CA2190610A1 - Inhibition of attachment of h. influenzae to human cells - Google Patents

Abstract

The attachment of H.influenza to human cells, such as oropharyngeal cells, may be inhibited by native human .beta.-casein, a recombinant form of human .beta.-casein, and hydrolysates of both. The human .beta.-casein or hydrolysate may be contained in a liquid enteral nutritional product such as an infant Formula. The enteral nutritional product may be used, for example, in the prevention and treatment of otitis media in infants. The human .beta.-casein or hydrolysate may also be administered as a throat spray or nasally using drops or a spray.

Description

WO9!jl32728 21 9061 0 r~ J.,,.,m~789 INHIBITION OF ATTACHMENT OF H. INFLUENZAE TO HUMAN CELLS
The present lnvention relates generally to inhibiting the attachment of Hdemophi1us inf1uenzde to human cells. and more specifically to the use of native or recombinant human ~-casein and hydrolysates thereof for inhibiting the attachment of Hdemophi1us inf1uenzde (H. inf1uenzde) to human cells .
Haemophilus are small, gram-negative, non-molotile, non-spore forming bacilli with complex growth requirements. Diseases caused by H. 7nf1uenzde usually begin as a nasopharyngitis, possibly precipitated by a viral lnfection of the upper respiratory tract. Morse, et al. "Haemophilus".
MICROBIOLOGY, FOURTH EDITION, published by J.B. Lipincott Company, pages 615-618 (1990).
H. inf1uenzde are spread from person to person by airborne respiratory droplets or direct contact with secretions. To colonize. H. inf1uenzde 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. inf1uenzde 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 Neisserid meningitis and Hdemophi1us inf1uenzdel~ REVIEWS OF INFECTIOUS DISEASES, 13:22-23 (1991). It has further been reported that H. inf1uenzde harbored in the nasopharynx are a key factor in the development of middle ear infections (otitis media), and that non-typable H. inf7uenzde adhere to nasopharyngeal and nasal mucosal cells. Harada et al., "Adherence of Hdemophi1us inf1uenzde 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. inf1uenzde 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 WO9!;/32728 ! 306 1 0 P~ 5g5/037R9 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 Qf 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 lnfants breast-fed for less than four months. Those investigators presented speculations that IgA, or micronutrients or Prostaglandin E, in breast mllk 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 fiTed 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 weiyht of at least 5,000 daltons for "therapeutic prophylactic, and/or diagnostic use in infections caused by S. pneumonde and/or H. inf7uenzden~ 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 ,B-casein and hydrolysates of both to inhibit H. 7nf1uenzde infections.

2 relates to a DNA sequence enc~ding human ,B-casein, but does not disclose the capacity of either native or recombinant human ~-casein to inhibit the attachment of H. 7nf1uenzde to human cells.
WO91/08675 di scl oses an i nfar.t formul a whi ch conta i ns recombi nant 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 ... . _ . . ... _ , . .. . .. ..... . . . .. . .. .... .. .... . . ... ....... ....

W095/32728 ~01~10 r~ 789 of H. tnf7uenzae to human ce]ls is not taught or suggested in said publ i cati on .
The two assays (a radiolabeled assay and an ELISA assay) which were used for determining the bioactivity of B-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
Hdemo~hi 1us inf1uenzde Hdemophi1us inf7uenzae (H. inf7uenzde) cultures (fimbrlated, 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 Fimbriation of Nontypable Hdemophi7us inf71lenzae and its Ability to Adhere to Chinchilla and Human Respiratory Epithelium", Infec~ion and ImmunitY. 56: 331-335 (1988) . The H. inf7uenzde 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 37C
in a 5% CO2 incubator for about 18 hours to obtain logrithmically growing cultures. The H. inf7uenzae was used in both the Enzyme Linked Immuno Sorbent Assay (ELISA) assay and the radiolabeling assay as described below.
Native Human B-Casein B-casein isolated from human milk was purchased from Symbicom AB, P.O.
Box 1451, S-901 24 Umea, Sweden.
Recombinant Human B-Casein Applicants obtained B-casein cDNA and the expression system from Symbicom AB, P.O. Box 1451, S-90124 Umea, Sweden. The human B-casein cDNA
used had been previously cloned and sequenced by Lonnerdal et al., Cloning and sequencing of a cDNA encoding human milk B-casein. (SEQ ID NO: 1: ) Federdtion of European Biochemicd7 Societies Letters 269, 153-156 (1990).
The recombinant human B-casein was obtained from E. co1i and purified according to the method of Hansson et al.,Expression of Human Milk B-Casein in Escherichid co7i: Comparison of Recombinant Protein with Native Isoforms.

wogsl32728 21 9G~ 1~ Pcr/U595/03789 Prote7n Expression dnd Purii'icdtion 4, 373-381 (1993). To express human ,B-casein in E.co1i. ,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 s~gnal sequence for extracellular expression. The procedure followed was substantially that described by Hansson et al.
Human ,B-casein cDNA was isolated by Hansson et al. as a l l-kb EcoRI
fragment from a human lambda gt mammary gland library, and was subcloned into pUCl9. 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.co1i codon usage, six synthetic oligonucleotides were constructed and ligated. Also, PstI and EcoRI sites were inserted in front of the NdeI site. The sequence of the syntheti c ~ragment was 5 ' -CTGCAGAATTCATATGCGT
GMMCCATCGMTCCCTGAGCTCGAGCGMGMTCGATCACCGMTACMAAMGTTGMAMGTTMMCACG
AGGACCAGGATCC-3'. ~SEQ ID NO: 2:) The protein encoding sequence ~s underlined. The synthetic fragment was cloned into PstI/Bdm~I-digested pUCl9 resulting in plasmid pS24. To insert ~he rest of the ,B-casein encod i ng sequence . a 303 -bp AccI IBg 1 I I fragment was i sol ated and cl oned i nto a pUC18 derivative and designated plasmid pS22. Four synthetic oligonucleotides contalning the sequence encoding the carboxy-terminal end and translation stop followed by BdmHI and EcoRI si~es were constructed resulting in the sequence 5'AGATCTACCCTGTGA
CTCAGCCACTTGCCCCAGTTCATMCCCCATTAGTGTCTMTMGGATCCGMTTC-3 ', .
(SEQ ID NO: 3 ) where the protein encodlng sequence is underlined. The synthetic fragment was cloned into Bg1II/EcoRI digested pS22, resulting in plasmid pS23. To obtain the recombinant modified ,~-casein encoding fragment, three fragments were ligated: an 89-bp PstI/AvdII fragment from pS24: a 197-bp AvdII/AccI fragment from pS21; and PstI/AccI digested pS23.
The resulting plasmid pS25 was digested with NdeI/BdmHI 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. co1i signal sequenc:e of the enterotoxin STII gene was introduced in front W095132728 2 1 ~ ~6 1 0 PCT/US95/03789 of the ~-casein encoding sequence. A modified STII sequence with NcoI- and NdeI-compatible ends and an internal C7dI site was obtained by using a synthetic oligonucleotide, 5'-CATGMAMGMTATCGCAI I ll:l li;l lliCATCGAI~
mCTATTGCTACAMTGCATATG-3' (SEQ ID NO: 4:). To insert the signal sequence in front of the ,B-casein encoding sequence, pS25 was digested with AvdI/EcoRI and a 619-bp fragment was isolated. This fragment was ligated wi th a syntheti c ol i gonucl eoti de fragment .
5 ' CATATGCACGTGMMCCATCGMTCCCTGAGCTCGAG - 3 ' ( SEQ I D NO: 5: ), and NdeI /EcoRI -digested pUCl9. The resulting plasmid was designated pS27. The final expression vector,pS28, was constructed by ligating three fragments: a 700-bp NdeI/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.co1i strains BL21(DE3), BL21(DE3)pLysS, and BL21(DE3)pLysE. The bacteria were grown in Luria Broth medium containing 50 IJg/ml carbenicillin, and when Bl21(DE3)pLysS and BL21(DE3)pLysE were used the medium was supplemented with 25 ~g/ml chl orampheni col .
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 (OD60~), then 0.4 mM IPTG was added to induce the T7 system. The cells were harvested about 90 minutes after induction.
Recombinant ,B-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 pelletted by centrifugation. The supernatant contained the periplasmic 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 ,B-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.co1i was found to comigrate with full-length, nonphosphorylated native human ~-casein, which is one of seven nati ve i soforms .
Recombinant human ,B-casein has also been expressed in S.cereviside using the pYES 2.0 vector (Invitrogen Corp., San Diego, CA), but the expression level was~approximately rO% of that obtained in E.co1i. However, Hansson et al. found that S. cerevisr~e appeared to express phosphorylated human milk ,B-casein.
~-Casein HYdrolYsates The human ,B-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 37C ir 0.1 M NH4HCO3, pH 7.8. These digests were dried and resuspended i n appropri ate buffers pri or to use i n the assays di scussed above .

WO 95132728 r~ 789 21S~6~0 RADIOLARFI Fn ASSAY
Detroit 562 cells - --Detroit 562 pharyngeal carcinoma cells were obtained from the American Type Cul ture Col l ecti on ( Rock vi l l e, Ma ryl and, U . S . A . ) . The use of th i s 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 RES_ARCH
COMMUNICATIONS, 182:1466-72. The Detroit 562 cells were cultured in Dulbecco's Modified Eagle Medium (GIBCO, ~rand 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, lmM 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 cells per well and maintained at 37C ir a 5% CO2 incubator for 8-10 days prov~ding a confluent monolayer for adhesion studies. Plates were washed three times with 200 ~l 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). Ill-Indium-oxine (Ill-In) (Amersham, Arlington Heights, Illinois, U.S.A.) a high energy, short lived tracer, was utilized to radiolabel the bacteria. (The Ill-In penetrates the cell membrane, where once inside the cell it dissociates resulting in the binding of Ill-Indium ions with cytoplasmic components.) The use of Ill-In labeling in other assays has been described in Ardehali, et al. ''Ill-lndium Labeling of Microorganisms to Facilitate the Investigation of Bacterial Adhesion", JOURNAL OF BI~MFnICAL MATERIALS RESEARCH, 27: 269-275 (1993). 50 ~Ci of an Ill-In solution was added to 2.5 ml of the bacterial suspension at 37C and ... .. . .. . .. . _ . . . . . ... . .. . . .. . ..... .

WO95/32728 ~ ) 6 ~ O r.~ 789 lncubated for ZO minutes The radiolabeled bacteria were then washed two times and resuspended in 5 ml HBS supplemented with 30 mM HEPES buffer.
25 ~l of the ~ In labeled bacterial suspension were preincubated with 25 of ~-casein in a polypropylene 96 well plate for 15 minutes at 37C to allow binding of ,B-casein to bacteria.
Radiolabeled bacterial adhesion measurement 25 ~l of ~he preincubation mixture containing both rad10labeled 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 37C 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 ~l of 1 N sodium hydroxide (NaOH) to disrupt the cell monolayer. The entire contents of each well was placed in a Cobra polypropylene tube (lZ mm in diameter and 75 mm in height) (Packard Meriden. Connecticut. U.S.A.) and counted on a Cobra ~amma counter (also from Packard). Results were calculated by averaginy 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.

wog5132728 2 1 ~6 1 0 r~ 789 RESULTS FROM RADIOLABELED ASSAY
The human and bovine ,B-casein solutions were prepared first in 20 mM
ethanol ami ne . 6 M urea . pH ~ . 5 and then washed twi ce i n PBS by ultrafiltration using Centricon membrane filters (Amicon, MA) with a cut-off of 3,000 daltons. After resuspending ln 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 50%
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 ~-casei n .
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 ~-casein against H. inf1uenzae.
.

WO 95132728 2 1 9 () 6 1 0 PCIIUS95/03 L~ , ~1 ~D UO~ ~OD ~ -~1~ '' =' ' x ~ ~ 2 C~J
I_ X
-- O
o O C r~ ~ ~ ~ ~
O
v q-- , Z .~
Z
D
O
C_7 C~
'~ O
D ,~
~1 .~ ~ c~ z ~- - 'I > Z

wo 95132728 2 ~ ~ ~6 1 0 r~ 789 ELISA ASSAY
H. inf1uenz~e Preparation Harvested H. inf7uenzae from chocolate agar plates were suspended in 7 ml PBS-BSA and biotinylated by incubation with 350 ul of biotin-caproate N-hydroxysuccinimide ester (0.01 9 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-3SA.
Oropharvnqeal cel l s Oropharyngeal (OP) cells were collected from donors and pooled in phosphate buffered saline (PBS). Cell suspensions washed one tlme 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 ,ul 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 mlnutes to sediment the suspended cell preparation, aspirated and incubated overnight at 37C 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 ul PBS-BSA before use ln adhesion assays.
Adhesion Measurement The biotinylated H. inf7uenzae were diluted 1:1 with ,B-casein or control buffer (PBS-BSA) and incubated at 37C in a shaking water bath for 30 mi nutes . 50 ul of the prei ncubati on mi xture was pl aced i nto the appropriate well of the OP cell assay plate and incubated for 60 minutes at WO 95/32728 2 ~ 9 0 6 ~ O ~ 89 37C. 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 ~1 of Extravidin-peroxidase conjugate (Sigma), diluted 100-fold in PBS-BSA, was added to each well and the plate incubated for 40 minutes at 37C. 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 ~1 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, U~S.A.) until the positive control wells containing OP
cells and biotinylated bacteria (no ,~-caseln) reached an OD60o of 2.5 to 3Ø
Binding results were calculated by averaging the results of three repl i cates .

w095132728 P~i/lJ,,,~.N'~789 ~ ~ q~

RESULTS FR~M 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-adheslon assay with normal human oropharyngeal cells from volunteers. Results from thls ELISA assay are shown in Table 2. Once again. native human milk ,B-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. A~ter 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. 7nf1uenzae to normal human oropharyngeal cells as well as human tumor cells.

WO95132728 ~4 ~ 0 r~ 789 ~I r7 ' Ir '~ o:
~ ~ O ~ ~ ~ O
x cl Z~¦ ~ '~ X¦ N
1~1 L~J
C~
. . ~ X ~ 0 C c~ * z ~ r~
X
N ~¦ ~ ~ X¦ Ln C~l O O
~ O ~ O
~, } ~ = o o ~ , o~ ~ o Z
E ~ E
~_ ~ 0 C ~ ~
~3 C) " o 3~ > 3~ ~r 11 z l_ ~
~1 ~

wo 9s/32728 2 ~ ~ ~ 6 1 3 P~ I, u~ ~ ~789 It has been concluded from the foregoing experiments that ~-casein isolated from human milk, a recombinant form of the,B-casein contained in human milk, and hydrolysates of both, inhibits the attachment of H.
inf1uenzde to human cells. Furthermore, inasmuch as H. inf1uenzde have been identified in the literature as being associated with otitis media, it has been concluded that the above identified forms of human ,B-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 i ngested human mi 1 k, conta i n i ng human ~- casei n upon otl ti s medi a, i t i s 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. inf1uenzde 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 ~-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. inf1uenzde to human cells because the interaction of the human ~-casein and H. inf1uenzde is believed to occur in the nasopharynx via direct contact rather than following digestion and absorption of the,B-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 WO 9513~728 ~ 1 9 U ~ 1 0 ~ u~ ~ . 789 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 thi s i nventi on .

W0 95/32728 ~ ~ 9 ~ o PCT/US95/03789 SEQUl~NCE LISTING
( 1 ) GENERAL INFORMATION:
(i) APPLICANT: Abbott Laboratories (ii) TITLE OF ~VENTION: Inhibition of Attach~nent of H ~fluen~ae to Hurnan Cells (iii) NUMBER OF SEOUENCES: 5 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LonnieR Drayer ROSS Products Division Abbott Laboratories (B) STREET: ~ 625 ClevelandAvenue (C) CITY: Columbus (D) STATE: Ohio (E) COUNTRY: United States of Ar~erica (F) ZIP: 43215 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.5 ~nch, 1.44 Mb storage (B) COMPUTER: IBM Cornpatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FIL~G DATE:
(C) CLASSIFICATION:

WO95/32728 2 ~ 9(~6 1 0 r~u n~789 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/249,556 (B) FILING DATE: 26-MAY-199~
(A) APPLICATION NUMBER: US 08/249,584 (B) FILING DATE 26-MAY-1994 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPE~ONE: (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 (n) MOLECULE TYPE: Cloned cDNA ~ 5~ the product of a humsm genomic DNA segment (A) DESCRIPTION: Hum~m milk ~eta-casein (iii) HYPOTHETICAL:
(iv) ANTI-SENSE
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Humarl (A) ORGANISM: Homo sapiens (B) STRAIN:

wog~/32728 ~ 61 0 r~o (C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE: Adult (E) HAPLOTYPE:
(F) TISSUE TYPE: Mamrnary gland (G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vn) IMMEDIATE SOURCE: Hur~an Mar~nary Gland (A) LIBRARY:
(B) CLONE:
(viii) POSmON IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENT~CATION METHOD: DNA sequencing and restrictior. analysis (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID
NO: I is the human rniLlc protenn, ~-casein.
- (x) PUBUBLICATION INFORMATION:
(A) AUTHORS: B. Lonnerdal, et al.
(B) TITLE Cloning and sequencing of a cDNA erlcoding hur~an miLIi beta-casein.
(C) JOURNAL Federation European Bior~ r~l Society Letters W095132728 ~ l)6 1 0 r~ 789 (D) VOLUME: 269 (E) ISSUE
(F) PAGES 153- 156 (G) DATE: 1990 (H) DOCUMENT N1~3ER:
(I) FrLlNG DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(~) SEQUENCE DESCRIPTION: SEQ ID NO: I

GCA AGG GAG ACC ATA GAa AGC CTT TCA AGC AGT -GAG GAA TCT ATT 9 0 ACA GAA TAC AAG AAA GTT GAG AAG GTT A~A CAT GAG GAC CAG CAG 13 5 CAA GGA GAG GAT GAA CZ~C.. CAG GAT AAA ATC TAC CCC TCT TTC CAG 13 0 CTG CCT GTC CCT CAG CCA~ GA~ ATA ATG GAA GTC CCT AAA GCT AAA 315 GAC ACT GTC TAC ACT AAG GGC AGA GTG ATG CCT GTC CTT A~A TCT 3 6 0 CAG CCC CTG TGG TCT GTT CCT CPG CCC A'A\A GTC CTG CCT ATC CCC 540 CAG CAA GTG GTG CCC. TAC CCT CAG AGA GCT GTG CCT GTT CAA GCC 535 GTC TAA GAA GAT TTC A~A GTT AAT TTT CCC TCC .TTA TTT TTG AAT 720 ATT TGA AAT TTG ACT CA~ GAA CTA TTT ACA TTT TCC AAA TCT TAA 9 0 0 TTC AAC TAG TAC CAC AG AGT TCA ATA CTC ATT TGG A~A TGC TAC 945 TAT TTT TAT TTC TTT Aa~G AAT CTA TTT CCT TTC CAG TCA TTT C~A 1035 TAA ATT ATT CTT AAG CaT AAA AAA AAA AAA 1065 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:

W095/32728 219~6~ pC",f/US9~/03789 (A) LENGTH: 105 base paifs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic nliE.v~ flr~l~idc (A) DESCRIPTION:
(iii) HYPOTHETICAL
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Synthetic O!;~g."" ~1r~,lif1f Sequence (A) ORGANISM:
(B) STRAlN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vu) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSmON IN GENOME:
(A) CHROMOSOME/SEGMENT:

wo 95/32728 ~ 1 9 () 6 1 0 F~ 07 (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 assures adaptation of translation sequence to E. coli codon usage.
(x) PUBUBLICATION INFORMATION
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Expression of Human ~Ik ~-casein in F~rh. rirhi~ coli: Comparison of R~, ' Protein with Native Isoforrns.
(C) JOURNAL: Protein Expression and Purification (D) VOLU~ = 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 ~AT GCG TGA AAC CAT CGA ATC CCT GAG CTC GAG 45 -.
CGA AGA ATC GAT CAC CGA l~TA CAA A~A AGT TGA A~A AGT TAA ACA 9 O
CGA GGA CCA GGA TCC _ - - 10 5 (2) INFORMATION FOR SEQ ID NO: 3 ~ wog5~32728 19~61G PcrluS9~/03789 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 71 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: Synthetic r~
(A) DESCRIPTION:
(iii) HYPOTHETICAL:
(iv) AN Il-SENSE
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Synthetic 01~ Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(E) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vu) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSmON IN GENOME:

W095/32728 L~ l (tU~ 1 0 r~l~o~ 789 (A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(iY) FEATURE:
(A) NAMEI~CEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER rNFORMATION: The synthetic f ~ PO~ P encodes the carbo.YY-terminal end and tranlation stop.
(x) PUBUBLrCATION INFORMATION:
(A) AUTHORS: L Hansson, et al.
(B) TITLE: EApression of Human Milk p-casein in F~hP il hi~ coli: Comparison of ~1 ' Protein with Native Isoforms.
(C) JOURNAL: Protein Expression amd Purification (D) VOLVME: 4 (E) ISSUE:
(F) PAGES: 373 - 381 (G) DATE: 1993 (H) DOCUME~T NUMBER:
(I~ FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESrDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3 WO 9S/32728 2 ~ 9 ~ 6 ~ ~ PCT/US95103789 (2) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Si~le (D) TOPOLOGY: Unknowrl (ii) MOLECULE TYPE: Synthetic n'i~ - ' ,Lidc (A) DESCRIPTION:
(iii) HYPOT~TICAL:
(i~) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Syuthetic O~ '^ Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDMDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDL4 TE SOURCE:
(A) LIBRARY
(B) CLONE:

W095132728 7 1 906 1 0 PCTI~IS95103789 (viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(iY) FEATURE:
(A) NAMEIKEY:
(B ) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: Modified enterotoxin STII sional sequence.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Ea~pression of Human Milk ~-casein in F~h~ril hi~ coli: Comparison of Rl ' Protein with Nati~e Isoforms.
(C) JOURNAL: Protenn Expression and Puriiication (D) VOLUME: 4 (E) ISSUE:
(F) PAGES: 3Z3 - 3~1 (G) DATE: 1993 (H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RES~)UES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 CAT GAA A~A GAA TAT CGC. ATT TCT TCT TGC ATC GAT GTT CGT TTT

~ W095/3272~ 1 9~1 0 r~"~ 789 (2) 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 oL6. F
(A) DESCRIPTION:
(iii) HYPOTHETICAL.
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Synthetic 0~ ;.lF Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISStJE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B ) CLONE:

WO 95/32728 2 1 ~ O ~ 1 0 PCT/US95/03789 ~

(~iii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B ) M.~P POSITION:
(C) UNTTS:
(iY) FEATURE:
(A) NAMEIKEY
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencino and restriction analysis (D) OTHER INFORMATION:
(Y) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Expression of l~uman MiL~ casein in Fcr1~ rirhi~ coh: COmParison of F~ ' Protein with Nati~e Isoforms.
(C) JOUT~NALmProtein E~ression 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:
(~n) SEQUENCE DESCRIPTION: SEQ ID NO: 5 CAT ATG CAC GTG AAA CC~TCG AAT CCC TGA GCT CGA G 3 7

Claims (14)

CLAIMS:

1. A liquid enteral nutritional product comprising at least one Drotein not contained in human milk in combination with at least one material selected from the group consisting of .beta.-casein isolated from humanmilk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the attachment of H inf1uenzae to human cells.

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

3. A liquid enteral nutritional product according to claim 1 where in the human cells are oropharyngeal cells.

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

5. 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 .beta.-casein isolated from humanmilk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both in a therapeutically effective amount which inhibits the attachment to human cells, said infant formula containing no other proteins which are found in human milk.

6. A liquid enteral nutritional formula according to claim 5 wherein the human cells are oropharyngeal cells.

7. A nasal1y administrable formulation comprising at least one material selected from the group consisting of .beta.-casein isolated from humanmilk, a recombinant form of the .beta.-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.

8. A throat spray formulation comprising at least one material selected from the group consisting of .beta.-casein isolated from human milk, a recombinant form of the .beta.-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.

9. 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 .beta.-casein isolated from human milk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.

10. 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 .beta.-casein isolated from human milk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.

11. A method of treating and preventing otitis media in a human by inhibiting the 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 a therapeutically effective amount of at least one material selected from the group consisting of .beta.-casein isolated from human milk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.

12. 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 .beta.-casein isolated from human milk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.

13. 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 .beta.-casein isolated from human milk, a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.

14. 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 .beta.-casein isolated from human milk. a recombinant form of the .beta.-casein contained in human milk and hydrolysates of both.