in human milk, for treating and preventing otitis media in a human infant by inhibiting the attachment of H. influenzae to human cells.

9. Use of at least one material selected from the group consisting of p-casein contained in human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both, in the preparation of a formulation for inhibiting the attachment of H. influenzae to human oropharyngeal cells.

10. A use as claimed in claim 5 wherein the formulation is formulated for nasal administration.

11. A use as claimed in claim 5 wherein the formulation is formulated as a throat spray.

12. A use as defined in any one of claims 1 to 9 substantially as herein described with reference to any example thereof. By the authorised averts INTELLECTUAL ffiLPERTY OFRUl OF N.z. " 6 JUU 2000 END