Note: Descriptions are shown in the official language in which they were submitted.
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.
WO93/04172 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
WO 95/32728 2 1 1 U 6 1 0 PCT/US95103789
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
789
L~ ,
~1 ~D UO~ ~OD ~ -
~1~ '' =' '
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O
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wo 95132728 2 ~ ~ ~6 1 0 r~ 789
11
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
12
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~
13
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
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Z~¦ ~ '~ X¦ N
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C~
. . ~ X ~ 0 C c~ * z ~ r~
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~, } ~ = o o ~ , o~ ~ o
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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
16
will be apparent to those skilled in the art that various changes and
modifications may be made therein without deviating from the spirit or scope
of thi s i nventi on .
W0 95/32728 ~ ~ 9 ~ o PCT/US95/03789
17
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
18
(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
19
(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
CGG ATG AAG GTC CTC ATC CTC GCC TGC CTG GTG GCT CTT GCT CTT 45
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
CCA CAG CCT CTG ATC TAT CCA TTC GTT GAA CCT ATC CCC TAT GGT 225
TTT CTT CCA CAA AAC ATT CTG CCT CTT GCT CAG CCT GCT GTG GTG 270
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
CCA ACG ATA CCC TTT TTT GAC CCT CAA ATC CCA AAA CTC ACT GAT 405
CTT GAA AAT CTG CAT CTT CCT CTG CCT CTG CTC CAG CCC TTG ATG 4 5 0
CAG CAG GTC CCT CAG CCT ATT CCT CAG ACT CTT GCA CTT CCC CCT 495
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
CTT CTG CTC AAC CAA GAA CTT CTA CTT AAC CCC ACC CAC CAG ATC 630
TAC CCT GTG ACT CAG CCA CTT GCC CCA GTT CAT AAC CCC ATT AGT 675
GTC TAA GAA GAT TTC A~A GTT AAT TTT CCC TCC .TTA TTT TTG AAT 720
TGA CTG AGA CTG GAA ATA TGA TGC CTT TTC CGT CTT TGT ATC ACG 7 6 5
TTA CCC CAA ATT AAG TAT GTT TGA ATG AGT TTA TAT GGA AAA AAT 810
GAA CTT TGT CCC TTT ATT TAT TTT ATA TAT TAT GTC ATT CAT TTA 8 5 5
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
AAA CAT ATC AAA CAT ATG TAT ACA AAT TGT TTC TGG AAT TGT GCT 9 9 0
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
24
(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
AGA TCT ACC CTG TGA CTC AGC CAC TTG CCC CAG TTC ATA ACC CCA 45
TTA GTG TCT AAT AAG GAT CCG AAT TC - 71
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
26
(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
TTC TAT TGC TAC AAA TGC ATA TG 6 8
~ 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 ~
28
(~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
