AU695101B2

AU695101B2 - Inhibition of attachment of (H. influenzae) to human cells

Info

Publication number: AU695101B2
Application number: AU21293/95A
Authority: AU
Inventors: Steven Neal Anderson; Linda Ann Harvey; Pradip Mukerji; Amanda Eun-Yeong Seo
Original assignee: Abbott Laboratories
Current assignee: Abbott Laboratories
Priority date: 1994-05-26
Filing date: 1995-04-18
Publication date: 1998-08-06
Anticipated expiration: 2015-04-18
Also published as: AU2129395A; EP0760673A1; WO1995032728A1; MX9605829A; JPH10500101A; NZ283281A; CA2190610A1

Description

1~1_ WO 95/32728 PCT/US95/03789 INHIBITION OF ATTACHMENT OF H. INFLUENZAE TO HUMAN CELLS The present invention relates generally to inhibiting the attachment of Haemophilus influenzae to human cells, and more specifically to the use of native or recombinant human #-casein and hydrolysates thereof for inhibiting the attachment of Haemophilus influenzae 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 influenzae", REVIEWS 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 non-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 4as$opharyngeal 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 WO 95/32728 PCT/US95/03789 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, 47% 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 40% 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., "Antiadhesive 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 S. 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 P-casein, but does not disclose the capacity of either native or recombinant human B-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 B-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 B-casein to inhibit the attachment I ii i L Li I L~I 3 Sof H. influenzae to human cells is not taught or suggested in said publication.

Accordingly, there is provided according to a first embodiment of the invention a liquid enteral nutritional composition comprising at least one protein not contained in human milk in combination with at least one material selected from the group consisting s of a hydrolysate of P-casein isolated from human milk and a hydrolysate of a recombinant form of P-casein contained in human milk in a tnerapeutically effective amount which inhibits the attachment of H. influenzae to human cells.

According to a second embodiment of the invention there is provided a liquid enteral nutritional infant formula comprising at least one protein not contained in human lo milk in combination with at least one material selected from the group consisting of a hydrolysate of P-casein is!ated from human milk and a hydrolysate of a recombinant form of P-casein contained in human milk in a therapeutically effective amount which inhibits the attachment of H. influenzae to human cells, said infant formula containing no other proteins which are found in human milk.

According to a third embodiment of the invention there is provided a nasally administrable formulation comprising at least one material selected from the group consisting of p-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the attachment of H. influenzae to human oropharyngeal cells.

According to a fourth embodiment of the invention there is provided a throat spray formulation comprising at least one material sel ;ted from the group consisting of P-casein isolated from human milk, a recombinant form of the P-casein contained in human mill- and hydrolysates of both in a therapeutically effective amount which inhibits the attachment of H. influenzae to human oropharyngeal cells.

According to a fifth embodiment of the invention there is provided a method of inhibiting the attachment 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 a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the ;30 p-casein contained in human milk and hydrolysates of both.

According to a sixth embodiment of the invention there is provided a method of inhibiting the attachment of H. influenzae to human cells in a human infant by enterally feeding to said human infant an infant formula comprising at least one protein not contained in human milk in combination with a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both.

According to a seventh embodiment of the invention there is provided a method of treating and preventing otitis media in a human by inhibiting the attachment of Sinfluenzae to human cells by feeding to said human an enteral nutritional product ho 1pris ing at least one protein not contained in human milk in combination with a ia I ,f'S 3 n INAUlB270l001l:SSC 1

I

3a therapeutically effective amount of at least one material selected from the group consisting of p-casein isolated from human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both.

According to an eighth embodiment of the invention there is provided a method of treating and preventing otitis media in a human infant by inhibiting the attachment of H. influenzae to human cells by feeding to said human infant an enteral formula comprising at least one protein not contained in human milk in combination with a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the 0-casein contained in human milk and hydrolysates of both.

According to a ninth embodiment of the invention there is provided a method of inhibiting the attachment of H. influenzae to human oropharyngeal cells by administering via a nasal passageway a formulation containing a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both.

According to a tenth embodiment of the invention there is provided A method of inhibiting the attachment of H. influenzae to human oropharyngeal cells by administering a throat spray formulation containing a therapeutically effective amount of at least one material selected from the group consisting of p-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both.

The two assays (a radiolabeled assay and an ELISA assay) which were used for determining the bioactivity of p-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 00 *s 0 0r 00 0 q~ 0 4 r 0001 eOO.

oA Ivjv o Haemophilus influenzae Haemophilus inluenzae 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 so described in Bakaletz, et al., "Frequency of Fimbriation 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 5% CO 2 incubator for about 18 hours to obtain logarithmically growing cultures. The H. influenzae was used in both the Enzyme Linked Immuno Sorbent Assay (ELISA) assay and the radiolabeling assay as described below.

ku IIJILVCtfl"'- IN :XL IB-_ZY;- 3b Native Human P-casein P-casein isolated from human milk was purchased from Symbicom AB, P.O. Box 1451, S-901 24 Umea, Sweden.

Recombinant Human p-casein Applicants obtained P-casein cDNA and the expression system from Symbicom AE, P.O. Box 1451, S-901 24 Umea, Sweden. The human p-casein cDNA used had been previously cloned and sequenced by Lonnerdal et al., Cloning and sequencing of a cDNA encoding human milk p-casein. (SEQ ID No: Federation of European Biochemical Societies Letters 269, 153-156 (1990). The recombinant human P-casein was obtained from E. coli and purified according to the method of Hansson et al., Expression of Human Milk P-casein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms.

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C3

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C(iNA LIM100 I:ss C t R$ts 47'.

l T (N\BZOO 1 :SS WO 95/32728 PCTJUS95/03789 4 Protein Expression and Purification 4, 373-381 (1993). To express human casein in E.coli, B-casein cDNA was cloned under control of a T7 promoter in two 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 by Hansson et al.

Human 1-casein cDNA was isolated by Hansson et al. as a 1.1-kb EcoRI 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, NdeI, 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 EcoRI sites were inserted in front of the Ndel site. The sequence of the synthetic fragment was

GAAACCATCGAATCCCTGAGCTCGAGCGAAGAATCGATCACCGAATACAAAAAAGTTGAAAAAGTTAAACACG

AGGACCAGGATCC-3'. (SEQ ID NO: 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/BglII 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 BamHI and EcoRI sites were constructed resulting in the sequence CTCAGCCACTTGCCCCAGTTCATAACCCCATTAGTGTCTAATAAGGATCCGAATTC-3', (SEQ ID NO: where the protein encoding sequence is underlined. The synthetic fragment was cloned into BgIII/EcoR digested pS22, resulting in plasmid pS 23 To obtain the recombinant modified #-casein encoding fragment, three fragments were ligated: an 89-bp Pstl/AvaII fragment from pS24; a 197-bp AvaII/AccI fragment from pS21; and Pstl/AccI digested pS23.

The resulting plasmid pS25 was digested with Ndel/BamHI 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 une•nd Th ytei rgetwscondit sIBmIdgse pUC1 _eutn _n plsmi pS4 oisr h rs fte^cs .i I. I-i WO 95/32728 PCT/US95/03789 of the f-casein encoding sequence. A modified STII sequence with Ncol- and NdeI-compatible ends and an internal C7al site was obtained by using a synthetic ol i gonucl eoti de, 5' CATGAAAAAGAATATCGCATTTCTTCTTGCATCGATGTTCGTTT TTTCTATTGCTACAAATGCATATG-3' (SEQ ID NO: To insert the signal sequence .in front of the #-casein encoding sequence, pS25 was digested with Aval/EcoRI and a 619-bp fragment was isolated. This fragment was ligated with a synthetic oligonucl eoti de fragment, 5'CATATGCACGTGAAACCATCGAATCCCTGAGCTCGAG-3' (SEQ ID NO: and Ndel/EcoRIdigested pUC19. The resulting plasmid was designated pS27. The final expression vector,pS28, was constructed by ligating three fragments: a 700bp Ndel/HindIII #-casein fragment isolated from pS27, the STII signal sequence, and a NcoI/HindIII-digested pACAT7 vector.

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

(OD

600 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 h inducible T7-based expression system resulted in high-level expression of recombinant B-casein. Bacteria were harvested and the cells pelletted by centrifugation. The supernatant contained the periplasmic proteins and the V 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 I 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 p-casein expressed in E.coli was found to comigrate with fulllength, nonphosphorylated native human /-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 WO 95/32728 PCT/US95/03789 6 expression level was approximately 10% of that obtained in E.coli. However, Hansson et al. found that S. cerevisiae appeared to express phosphorylated human milk #-casein.

B-Casein Hvdrolysates 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 0 C in 0.1 M NH 4

HCO

3 pH 7.8. These digests were dried and resuspended in appropriate buffers prior to use in the assays discussed above.

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WO 95/32728 PCT/US95/03789 7 RADIOLABELED ASSAY Detroit 562 cells Detroit 562 pharyngeal carcinoma cells were obtained from the American Type Culture Collection (Rockville, Maryland, The use of this type of cell in assays has been described in Takahashi, et al. "Phosphorylation Of A Surface Receptor Bound Urokinase-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 cm 2 flasks (Costar, Cambridge, Massachusetts, using Trypsin-EDTA (0.25% trypsin, ImM EDTA 'Ethylenediaminetetraacetic acid), (GIBCO) to detach cells. Cell subculture passages 48-75 were utilized for adnesion studies. Cells were seeded into 96 well plates (Costar) at a density of 20,000 cells per well and maintained at 37 0 C in a 5% CO, incubator for 8-10 days providing a confluent monolayer for adhesion studies. Plates were washed three times with 200 pI 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.

Radiolabelinq of bacterium Harvested bacteria in phosphate buffered saline supplemented with 0.05% 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). i"-Indium-oxine (Amersham, Arlington Heights, Illinois, a high energy, short lived tracer, was utilized to radiolabel the bacteria. (The penetrates the cell membrane, where once inside the cell it dissociates resulting in the binding of Il-Indium ions with cytoplasmic components.) The use of labeling in other assays has been described in Ardehali, et al. ""'-Indium Labeling of Microorganisms to Facilitate the Investigation of Bacterial Adhesion", JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, 27: 269-275 (1993). 50 pCi of an solution was added to 2.5 ml of the bacterial suspension at 37 0 C and ;i WO 95/32728 PCT/US95/03789 8 incubated for 20 minutes. The radiolabeled bacteria were then washed two times and resuspended in 5 ml HBS suppiemented with 30 mM HEPES buffer.

pl of the "1-In labeled bacterial suspension were preincubated with pl of f-casein in a polypropylene 96 well plate for 15 minutes at 37 0 C to allow binding of f-casein to bacteria.

Radiolabeled bacterial adhesion measurement pl of the preincubation mixture containing both radiolabeled bacteria and B-casein was pipetted into each well of the assay plate containing Detroit 562 ,ells. 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 ul of 1 N sodium hydroxide (NaOH) to disrupt the cell monolayer. The entire contents of each well was placed in z, Cobra polypropylene tube (12 mm in diameter and 75 mm in height) (Packard, Meriden, Connecticut, and counted on a Cobra gamma counter (also from Packard). Resuits 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 wel's.

WO 95/32728 PCT.JS95/03789 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 B-casein exhibited an inhibition of 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 R-casein was not significantly active even at 1.5 mg/ml. These results indicated that Pcasein from human milk has different bioactivity compared to the bovine milk /-casein.

Hydrolysate of human -casein obtained with GLU-C enzyme (prepared as described above) was also active inhibition) at concentrations of 0.75 mg/ml or higher. When the GLU-C hydrolysate of purified recombinant Pcasein was tested at 3.0 mg/ml, it exhibited activity similar to that of the human milk f-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 casein against H. influenzae.

TABLE 1 PERCENT INHIBKTION OF H.influenzae TO DETROIT 562O CELLS

ATTACHMENT

AGENT

fl-Casein Isolated from Human Milk Hydrolysate of fl-Casein Isolated from Human Milk Recombinant Human fl-Casein Hydrolysate Bovine fl-Casein

CONCENTRATION

MG/ML

3 0.75 3 0.75 EXP 1 67 71 60

ND*

EXP 2

ND*

34

ND*

27 EXP 3

ND*

45

ND*

28

AVERAGE

67 28 ND NOT DONE rllrr' i I -r I i I i; Y WO 95/32728 PCT/US95/03789 11 ELISA ASSAY H. influenzae Preparation Harvested H. inf7uenzae from chocolate agar plates were suspended in 7 ml PBS-BSA and biotinylated by incubation with 350 I1 of biotin-caproate N-hydroxysuccinimide ester (0.01 g biotin/lml dimethyl sulfoxide; Sigma, St.

Louis, Missouri, for 15 minutes at room temperature. The Jbiotinylated 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 OD 600 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, 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 pl 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 0 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 p1 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 0 C in a shaking water bath for minutes. 50 pl of the preincubation mixture was placed into the appropriate well of the OP cell assay plate and incubated for 60 minutes at i t.

1 t p I Il WO 95/32728 PCT/US95/03789 37 0 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 pl 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 0 C. This conjugate binds to the biotinlabeled bacteria. Excess unbound conjugate was removed by washing the assay plate three times with PBS-BSA and 100 pl of peroxidate substrate 2,2'- Azino-bis(3-Ethylbenzthiazoline-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, until the positive control wells containing OP cells and biotinylated bacteria (no /-casein) reached an ODRoo of 2.5 to Binding results were calculated by averaging the results of three replicates.

4 Ix. :-i WO 95/32728 PCT/US95/03789 RESULTS FROM ELISA ASSAY 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 R-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 #-casein and its hydrolysate inhibit attachment of H. influenzae to normal human oropharyngeal cells as well as human tumor cells.

e

I-

1; i' a TABLE 2 PERCENT INHIBITION OF H. influenzae ATTACHMENT TO HUMAN OROPHARYNGEAL CELLS SET I AGENT #-Casein Isolated from Human Milk Recombinant Human #-Casein Bovine #-Casein

AGENT

#-Casein Isolated from Human Milk

CONCENTRATION

MG/ML

0.5 0.5 0.5

CONCENTRATION

MG/ML

1 0.25 1 0.5 EXP 1 10 49 0 EXP 1 53 12 20 70 EXP 2 45 58 0 EXP 2 88

ND*

48 73 EXP 3 56 53 0 EXP 3 68 82

ND*

EXP 4 45 EXP 5 31

AVERAGE

37 0 SET II AVERAGE 47 Hydrolysate of #-Casein Isolated from Human Milk ND Not Done

.L

WO 95/32728 PCT/US95/03789 It has jeen concluded from the foregoing experiments that #-casein isolated from human milk, a recombinant form of the P-casein contained in human milk, and hydrolysates of both, inhibits the attachment of H.

influenzae to human cells. Furthermore, inasmuch as H. infiuenzae 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 B-casein upon otitis media, it is concluded that the above identified forms of human #-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 B-casein described in the preceding paragraph. It is further concluded that the attachment of H. influ-.r' to human oropharyngeal cells may be inhibited by administering via a nuL^ passageway, or as a throat spray, a formulation containing a therapeutically effective amount of at least one of the forms of human f-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 P-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 B-casein may be incorporated into any standard or specialized enteral liquid nutritional Sproduct 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 Ithereof 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.

'I While preferred embodiments of the invention have been disclosed, it and the radiolabeling assay as described below.

IN:\LIBZZO00011;SSC WO 95/32728 PCT/US95/03789 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.

i 1 i I WO 95/32728 PCT/US95/03789 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Abbott Laboratories (ii) TITLE OF INVENTION: Inhibition of Attachment ofH. Influenzae to Human Cells (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Lonnie R. Drayer ROSS Products Division Abbott Laboratories STREET: 625 Cleveland Avenue CITY: Columbus STATE: Ohio COUNTRY: United States of America ZIP: 43215 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage COMPUTER: IBM Compatible OPERATING SYSTEM: MS-DOS Version 6.21 SOFTWARE: WordPerfect Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE:

CLASSIFICATION:

L t. co17 signal sequence of the enterotoxin STII gene was introduced in front WO 95/32728 PCT/US95/03789 18 (vii) PRIOR APPLICATION DAT';: APPLICATION NUMBER: US 08/249,556 FILING DATE: 26-MAY-1994 APPLICATION NUMBER: US 08/249 584 FILING DATE: 26-MAY-1994 (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (614) 624-3774 TELEFAX: (614) 624-3074 TELEX: None INFORMATION FOR SEQ ID NO: 1 SEQUENCE CHARACTERISTICS: LENGTH: 1065 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Unknown (ii) MOLECULE TYPE: Cloned cDNA representing the product of a human genomic DNA segment DESCRIPTION: Human milk peta-casein S(iii)

HYPOTHETICAL:

(iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Human ORGANISM: Homo sapiens

STRAIN:

using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA), Dut tne No I WO 95/32728 PCT/US95/03789 19 INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE: Adult

HAPLOTYPE:

TISSUE TYPE: Mammary gland CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE: Human Mammary Gland

LIBRARY-

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: DNA sequering and restriction analysis a OTHER INFORMATION: The encoded product ofnucleotide SEQ ID NO: 1 is the human milk protein, p-casein.

PUBUBLICATION INFORMATION: AUTHORS: B. Lonnerdal, et al.

TITLE: Cloning and sequencing of a cDNA encoding human milk betacasein.

JOURNAL: Federation European Biochemical Society Letters h i n WO 95/32728 WO 9532728PCTIUS95/03789 VOLUME: 269

ISSUE:

PAGES: 153 156 DATE: 1990 DOCUMENT NUMB3ER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ lID NO: 1

CGG

GCA

ACA

CAA

CCA

TTT

CTG

GAC

CCA

CTT

CAG

CTT

TAC

GTC

TGA

TTA

GAA

ATT

TTC

AAA

TAT

TAA

ATG

AGG

GAA

GGA

CAG

CTT

CCT

ACT

ACG

GAA

CAG

CCC

CAA

CTG

CCT

TAA

CTG

CCC

CTT

TGA

AAC

CAT

TTT

ATT

AAG

GAG

TAC

GAG

CCT

CCA

GTC

ATA

AAT

GTC

CTG

GTG

CTC

GTG

GAA

AGA

CA.A

TGT

AAT

TAG

ATC

TAT

ATT

GTC

ACC

AAG

GAT

CTG

CAA

CCT

TAC

CCC

CTG

CCT

TGG

GTG

AAC

ACT

GAT

CTG

ATT

CCC

TTG

TAC

AAA

TTC

CTT

CTC

ATA

GAA

ATC

AAC

CAG

ACT

TTT

CAT

CAG

TCT

CCC

CAA

CAG

TTC

GAA

AAG

TTT

ACT

CAC

CAT

TTT

AAG

ATC

GAA

GTT

CAC

TAT

ATT

CCT

AAG,

TTT

CTT

CCT

GTT

TAC

GAA

CCA

AAA

ATA

TAT

ATT

CAT

AGA

ATG

AAG

CAT

CTC

AGC

GAG

CAG

CCA

CTG

GAA

GGC

GAC

CCT

ATT

CCT

CTT

GTT

TGA

GTT

TAT

GAA

AGT

TAT

AAT

AAA

GCC

CTT

AAG

GAT

TTC

CCT

ATA

AGA

CCT

CTG

CCT

CAG

CTA

GCC

AAT

TGA

TTT

CTA

TCA

ACA

CTA

AAA

TGC

TCA

GTT

AAA

OTT

CTT

ATG

GTG,

CAA

CCT

CAG

CCC

AGA

CTT

CCA

TTT

CTT

ATG

ATA

TTT

ATA

AAT

TTT

AA

CTG

AGC

AAA

ATC

GAA

GCT

GAA

ATG

ATC

CTG

ACT

AAA

AAC

GTT

CCC

TTC

AGT

TAT

ACA

CTC

TOT

CCT

GTG

AGT

CAT

TAC

CCT

CAG

GTC

CCT

CCA

CTC

CTT

GTC

GTG

CCC

CAT

TCC

CGT

TTA

TAT

TTT

ATT

TTC

GCT

GAG

CCC

ATC

CCT

GTC

AAA

CAG

GCA

CTG

CCT

ACC

AAC

TTA

CTT

TAT

GTC

TCC

TGG

CAG

CTT

GAA

GAC

TCT

CCC

GCT

AAA

CTT

CTC

CCC

CTT

CCT

OTT

CAC

CCC

TTT

TOT

GGA

ATT

AAA

AAT

TCA

OCT

TCT

CAG

TT C

TAT

GTG

GCT

AAA

ACT

TTG

CCC

ATC

CAA

CAG

ATT

TTG

ATC

AAA

CAT

TCT

TGC

TGT

TTT

CTT

ATT

CAG

GT

GTG

AAA

TCT

GAT

ATG

CCT

CCC

GCC

ATC

AGT

AAT

ACG

AAT

TTA

TAA

TAC

GCT

CAA

135 180 225 270 315 360 405 450 495 540 585 630 675 720 765 810 855 900 945 990 1035 1065 INFORMATION FOR SEQ ID NO: 2 SEQUENCE CHARACTERISTICS: "-In solution was added to 2.5 ml of the bacterial suspension at 37 0 C and

~CY~

m WO 95/32728 21 LENGTH: 105 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide

DESCRIPTION:

(iii) HYPOTHETICAL: (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOUTRE: Synthetic Oligonucleotide Sequence

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

EL1CU 95/03789 *Z i 4 U WO 95/32728 PCT/US95/03789 22 MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD. DNA sequencing and restriction analysis OTHER INFORMATION: The synthetic oligonucleotide assures adaptation of translation sequence to E. coli codon usage.

PUBUBLICATION

INFORMATION:

AUTHORS: L. Hansson, et al.

TITLE: Expression of Human Milk P-casein in Escherichia coli: Comparison of Recombinant Protein with Native Isoforms.

JOURNAL: Protein Expression and Purification VOLUME: 4

ISSUE:

PAGES: 373 381 DATE: 1993 DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 CTG CAG AAT TCA TAT GCG TGA AAC CAT CGA ATC CCT GAG CTC GAG CGA AGA ATC GAT CAC CGA ATA CAA AAA AGT TGA AAA AGT TAA ACA CGA GGA CCA GGA TCC 105 INFORMATION FOR SEQ ID NO: 3 i P1' 7 WO 95/32728 PCT/US95/03789 23 SEQUENCE CHARACTERISTICS: LENGTH: 71 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide

DESCRIPTION:

(iii) HYPOTHETICAL: (iv) ANTI-SENSE: FRAGMENT TYPE: (vi) ORIGINAL SOURCE: Synthetic Oligonucleotide Sequence

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

(viii) POSITION IN GENOME: WO 95/32728 PCT/US95/03789 24

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: DNA sequencing and restriction analysis OTHER INFORMATION: The synthetic oligonucleotide encodes the carboxyterminal end and tranlation stop.

PUBUBLICATION INFORMATION: AUTHORS: L. Hansson, et al.

JOURNAL: Protein Expression and Purification VOLUME: 4

ISSUE:

PAGES: 373-381 DATE: 1993 DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 AGA TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA TTA GTG TCT AAT AAG GAT CCG AAT TC 71 WO 95/32728 PCT/US95/03789 INFORMATION FOR SEQ ID NO: 4 SEQUENCE CHARACTERISTICS: LENGTH: 68 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide

DESCRIPTION:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

ila WO 95/32728 PCT/UST 5/03789 26 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: DNA sequencing and restriction analysis OTHER INFORMATION: Modified enterotoxin STII signal sequence.

PUBUBLICATION INFORMATION: AUTHORS: L. Hansson, et al.

JOURNAL: Protein Expression and Purification VOLUME: 4

ISSUE:

PAGES: 373 381 DATE: 1993 DOCUMENT NUMBER: 4 FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 CAT GAA AAA GAA TAT CGC ATT TCT TCT TGC ATC GAT GTT CGT TTT TTC TAT TGC TAC AAA TGC ATA TG 68 WO 95/32728 PCTIUS95/03789 27 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: Nucleic acid STRANDEDNESS: Single TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic oligonucleotide

DESCRIPTION:

ORGANISM:

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE:

LIBRARY:

CLONE:

L WO 95/32728 PCT/US95/03789 28 (viii) POSITION IN GENOME:

CHROMOSOME/SEGMENT:

MAP POSITION:

UNITS:

(ix) FEATURE:

NAME/KEY:

LOCATION:

IDENTIFICATION METHOD: DNA sequencing and restriction analysis OTHER INFORMATION: PUBUBLICATION INFORMATION: AUTHORS: L. Hansson, et al.

JOURNAL: Protein Expression and Purification VOLUME: 4

ISSUE:

PAGES: 373 381 DATE: 1993 DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CAT ATG CAC GTG AAA CCA TCG AAT CCC TGA GCT CGA G 37 I I

Claims

1. A liquid enteral nutritional composition comprising at least one protein not contained in human milk in combination with at least one material selected from the group consisting of a hydrolysate of p-casein isolated from human milk and a hydrolysate of a recombinant form of p-casein contained in human milk in a therapeutica., effective amount which inhibits the attachment of H. influenzae to human cells. J

2. A liquid enteral nutritional composition according to claim 1 wherein the I composition is an infant formula. 1

3. A liquid enteral nutritional composition according to claim 1 wherein the o1 human cells are oropharyngeal cells.

4. A liquid enteral nutritional composition according to claim 2 wherein the human cells are oropharyngeal cells.

A liquid enteral nutritional composition according to any one of claims 1-4 wherein the hydrolysate is a GLU-C hydrolysate. is

6. A liquid enteral nutritional infant formula comprising at least one protein not contained in human milk in combination with at least one material selected from the group consisting of a hydrolysate of P-casein isolated from human milk and a hydrolysate of a recombinant form of P-casein contained in human milk in a therapeutically effective amount which inhibits the attachment of H. influenzae to human cells, said infant formula containing no other proteins which are found in human milk.

7. A liquid enteral nutritional formula according to claim 6 whe.ein the human S cells are oropharyngeal cells. S:

8. A liquid enteral nutritional infant formula according to claim 6 or 7 wherein the hydrolysate is a GLU-C hydrolysate. 25

9. A nasally administrable formulation comprising at least one material selected from the group consisting of p-casein isolated from human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the attachment of H. influenzae to human oropharyngeal s cells. 30

10. A nasally administrable for 'ulation according to claim 9 wherein the material a is P-casein isolated from human milk. S"

11. A nasally administrable formulation according to claim 9 wherein the material is a recombinant form of the p-casein contained in human milk.

12. A nasally administrable formulation according to claim 9 wherein the material is a hydrolysate of 0-casein.

13. A nasally administrable formulation according to claim 9 wherein the material is a GLU-C hydrolysate.

14. A throat spray formulation comprising at least one material selected from the P. s group consisting of 0-casein isolated from human milk, a recombinant form of the IN:\LIBZZ100o11 :SSC P-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the attachment, of H. influenzae to human oropharyngeal cells.

A throat spray formulation according to claim 14 wherein the material is P-casein isolated from human milk.

16. A throat spray formulation according to claim 14 wherein the material is a recombinant form of the P-casein contained in human milk.

17. A throat spray formulation according to claim 14 wherein the material is a hydrolysate of P-casein.

18. A throat spray formulation according to claim 14 wherein the material is a o1 GLU-C hydrolysate.

19. A method of inhibiting the attachment 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 a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates of both.

A method of inhibiting the attachment of H. influenzae to human cells in a human infant by enterally feeding to said human infant an infant formula comprising at least one protein not contained in human milk in combination with a therapeutically effective amount of at least one material selected from the group consisting of P-casein 20 isolated from human milk, a recombinant form of the P-casein contained in human milk S and hydrolysates of both.

21. A method of treating and preventing otitis media in a human by inhibiting the S attachment of H. influenzae to human cells by feeding to said human an enteral nutritional product comprising at least one protein not contained in human milk in combination with 25 a therapeutically effective amount of at least one material selected from the group consisting of P-casein isolated from human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both.

22. A method of treating and preventing otitis media in a human infant by inhibiting the attachment of H. influenzae to human cells by feeding to said human infant S 30 an enteral formula comprising at least one protein not contained in human milk in S* combination with a therapeutically effective amount of at least one material selected from S the group consisting of p-casein isolated from human milk, a recombinant form of the P3-casein contained in human milk and hydrolysates of both.

23. A method of inhibiting the attachment of H. influenzae to human oropharyngeal cells by administering via a nasal passageway a formulation containing a therapeutically effective amount of at least one material selected from the group consisting of p-casein isolated from human milk, a recombinant form of the P-casein contained in human milk and hydrolysates cf both.

24. A method of inhibiting the attachment of H. influenzae to human S,/-'q4,,oropharyngeal cells by administering a throat spray formulation containing a -g l Q IN:\LIBZZ1001 :SSC Ir 31 therapeutically effective amount of at least one material selected from the group consisting of 0-casein isolated from human milk, a recombinant form of the p-casein contained in human milk and hydrolysates of both.

The method of any one of claims 19-24 wherein the material is P-casein isolated from human milk.

26. The method of any one of claims 19-24 wherein the material is a recombinant form of the P-casein contained in human milk.

27. The method of any one of claims 19-24 wherein the material is a hydrolysate of p-casein.

28. The method of any one of claims 19-24 wherein the material is a GLU-C hydrolysate. Dated 5 June, 1998 Abbott Laboratories Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 4 I. 1. i S i :A" S[N:IBZZIO001 1:SS TO* C i-