CA2190609A1 - Inhibition of infection of mammalian cells by respiratory syncytial virus - Google Patents

Abstract

The infection of mammalian cells by RSV 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 respiratory tract infection in infants. The human .beta.-casein or hydrolysate may also be administered as a throat spray or nasally using drops or a spray.

Description

WO 95/32727 I ~ '103~

INHIBITION OF INFECTION OF MAMMALIAN CELLS BY RESPIRATORY SYNCYTIAL VIRUS
The present i nventi on rel ates genera l l y to i nh i bi ti ng the i nfecti onof mammalian cells by Respiratory Syncytial Virus, and more specifically to the use of native or recombinant human ,B-casein and hydrolysates thereof for inhibiting the infection of mammalian cells by Respiratory Syncytial Virus.
Respiratory Syncytial Virus ~RSV) is the single most frequent cause of acute respiratory tract infection in infants and children. Infants less than six months of age are most frequently and seriously affected. In most immunologically normal subjects, infection with RSV is limited to the respi ratory mucosa, and i s associ ated wl th the devel opment of bronchi ol i ti s, pneumonia and reactive airway disease. RSV infection in immunocompromised subjects has until recently been associated with increased mortality in infants and increased morbidity in other age groups. It has recently been reported in PEnIATRIC NOTES, Vol. 18, No. 14. January 27, 1994, that periods of high incidence of acute respiratory disease and numbers of deaths in elderly people were followed within 2-3 weeks by reports of high numbers of RSV or influenza virus isolates. The analyses indicate that RSV is as important as influenza viruses in causing morbidity and deaths among the el derly .
It has been reported that some respi ratory di sease may be prevented by breast feeding, and that "bronchiolitis of infants due to respiratory syncytial virus is less frequent in breast fed than in artificially fed infants". While human breast milk can contain antibodies to RSV, it has been found that milk also has antiviral activity that is not due to anti bodi es . It has been theori zed that thi s effect "may be produced by certain polysaccharides which are found on a number of different molecular constituents of milk." Tyrrell, "BREAST FEEDING AND VIRUS INFECTIONS", THE
IMMUNOLOGY OF INFANT FEEDING, edited by A. W. Wilkinson, Plenum Press, New York, NY pages 55-62 (1981).
Okamato, et a l . . "Anti vi ral Factors i n Human Mi l k: Impl i cati ons i n Respiratory Syncytial Virus Infection", ACTA PAEDIATRICA SCANDANAVICA
SUPPLEMENT, 351:137-143 (1989) disclose that while ,the mechanisms of protective immunity to RSV had not been clearly defined, immunity acqu~red WO 95132727 2 1 9 0 6 0 9 PCTII~S9~/03628 transplacentally or via breast feeding has been suggested to reduce the risk of lower respiratory tract disease. However, this publication focuses upon the role of antibodies transmitted in breast milk or the possible role of breast milk in modulating an infant's RSV immune response.
Laegreid et al.. "Neutralizing Activity in Human Milk Fractions against Respiratory Syncytial Virus", ACTA PAEDIATRICA SCANDANAVICA, 75:6g6-701 (1986) reports a study which confirms that human milk may contain RSV-neutralizing activity b~a non-immunoglobulin nature as well as RSV-specific antibody. However, the identity and mechanism of the non-immunoglobulin anti-RSV component of human milk is not identified. It is important though to note that Laegreid et al. disclose that RSV-neutralizlng components from breast milk may reach an infant's respiratory tract directly as a result of regurgitation and inhalation of milk during and after feeding. The mucosa of the respiratory tract may gain direct protection in this way.
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. inf7uenz~e" 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 RSV infections.

2 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 RSV to human cells.
IIO91/08675 discloses an infant formula which contains recombinant forms of both human alpha-lactalbumin and human ~-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 of RSV to human cells is not taught or suggested ln said publication.
The two assays (a HEp-2 cell assay and a LLC-MK2 cell assay) which were used for determining the bioactivity of ,~-casein are described below.
These assays have not been published heretofore, although the HEp-2 cell WO9513t727 2 i 9060~ P~

assay was based upon established methodology.
MATERIALS USED IN BOTH ASSAYS
Native Human B-Casein B-casein isolated from human milk was purchased from Symbicom AB, P.O.
Box 1451, S-9OI 24 Umea, Sweden.
Recombinant Human B-Casein Applicants obtained B-casein cDNA and the expression system from Symbicom AB, P.O. Box 1451, S-901 24 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:) Federation of Europedn Biochen1ica7 Societies Letters 269, 153-156 (1990).
The recombinant human ~7-casein was obtained from E. co1i and purified according to the method of Hansson et al., "Expression of Human Milk 6-Casein in Escherichia co1i: Comparison of Recombinant Protein with Native Isoforms". Protein Expression and Purification 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 signal 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 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, Ndel, a translational start, was inserted in front of the sequence encoding mature human B-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 synthetic fragment was 5'-CTGCAGAATTCATATGCGT
GMMCCATCGMTCCCTGAGCTCGAGCGMGMTCGATCACCGMTACAAAAAAGTTGMAMGTTMMCACG
AGGACCAGGATCC-3'. (SEQ ID NO: 2:) The protein encoding sequence is underlined. The synthetic fragment was cloned into PstI/Ban1HI-digested pUC19 resulting in plasmid pS24. To insert the rest of the B-casein woss/32727 2 ~ 9 06 09 P~
encoding sequence, a 303-bp AccI/Bg1II 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 BdmHI and EcoRI sites were constructed resulting in the sequence 5'AGATCTACCCTGTGA
CTCAGCCACTTGCCCCAGTTCATMCCCCATTAGTGTCTMTMGGATCCGMTTC - 3,', (SEQ ID NO: 3:) where the protein encoding sequence is underlined. The synthetic fragment was cloned into Bg1II/EcoRI dlgested pS22, resulting in plasmid pS23. To obtain the recombinant modified B-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/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. co7t signal sequence of the enterotoxin STII gene was introduced in front of the ,B-casein encoding sequence. A modified STII sequence with NcoI- and NdeI-compatible ends and an internal C7dI site was obtained by using a synthetic oligonucleo~ide, 5' -CATGAMMAGAATATCGCAI I I~I I(;I IbCATCGATGTTCGm mCTATTGCTACMMTGCATATG-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 f ragment, 5'CATATGCACGTGAAACCATCGMTCCCTGAGCTCGAG-3' (SEQ ID NO: 5: ), and NdeI/EcoRI-digested pUC19. The resulting plasmid was designated pS27. The final expression vector,pS28, was constructed by ligating three fragments: a 700-bp NdeI/HindIII ,B-casein fragment isolated from pS27, the STII signal sequence, and a NcoIlHindIII-digested pACAT7 vector.
The expression vectors pS26 and pS28 were used to transform E.co7i strains BL21(DE3), BL21(DE3)pLysS, and BL21(DE3)pLysE. The bacteria were grown in Luria Broth medium containing 50 ~g/ml carbenicillin, and when Bl21(DE3)pLysS and BL21(DE3)pLysE were used the medium was supplemented with 25 /lg/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 (OD600), then 0.4 mM IPTG was added to induce the T7 system. The cells were W095/32727 2~ 9~6~ P(l/~).. '.'Q~
harvested about 90 min~tes after inductlon.
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 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 ,~-casein were compared by standard biochemical techniques comprising SDS-PAGE, Western blotting, amino acid analysis,peptide mapping, phosphate analysis, and mass spectrometry.
Recombinant ,B-casein expressed in E.co1i was found to comigrate with full-length, 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 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.

21 9(~609 B-Casel n Hvdrol Ysates The human B-casein (both native and recomblnant) 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~O.1 M NH4HCO3, pH 7.8. These digests were dried and resuspended in appropriate buffers prior to use in the assays discussed above .

WO95132727 2 1 ~Q9 PCT/I~S9~103628 INHIBITIQN OF HUMAN RSV INFECTION OF HE~-2 ~FI I S
The Long straln of RSV was grown in HEp-2 cells under Eagle minimal essential medium (MEM) with 2% fetal bovine serum (FBS). The virus was harvested at a cytopathic effect (CPE) of 3 to 4+, sonicated for 10 seconds at 50% power with a Microson ultrasonic bell disrupter (Heat Systems -Ultrasonics, Inc., Farmlngdale, N.Y.), divided into portions and stored at -70C. The same preparation of virus was used for a series of tests, but a fresh sample was used for each test run.
The neutralization tests were performed in 96-well, flat-bottomed, tissue culture, microtiter plates (catalog no. 3596: Costar. Cambridge, Mass.). Serum or a monoclonal antibody (MAb) in the form of ascites fluid.
which had been heat inactivated at 56C for 30 min., was added to duplicate wells and serial fourfold dilutions were performed in the microtiter plates.
All dilutions were in MEM-2~ FBS, and the final volume was 75 I~l per well.
Approximately 60 50% tissue culture infective doses of virus in 25 Ill of MEM-2% FBS then were added to each well. and the mixture was incubated for 2 h at 4C.
Approximately 15,000 HEp-2 cells in 10011l of MEM-5% FBS were added to each well, and the plates were wrapped in cellophane and incubated at 35.5C in a humidified CO2 incubator for 3 days. The plates were fixed by aspirating the contents of the wells, washing three times with phosphate-buffered saline (PBS) at pH 7.2 with 0.5% Tween 20, adding 75 Ill of an 80%
(vol/vol) solution of acetone-PBS, and incubating for 15 min at 4D. After the incubation period. the contents were aspirated, and the plates were air dri ed .
The Enzyme Linked Immuno Sorbent Assay (ELISA) was performed on the same day as the fixation, or the plates were stored overnight at 4C and the ELISA was performed on the next day. For the ELISA, the wells were precoated with 200 ~l of PBS with 0.5% gelatin for 30 min at 35C, the contents were aspirated, the wells were washed with PBS (pH 7.2)-0.5% Tween 20 and 75 ~l o~ bovine anti-RSV serum (BaRSV) Burroughs Wellcome Co., Research Triangle Park. N.C.) diluted in PBS 0.5% gelatin plus 0.5% Tween 20 and 2~ normal goat serum was added and incubated for 1 hour at 35.5C.
The contents were aspirated, the wells were washed, and 75 ,ul of peroxidase-conjugated, goat anti-bovine immunoglobulin G (IgG) (Kirkegaard and Perry . . , . _, ......

WO 95132727 1/' '7~ 1--Laboratories. Inc. . Gaithersburg, MD) diluted in PBS-0.5% gelatin-0.5% Tween 20-2% normal goat serum was added and incubated for 1 hour at 35.0C. The contents of the wells were aspirated again. the wells were washed. and 125 of substrate (0.4 m~ of o-phenylenediamine dihydrochloride per ml 0.015%
H202) in 0.15 M citrate phosphate buffer (pH 5.5) was added and incubated at room temperature for 40 to 45 min. The reaction was.,stopped with 3.5 M
HCl, and the A490 was read wi th a mi cropl ate reader . Each di l uti on of antibody was run in duplicate. and each plate included control wells with uninfected cells. a back titration. i.e. titration of the virus inoculum in MEM-2% FBS. and a titration of a preimmune or nonneu~ralizing antibody. An absorbance reading of greater than or equal to 3 standard deviations above the mean of 15 control wells~was considered to be evidence of virus replication. The dilutions of BaRSV (1:1.000) and goat anti-bovine IgG
(1:5,000) used through the study initially was determined by checkerboard titrations .
This assay was based upon the disclosure of Anderson et al..
"Microneutralization Test for Respiratory Syncytial Virus Based on an Enzyme Immunoassay". JOURNAL OF CLINICAL MICROBIOLOGY, December 1985, pages 1050-1052.

WO 95/32727 2 ~ 9 ~ 6 ~ 9 PCTIUS95103628 .

RESULTS FROM HEp-2 CELL ASSAY
The human and bovine,B-caseln solutions were prepared first in 20 mM
ethanol ami ne . 6 M urea, pH 9 . 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 in appropriate buffer for the HEp-2 cell 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 caused an inhibition of infection/virus replication of 50% or more at concentrations of 0.4 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 sign~ficantly active even at 1.6 mg/ml.
These results indicated that ~-casein from human milk has different bioactivity compared to the bovine milk ~-casein.

21 9060q INHIBITIQN OF HUMAN RSV INFECTION OF HEp-2 CELLS
AGENT USED CONC. PERCENT INHIBITION
(mg/ml ) ~-Casein Isolated 1.6 >90 from Human Milk 0 . 8 >90 0.4 >90 Bovine ~-casein 1.6 0 0.8 0 0.4 0 Data shown are average of three replicates.

WO95/32727 2~ 9~9 r~".l~ /Q~
INHIBITION OF HUMAN RSV INFECTION OF LLC-MK2 r~F~
The RSV inhibition assay quantitatlvely determines the ability of a test reagent (antibody or other bioactive compound) to inhibit the infection of monkey kidney cells (LLC-MK2) (ATCC CCL 7) in microtiter plates. Infected cells were identified using an immunoperox1dase method.
The method is described briefly below.
LLC-MK2 cells were seeded into fibronectin treated Costar microtiter plates (5.0 X 103 cells per well) and incubated for 3-4 days prior to use in the infectivity reduction assay. On the day of assay the Long stra1n of RSV was diluted in MEM to 10-20.000 infected cell units (ICU/mL). and added to an equal volume (200,uL) of serially diluted sample preparations at suitable concentration (ex. 0.5, 1.0, and 2.0 mg casein/mL). Mixtures of diluted test samples and virus were then incubated for 2 hours at 4ac prior to adding to LLC-MK2 cells. Prior to addition of the diluted sample-virus mixtures to microtiter plates. culture medium was removed and the monolayers rinsed one time with MEM. All diluted sample-virus mixtures were tested in tr1pl1cate wells. The diluted sample-virus mixtures were allowed to absorb to LLC-MK2 monolayers for 2 hours at 37C in a humid1fied CO2 incubator. Following incubation. 150 ~l of MEM was added to all wells and the pl ates i ncubated at 37C for 16 hours i n the COz i ncubator . After overnight incubation. the culture medium was removed and the monolayers fixed with cold ethanol. After fixing. microtiter plates were rinsed once with 200 1~1 Dulbecco s PBS per well. and bovine anti-RSV antibody (200 ~l) was added to all wells. Following a 30 minute incubation at room temperature and three rinses wlth PBS/0.5% chick albumen (PBS/CEA) peroxidase labeled rabbit anti-bovine IgG was added to all wells and incubated at room temperature for 30 minutes. Microtiter plates were then rinsed 3 times with PBS/CEA and diaminobenzadine substrate added and incubated for 20 minutes. Plates were then rinsed as above with PBS/CEA.
and the number of stained RSV-infected cells per well determined using an i nverted mi croscope.

WO 95/32727 2 1 9 0 6 ~ 9 PCTIUS95103628 The proteins described in Table l were also tested in this assay for inhibition of RSV infection. Results from this assay are shown in Table 2.
Once again. native human milk ,B-casein was found to be active at concentrations of 1 mg/ml or greater while bovine~ ,B-casein was not significantly active. The GLU-C hydrolysates of both native and recombitant human ~-casein were active at concentrations of 0.75 mg/ml and higher.
Hence these results indicated that the recombinant human ,~-casein native human ,B-casein and their hydrolysates inhibit RSV infectlon of both HEp-2 mammalian cells and LLC=MK2 mammalian cells.

W095132727 2 i ~6~q r~l,u~ 6~

INHIBITION OF HUMAN RSV INFECTION OF LLC-MK2 CELLS . -.
AGENT U~ED CONC. PERCENT INHIBITION
(mg/ml ) -Casein Isolated from 1.5 87 Human Mi l k 0 . 75 33 O . 38 20 Bovi ne ,~-casein 1 21 0.5 23 0.25 6 Hydrolysate of ~-casein 1.5 99 Isola~ed from Human Milk 0 . 75 77 O . 38 60 Hydrolysate of Recombinant Human,B-Casein 1.5 84 0 . 75 42 O . 38 25 GLU-C Enzyme Control .025 64 . 0125 27 . 0068 19 Data shown are average of four replicates.

w09~132727 2 l 9 0 6 0 9 P._l/I.lJ, ~/Q~f.7f It has been concluded from the foregping experiments that ~-casein isolated from human milk, a recombinant form of the ~-casein contained in human milk, and hydrolysates of both. inhibits the infection of mammalian cells by RSV. Furthermore, inasmuch as RSV has been identified in the literature as being associated with respiratory tract infection, it has been concluded that the above identified forms of human ,B-casein may be employed in the preventlon and treatment of respiratory tract infection in humans, especially in human infants. In view of the therapeutic effect of enterally ingested human milk cQntaining human ,B-casein upon respiratory tract infection, it is concluded that the above identified forms of human ,B-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 infection of mammalian cells by RSV 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 ,B-casein identified in the preceding paragraph. Such a nasally administered formulation may be in the form of either drops or a spray.
The enteral nutritional, throat spray and nasal products and methods are believed to be effective in inhibiting the infection of mammalian cells by RSV because the interaction of the human ~-casein and RSV is believed to occur 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 i ncorporated i nto any standard or speci al i zed enteral 1 i qui d nutri ti onal 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 preventipn of respiratory tract infection in human infants.
While preferred embodiments of the invention have been disclosed~ it will be apparent to those skilled in the art that various changes and WO 95/32727 2 ~ 9 ~ 6 ~J q r~ ;?12 modiflcations may be made therein w~thout deviating from the spirit or scope of this invention.

WO 95/32727 2 ~ 9 1) 6 0 9 P~ 1 / u~ ~ ~7~ ~

SEQUENCE LIST[NG
( I ) GENERAL INFORMATION:
(i) APPLICANT: Abbùtt T.~hl~ratnri~c (ii) TITLE OF ~NVENTION: Inhibition of Infection of Marnm~lian Cells b Respiratory Syncytial Vilus (ii~i) NUMBER OF SEQUENCES: 5 (i~) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: LonnieR Drayer ROSS Products Division Abbott I .~hnr~toripc (B) STREET: 625 Cleveland Avenue (C) CITY: Colurnbus (D) STATE: Obio (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 Cornpaùble (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FTLING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:

Wo 9s/32727 ~ 1 9 () 6 0 9 P~
(A) APPLICATION NUMBER: US 08/249,554 (B) FILlNG DATE: 26-MAY-1994 (A) APPLICATION NUMBER: US 08/249,555 - --(B) FILINGDATE: 26-MAY-1994 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (614) 624-3774 (B) TELEFAX: (614) 624-3074 (C) TELEX: None (2) INFORMATION FOR SEQ rD NO: I
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1065 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: SDIgle (D) TOPOLOGY: UDkDowD
(ii) MOLECULE TYPE: Cloned cDNA ~ , the product of a humaD geDor~ic DNA segmeDt (A) DESCRIPTION: HumaD miLk ~eta-case (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE: ~~
(vi) ORIGINAL SOURCE: Human (A) ORGANISM: Homo sapieDs (B) STRAIN:
(C) rNDlVIDUAL ISOLATE:

wogs/32727 2 ~ 90609 (D) DEVELOPMENTAL STAGE: Adult (E) HAPLOTYPE:
(F) TISSUE TYPE: Man~mary 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) NAMElKEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID
NO: I is the hunlan milk protein, ,~-casenn.
(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 P ~ I ' SocietyLetters (D) VOLUME: 26g wo g5132727 2 1 q 0 6 0 9 PcTIUssS103628 (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: I

GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT ~ 0 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 A~A TCT 3 6 0 CAG CAG GTC CCT CAG CCT ATT CCT CAG ACT CTT GCA CTT CCC CCT . 4 9 5 CAG C~A GTG GTG CCC TAC CCT CAG AGA GCT GTG CCT GTT CaA GCC 5 Z 5 CTT CTG CTC AAC CaA GAA CTT CTA CTT AAC CCC ACC CAC CAG ATC 63 0 GTC TAA GAA GAT TTC A~A GTT AAT TTT CCC TCC TTA TTT TTG AAT 720 GAA CTT TGT CCC TTT ATT TAT TTT ATA TAT TAT GTC ATT CAT TTA ~55 A~A CAT ATC AAA CAT ATG TAT ACA AAT TGT TTC TGG AAT TGT GCT 9 9 0 TAA ATT ATT CTT AAG CAT AAA AAA AAA A~A 1065 (2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 105 base pairs Wo9S/32727 2 ~ 906 0q P~ 7~ ~

(B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unkuown (ii) MOLECULE TYPE: Synthetic 'i,, (A) DESCRIPTION:
(iii) HYPOTHETICAL
~ ) ANTI-SENSE:
(~) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Synthetic O'i~ Sequence (A) ORGANISM:
(B) STRAIN:
(C) rNDlVlDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANEILE:
(vii) IMMEDL9TE SOURCE: _ (A) LIBRARY:
(B) CLONE:
(vui) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:

wo gsl32727 2 ~ ~ ~ 6 0 9 1~ L L9.
(C) UNlTS:

(rx) FEATURE:

(A) NAMEIKEY:

(B) LOCATION:

(C) lDENTrFlCATlON METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The synthetic n~j~ ^IP~ assures adaptation of translation sequence to E. coh codon usage.
(x) PUBUBLICATION rNFORMATlON:

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

(B) TITLE: Expression of Human Milk ~-casein in Escherichia coli: Comparison of Rf - ' Protern witb Natrve Isoforms.
(C) JOURNAL: Protern Expression and Purification (D) VOLUME: 4 (E) rssuE:
(Fl PAGES: 373 - 381 (G) DATr~`: 1993 (H) DOCUMENT NUMBER:
(I) FlLrNG DATE:
(J) PUBLICATION DATE:
(K) Rr LEVANT RESrDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 CGA AGA ATC GAT CAC CGA ATA CA~ A~A AGT TGA A~A AGT TAA ACA ~ O

(2) rNFORMATlON FOR SEQ rD NO: 3 (i) SEQUENCE CHAr~ACTERlSTrCS:

Wo 95/32727 2 1 9 0 6 ~ ~ r~l,o~
(A) LENGTH: 71 base pairs (B) TYPE: Nucleicacid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unknown (ii) MOLECULE TYPE: S~ntbetic rli, ' '-(A) DESCRIPTION:
(iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGlNAL SOURCE: Syntbetic O~ '~ Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(~) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBR~RY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:

2 1 9060q Wo 9~/32727 r~ Jsg~lu~628 (B) MAP POSmON:
(C) UNITS:
(rx) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) rDENTlFICATlON METHOD: DNA sequencing and restriction analysis (D) OTHER INFORMATION: The synthetic ~ encodes the carbox~-terminal end and tranlation Stop.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: Expression of Human Milk ~-casenn in Escherichia coh: Comparison of Protenn with Native Isoforms.
(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE:
(F) PAGES: 373 - 381 (G) DATE: 1993 (H) DOCUMENT NUI\~BER:
(I) FILTNG DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQIJENCE DESCRIPTION: SEQ ID NO: 3 AGA TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA 45 - =~

(2) INFORMATION FOR SEQ ID NO: 4 _ . . . . . . .

wogs/32727 2190609 r~llu~ fi7~ ~

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 68 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Unlmown (ii) MOLECULE TYPE: Synthetic (A) DESCRIPTION:
(iii) HYPOTHETICAL:
ANTI-SENSE:
(v) FRAGMENT TYPE, (vi) ORIGINAL SOURCE: Syn~etic O'i,, ' '- Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDIV~DUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(~I) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:

wo 95/32727 2 ~ 9 1) 6 0 9 ~ s~ x (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: Ex,oression of Human MiLk ~-casein in F~r~Tirhi~ coli: Co~arison of Protein with Native Isofor~ns.
(C) JOURNAL: Protein Expression and Purification (D) VOLUME: 4 (E) ISSUE:
(F) PAGES: 373 - 3x 1 (G) DATE: 1993 (H) DOCUMENI NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ rD NO: 4 TTC TAT TGC TAC A~A TGC ATA TG 68 (2) INFORMATION FOR SEQ ID NO: 5 WO9~/32727 2 ~ 9 0609 r~ q~

(i) SEQUENCE C~iARACTERlSTlCS:
(A) LENGTH: 37 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Singie (D) TOPOLOGY~ Unknown (ii) MOLECULE TYPE Synthetic (A) DESCRIPTION:
(iii) HYPOTHETICAL:
(i~) ANTI-SENSE: :
(v) FRAGMENT TYPE:
(vi) ORiGlNAL SOURCE: Synthetic O'i" I ' Sequence (A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LrNE:
(I) ORGANELLE:
(vii) IMMEDiATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:

~ woss/32727 21 q0609 r~l,o~s~
(A) CHROMOSOMEISEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE: =
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD: DNA seqnencing and restriction analysis (D) OTHER INFORMATION:
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE E~ression of Human Milk ~-casein nn Escherichia coli: Cornparison of R~ ' Protein with Native Isofornns.
(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:
CAT ATG CAC GTG A~A CCA TCG AAT CCC TGA GCT CGA G 3 7 - -~

Claims (11)

CLAIMS:

1. A liquid enteral nutritional product 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 infection of mammalian cells by RSV.

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

3. 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 infection of mammalian cells by RSV. said infant formula containing no other proteins which are found in human milk.

4. A nasally 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 infection of mammalian cells by RSV.

5. 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 infection of mammalian cells by RSV.

6. A method of inhibiting the infection of mammalian cells by RSV 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.

7. A method of inhibiting the infection of mammalian cells by RSV 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.

8. A method of treating and preventing respiratory tract infection in a human by inhibiting the infection of mammalian cells by RSV 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.

9. A method of treating and preventing respiratory tract infection in a human infant by inhibiting the infection of mammalian cells by RSV 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.

10. A method of inhibiting the infection of mammalian cells by RSV
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.

11. A method of inhibiting the infection of mammalian cells by RSV
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.