CA2199707A1 - Inhibition of human rotavirus infection - Google Patents

Abstract

An enteral nutritional product containing either bovine -casein or human -casein at a concentration greater than that found in human or bovine milk and sufficient to inhibit infection of mammalian cells by human rotavirus.

Description

V096t08269 2 1 9 ~ 7 0 7 PCT~S95/05676 INHIBITION OF HUMAN ROTAVIRUS INFECTION

BACKGROUND OF THE INVENTION
Field of the Invention This invention relates to a secretory protein, kappa-casein (~-casein), which inhibits the attachment of human rotavirus (HRV) to m~mm~lian cells.
DescriPtion of the Prior Art Rotaviruses are the most important viral agents causing gastroenteritis in children living in both developing and developed countries (Yolken et al, "Human Milk Mucin Inhibits Rotavirus Replication and Prevents Experimental Gastroenteritis", Journal of Clinical Investigation 90, 1984-l991, 1992).
Rotaviruses also cause diarrhea in nursing homes and day care centers, among travelers, in adults who have contact with children, and in immunocompromised patients.
Breast feeding provides some protection against enteric infections by pathogens when breast-fed infants are compared with bottle-fed babies. Studies of children living in developing and developed countries have shown that breast-fed infants have fewer episodes of gastroenteritis than bottle-fed infants. Breast feeding can also lessen the severity of diarrhea and vomiting associated with enteric diseases. However, breast-feeding does not provide total protectiGn against infection and rotavirus infection has been observed in breast-fed infants.
No single factor has been found to be entirely responsible for the protective effect of breast feeding. It is believed that antibodies play a role, however, the level of anti-rotavirus antibody in human milk does not correlate with the degree of protection afforded by the milk. This suggests that non-immunoglobulin factors contribute to the protective effect of breast milk, and investigations have been undertaken to identify these factors.
Yolken et al. report a nonimmunoglobulin natural substance, milk mucin, that inhibits the replication of rotavirus. Mucin is a sialic acid-containing glycoprotein. In this report, Yolken et W 0 96/08269 2 1 99707 PC~rAUS95/05676 al. demonstrate that mucin and mucinous components found in human milk show anti-rotaviral activity. Using purified milk mucin components in a solid-phas~ binding assay, Yolken et al. first showed mucin binding to African Green Monkey Kidney (MA-104) cells infected with human and simian strains of rotavirus. Using tissue culture techniques they next showed that a fraction of human milk, subsequently identified as human milk mucin complex, inhibited rotavirus replication. This was further demonstrated by showing that this fraction bound specifically to cells infected with rotavirus. When the active fraction was desialylated by chemical hydrolysis, there was a substantial decrease in binding to the rotavirus infected cells. This indicates that in the antiviral system reported here sialic acid is necessary for anti-rotaviral activity. Yolken et al. did not find evidence of rotavirus inhibitory activity in the lipid fractions of milk.
Yolken et al. also demonstrated the efficacy of mucin-containing milk components in preventing experimental rotavirus gastroenteritis in suckling mice.
Wo 94/09651 (Newburg et al.,"Anti-Diarrheic Product and Method of Treating Rotavirus-Associated Infection") discloses a product containing an anti-diahrreal agent, such as the milk mucin complex and its components, which can bind rotavirus, and a method of inhibiting rotavirus infection in mammalian cells by exposing them to these agents.
U.S. Patent No. 5,147,853 (Dosako et al., "Infection Protectant") discloses a method for preventing adhesion of E.
coli to human epithelial cells by treating them with K-casein, a sialic acid-conjugated protein derived from cow's milk.
The prevalence of rotavirus infection in the groups at risk, especially infants and children, and the seriousness of its effects attests to the need for an effective treatment. It is of particular importance to develop methods for prevention and treatment of rotavirus infection that have no adverse side effects.

~096t08269 2 1 9 9 7 0 7 PCT~S9510S676 BRIEF DESCRIPTION OF THE INVENTION
In the present invention it is disclosed that a secretory protein, K-casein, which can be purified from human or bovine milk, or manufactured in recombinant form using cDNA, inhibits the attachment of human rotavirus to mammalian cells. In an assay, 60-70~ inhibition of human rotavirus binding was obtained when the cells were treated with either human or bovine K-casein.
K-casein, which is a mammary product believed to be without adverse side effects, can be used for the prevention and treatment of rotavirus infection.

BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows the method of preparation of radioactive iodine labeled human rotavirus.
Fig. 2 is a graph showing the extent of inhibition of binding by human rotavirus to MA-104 cells by human and bovine K-caseins, Fig. 3 shows the dose-dependent inhibition of human rotavirus to MA-104 cells by human K-casein.
Fig. 4 shows that human K-casein retained its ability to inhibit binding by human rotavirus to MA-104 cells after being treated with either neuraminidase, fucosidase or both.
Fig. 5 shows that bovine K-casein retained its ability to inhibit binding by human rotavirus to MA-104 cells after being treated with neuraminidase.
Fig. 6 shows the effect of digestion by PronaseX on the ability of bovine K-casein to inhibit binding by human rotavirus to MA-104 cells.
Fig. 7 shows the dose-dependent neutralization of human rotavirus as measured by the ability of bovine K-casein to inhibit infection of MA-104 cells.
Fig. 8 shows the DNA sequence and the amino acid sequence of human K-casein.
Fig. 9 shows the amino acid sequence which has the biological activity of human K-casein.

W096/08269 2 1 9 9 7 0 7 PCT~S95/05676 DETAILED DESCRIPTION OF THE INVENTION
This invention results from a research effort directed toward improving methods for preventing and treating enteric infections caused by rotavirus in mammals, especially in humans.
The invention demonstrates the effectiveness of K-casein in inhibiting the attachment of human rotavirus (HRV) to simian cells, and thereby preventing the virus from entering the cells and causing infection. The agent of this invention, K-casein, is particularly useful in the treatment of infants and children as it is a normal constituent of human milk and is present in the human diet in bovine milk. It is, thus, unlikely to cause toxic or allergic reactions in treated subjects.
Bovine K-casein is present in bovine milk at a concentration of about 3.3 grams/liter. It can be added to food preparations and made available to both adults and children at risk for rotavirus infection with little, if any, risk of immunological reaction.
What is disclosed in this application is a method of preventing or retarding the onset of or treating an infection of a mammalian cell caused by human rotavirus comprising contacting the cell with an effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein. Also disclosed is a method of preventing or retarding the onset of or treating human rotavirus infection of a mammal's cells comprising administering to the mammal an enteral nutritional composition comprising an anti-rotaviral infection effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein at a concentration exceeding that found in human or bovine milk.
Specific examples will now be provided to describe the methods of preparation of K-casein and the two assays employed to measure inhibition of HRV-binding ability by human and bovine K-casein. The examples are provided for purposes of illustration ~096/08269 2 1 9 9 7 0 7 PCT~S95/05676 only and are not intended to be limiting.

Example 1: Preparation of Human Milk Kappa-casein Approximately 0.5 1 of frozen human milk was thawed, centrifuged at 15,000 x g for 1 hour at 40C and the fat pad, which is the top layer, was removed. The supernatant and pellet were homogenized and the pH of the resulting material was adjusted to 4.3 by the addition of hydrochloric acid. Calcium chloride was added to a final concentration of 60 mM. The mixture was stirred at room temperature for 1 hour and the precipitated caseins were then recovered by centrifugation at 18,000 x g for 90 minutes at 40C. Casein pellets were then dissolved in approximately 90 ml of 6M urea and 20 mM
ethanolamine at a pH of 9.5 and extracted three times with 4 volumes of hexane to reduce the fat content further. The aqueous phase was dialyzed three times against 4 1 of water using a 12,000 molecular weight cut-off membrane and lyophilized. This crude casein (yield was approximately 2-3 g/l) was dissolved in 6M urea, 20 mM n-2-hydroxyethylpeperazine-n'-2-ethane sulfonic acid (HEPES) (Sigma Chemical Co., St. Louis, Mo) with 0.5% (V/V)

2-mercaptoethanol (2-ME) at Ph 7.5 and stirred at 40C for 1 hour.
This solution was chromatographed in a 2.6 x 13 cm Macroprep-S0 column (Bio-Rad, Hercules, CA) and equilibrated in the same buffer except that 0.1% (V/V) 2-ME was used. The washing buffer was 6M urea, 20 mM HEPES and 0.1% (V/V) 2-ME at pH 7.5. The eluant was monitored at a wavelength of 280 MM. Elution of the bound material was accomplished by application of a linear gradient from 0 to 0.3M sodium chloride in 100 minutes at a flow rate of 2 ml/minute. Fractions of 7 ml were collected and ~-casein was detected either by comparison with human K-casein obtained from SYMBICOM AB, Sweden used as a standard in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or by immunoblot analysis. The fractions containing ~-casein were then pooled and dialyzed three times in 4 1 of water as described above followed by lyophilization. This material was dissolved in W096/08269 PCT~S95/05676 6M urea, 20 Mm HEPES and 0.5% (V/V) 2-ME at pH 7.0 at a concentration of approximately 10 mg/ml at 4OC and loaded on the same column. The column was washed with 6M urea, 20 mM HEPES and 0.1% (V/V) 2-ME at pH 7.0 and then eluted with a linear gradient of 0 to 0.6M sodium chloride in 140 minutes at a flow rate of 2 ml/minute. The fractions containing the major peak at 280 MM
were pooled, dialyzed as before and then lyophilized. The material was estimated to be of greater than 90% purity.

Example 2: Source of bovine Kappa-casein Bovine ~-casein was purchased from Sigma (catalog #CO406) and had a purity of greater than 80%.
xamPle 3: Procedure for human rotavirus cell-binding inhibition assay MA-104, African Green Monkey Kidney, cells (American Type Culture Collection, MD) were grown on 24-well tissue culture plates to 100% confluency. The use of Ma-104 cells is described in Kitamoto et al., "Comparative Growth of Different Rotavirus Strains in Differentiated Cells", Virology 184:729-737 (l991).
Experiments reported by Kitamoto et al. show that human rotaviruses, including the Wa strain, grow and produce antigen in MA-104 cells. The cells were washed twice with Hanks' medium (Sigma) and fixed with 1% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 15 minutes. This was followed by three washes with tris-buffered saline (TBS).
Solutions of potential inhibitors in TBS containing 5 mg/ml of bovine serum albumin (BSA) were added to the wells prior to the addition of 12sI-labeled human rotavirus (HRV) suspensions.
Radiolabeled virus was prepared as described in Fig.1. The plates were incubated for 90 minutes at 37OC and washed three times with TBS. The cells were then lysed by adding 0.2% sodium hydroxide and 2% SDS solution. Counts per minute (cpm) in the lysates were determined in a scintillation counter and used as indicators of cell-bound HRV.

~096/08269 2 1 9 9 7 0 7 PCT~S95tO5676 ExamPle 4: Determination of Inhibition of Binding of Human Rotavirus Strain Wa to MA-104 Cells by Human and Bovine Kappa-Caseins In an experiment shown in Fig. 2, five agents were tested for inhibitory potential at a concentration of 1 mg/ml. Human K-casein inhibited binding by HRV strain Wa to MA-104 cells by more than 70% and bovine ~-casein (Sigma) inhibited binding by more than 60% when compared with control cells which did not receive any potential inhibitory agent after being exposed to HRV.
Bovine beta-casein (Sigma) and human beta-casein (Symbicom AB, Sweden) exhibited much less ability to inhibit binding by HRV Wa.
Bovine beta-casein showed 45% inhibition and human beta-casein showed 25% inhibition. Bovine submaxillary mucin (Sigma) was tested in the same experiment and showed 50% inhibition, however, further experimentation showed that this inhibition was not dose-dependent and, therefore, not specific. This is illustrated in Fig. 3. By contrast, human K-casein was shown to be dose-dependent in the same experiment.

Example 5: Inhibition of Human Rotavirus Infection by Human or Bovine Kappa-Casein - Effect of Sialic Acid In another experiment using the procedure described in ple 3, human K-casein was treated with neuraminidase to remove sialic acid from the glycan chains of the protein molecules. As illustrated in Fig. 4 the desialylated human K-casein showed similar activity in the rotavirus inhibition assay to the intact human ~-casein. This indicates that in this experiment the sialic acid residues are not required for the inhibitory effect of human ~-casein. Intact and desialylated human K-casein were then treated with fucosidase. Both treated agents retained their inhibitory activity, which is indicated in Fig. 4 This shows that under the conditions of this assay fucose residues on human K-casein are not required for inhibitory activity against HRV.
When bovine ~-casein was treated with neuraminidase to W096/08269 2 1 9 9 7 0 7 PCT~SsS/05676 remove sialic acids, the resulting desialylated bovine ~-casein was highly active in this assay, as shown in Fig. 5. It is concluded that sialic acid residues on bovine ~-casein are also not required for activity against HRV.

Example 6: Inhibition of Human Rotavirus Infection by Bovine Kappa-Casein - Effect of Hydrolysis Bovine K-casein was digested in 100-fold excess with a protease, Pronase~ (Calbiochem, La Jolla, CA), for 16 hours at 37OC in O.lM tris buffer at pH 8Ø Fig. 6 shows that the dose-dependent activity against HRV of the hydrolyzed protein was significant in this assay. The doses in this experiment were determined by the molar amounts of hexose present in the glycan chains of bovine ~-casein. Although 7-8 fold more of the hydrolyzed bovine K-casein than native bovine ~-casein was required to achieve the same degree of inhibition, inhibitory activity against HRV was retained in the hydrolyzed protein under the conditions of this assay.

ExamPle 7: Inhibition of Human Rotavirus Infection - Second Assay Method - MA-104 Cells and Wa Strain of Human Rotavirus Another set of experiments was performed to measure the ability of bovine ~-casein to inhibit infection of cells by HRV.
The assay procedure employed is as follows.
MA-104, African Green Monkey Kidney Cells whose use in rotavirus assays is described in Kitamoto et al, "Comparative Growth of Different Rotavirus Strains in Differentiated Cells", Virology 184:729-737 (1991), were obtained from BioWhittaker, Inc. (Walkersville, MD). Experiments reported by Kitamoto et al.
show that human rotaviruses, including the Wa strain, grow and produce antigen in MA-104 cells. The cells were cultured in Basal Medium Eagle (BME) (BioWhittaker) supplemented with 10%
heat inactivated Fetal Bovine Serum (FBS) (Hyclone Laboratories, Logan, UT) and 2 mM L-glutamine (BioWhittaker). Cells were ~096/08269 PCT~S95/05676 routinely subcultured in 75 cm2 flasks using trypsin-EDTA (0.25 trypsin, 1 Mm EDTA) (BioWhittaker) to detach cells.
Assay plates were treated with 1 ~g/well of fibronectin (Sigma) to aid in the adhesion of cells to the culture substrate and to ensure their subsequent attachment during the wash steps of the virus neutralization assay. Fibronectin, solubilized in distilled water, was dispensed into each well and incubated for 1-3 hours at room temperature to allow binding to the plastic wells. Prior to dispensing the cells into the wells of the assay plate, the residual fibronectin was aspirated. Cells seeded into 96-well plates at a density of 10,000 cells per well with growth medium were maintained at 37OC in a 5% CO2 incubator for 3-4 days providing a confluent monolayer for virus neutralization studies.
Serotype 1 Wa strain of human rotavirus was obtained from Dr. Linda J. Saif (Ohio Agricultural Research and Development Center and was originally acquired from Dr. R. Wyatt (National Institutes of Allergy and Infectious Diseases). The virus stock used in these studies was a passage 5 viral harvest. Before use in these assays, HRV was activated with 10 IU/ml trypsin per 1 ml HRV and incubated for 30 minutes at 3 60 C. The virus was diluted with BME medium supplemented with 2 mM glutamine, 50 ~g/ml gentamicin sulfate (BioWhittaker) and 1~ penicillin-streptomycin solution (Sigma) to achieve a 1:250 dilution, which is twice the final desired virus concentration.

ExamPle 8: Inhibition of Human Rotavirus Infection - Second Assay Method - Virus Neutralization Equal volumes of test agent and trypsin-activated HRV were mixed and preincubated for 1 hour at room temperature to permit the test agent to bind to the virus. During the HRV+test agent preincubation period, plates containing MA-104 cells were washed

3 times with 15 ~1 diluent medium (150 ~l/well/wash) after which another 50 ~1 of diluent medium was added to each well. MA-104 plates were then incubated at 37OC in 5% CO2 until the preincubation period for the HRV+test agent was completed. A

W096/08269 2 1 9 9 7 0 7 PCT~S95/05676 control sample or 100 ~l of the HRV+test agent sample was pipetted into appropriate wells and mixed with 50 ~l of diluent medium. The plates were incubated for an additional 12-14 hours at 37OC to allow the virus to propagate within the cells. Longer incubation time would result in infection of MA-104 cells by progeny virus, which is not desirable. Three types of control wells were included in each assay; (1) wells containing MA-104 cells, HRV, but no test agent, (2) wells containing MA-104 cells, HRV, and HRV antibodies, which are known HRV inhibitors and (3) wells containing MA-104 cells, but without virus. Each concentration of test agent or antibody was tested in triplicate.
After the incubation period, the assay plates were washed twice with Dulbecco's phosphate buffered saline (PBS) (BioWhittaker) 150 ~l of cold 70% ethanol was added to each well and 95 ~l was removed to prevent air from drying the cell monolayer. Cold absolute ethanol (190 ~l) was added to the 70%
alcohol layer and the plates were rocked and refrigerated overnight. The alcohol was then removed and 150 ~l of PBS
containing 0.05% chick egg albumin (PBS-CEA) (Sigma) was added to each plate to block nonspecific binding sites.
To stain the plates, the PBS-CEA was removed and 200 ~l of anti-rotavirus IgG was added to each well at a dilution of 1:2500. The plates were incubated for 30 minutes at room temperature. The plates were washed three times with PBS-CEA and 200 ~l of peroxidase conjugated to anti-bovine IgG (Cappel Organon Teknika, Durham, NC) was added to each well at a dilution of 1:2000. The plates were covered and incubated for 30 minutes at room temperature. They were then washed three times with PBS-CEA and 100 ~l of diaminobenzidine (Sigma) substrate was added to each well. The plates were incubated at room temperature for 20 minutes, washed twice with PBS-CEA and then washed with distilled water.

Example 9: Inhibition of Human Rotavirus Infection by Bovine Kappa-Casein - Second Assay Method - Counting Infected Cells ~096/08269 2 1 9 9 7 0 7 PCT~S9S/OS676 Plates which had been prepared according to the method of examples 7 and 8 were rehydrated by the addition of 150-200 ~l of distilled water per well prior to microscopic Px~m;nation. The plates were examined with a light microscope at 100%
magnification. Cells which were darkly stained and contained characteristic brown and granular cytoplasmic regions were counted as virus-infected cells. A representative infected cell count was obtained for each well by counting all the stained cells within a uniform area which represented approximately 20%
of the surface area of each well. Control wells, to which no virus had been added, were examined to ascertain that they were free of infected cells. Results were reported as percent inhibition obtained by comparing the average number of infected cells in the population which had been treated with test agent+HRV with the average number of infected cells in the population which had been exposed to virus alone. Results are shown in Fig. 7. Bovine ~-casein, the test agent, neutralized human rotavirus in a dose-dependant manner. Increased concentrations of bovine K-casein resulted in correspondingly reduced levels of infection up to a concentration of 1.6 mg/ml where infection was inhibited by almost 90% and only 10% of cells were infected.
xample 10: Inhibition of Human Rotavirus Infection by Bovine Kappa-Casein - Second Assay Method-Effect of Hydrolysis Bovine ~-casein was digested with Pronase~ (Calbiochem), a protease, for either 4 hours or 20 hours and at the concentrations indicated in Tables 1 and 2, which show experiments run on different days. The plates were incubated at 370C according to the method of Example 8, and stained and counted according to the method of Example 9 to determine percent inhibition. The results of experiments performed on different days shown in Tables 1 and 2 indicate that hydrolysis of bovine W096/08269 2 1 9 9 7 0 7 PCT~S95/05676 ~-casein with the protease eliminated inhibition of infection of MA-104 cells by HRV. Undigested bovine K-casein showed between 86% and 93% inhibition of viral attachment to Wa cells.
Hydrolyzed bovine K-casein showed either no inhibition or very slight inhibition in this assay. Pronase~ alone, without the addition of bovine K-casein to the plates, resulted in variable amounts of inhibition which were not dose-related.
In this assay, bovine K-casein with Pronase~ or Pronase~
alone were boiled after the period of digestion to denature the protein and abolish its activity. Therefore, all inhibition of HRV which has been observed can be attributed to the action of the bovine K-casein and not to the PronaseX. In the assay described in Example 6, Pronase~ was not boiled sufficiently boiled after the initial digestion period. The results of that assay showed that hydrolysis did not abolish inhibition. It is likely that in that case the protease had not been completely inactivated and the inhibition observed may be attributed to residual Pronase~ rather than to hydrolyzed bovine ~-casein.

W096/08269 2 1 9 9 7 0 7 PCT~S95/05676 Table 1 INHIBITION OF HUMAN ROTAVIRUS INFECTION BY BOVINE KAPPA-CASEIN:

TREATMENT CONCENTRATION mg/ml PERCENT INHIBITION

BKC2 20hrs/370C 1.500 93 BKC 20hrs/370C 0.750 97 BKC 20hrs/370C 0.375 92 BKC 4 hrs/370C 1.500 86 BKC 4 hrs/370C 0.750 89 BKC 4 hrs/370C 0.375 87 BKC+Pronase~ 1.500 -19 20hrs/370C boiled BKC+Pronase~ 0.750 -6 20hrs/370C boiled BKC+Pronase~ 0.375 -7 20hrs/370C boiled BKC+Pronase~ 1.500 -4

4 hrs/370C boiled W096/08269 2 1 99707 PCT~S9S/OS676 Table 1 (continued) BKC+Pronase~ 0.750 -7 4 hrs/370C boiled BKC+Pronase~ 0.375 -17 4 hrs/370C boiled Pronase~ 0.150 -29 20hrs/370C boiled self-digested Pronase~ 0.075 -21 20hrs/370C boiled self-digested Pronase~ 0.037 -22 20hrs/370C boiled self-digested Pronase~ 4 0.150 -8 hrs/370C boiled self-digested Pronase~ 4 0.075 -34 hrs/370C boiled self-digested Pronase~ 4 0.037 -64 hrs/370C boiled self-digested Comparison of inhibition by bovine ~-casein, hydrolyzed ~-casein, and protease (Pronase~) alone.
2Bovine kappa-casein W096/08269 2 1 9 ~ 7 0 7 PCT~S95/OS676 Table 2 INHIBITION OF HUMAN ROTAVIRUS INFECTION BY BOVINE KAPPA-CASEIN:

TREATMENTCONCENTRATION PERCENT
mg/ml INHIBITION

BKC+Pronase~ 1.5000 10 20hrs/370C boiled BKC+Pronase~ 0.7500 6 20hrs/370C boiled BKC+Pronase~ 0.3750 -6 20hrs/370C boiled Pronase~ 20hrs/370C 0.1500 54 boiled self-digested Pronase~ 20hrs/370C 0.0750 13 boiled self-digested Pronase~ 20hrs/370C 0.0375 8 boiled self-digested Pronase~ 20hrs/370C 0.0188 8 boiled self-digested Pronase~ 20hrs/370C 0.0094 15 boiled self-digested Comparison of hydrolyzed bovine ~-casein and protease (Pronase~) alone.

W096/08269 2 1 9 9 7 0 7 PCT~S95/OS676 EXAMPLE 11: Utility and Administration of Kappa-Casein Human K-casein can be extracted from human milk or can be obtained by recombinant DNA methods and cloning in prokaryotic or eukaryotic cells or from transgenic mammals using the DNA
sequence and expression system of Hansson et al. Bovine K-casein can be extracted from bovine milk where it is present at a concentration of approximately 3.3 grams/liter.
A DNA sequence encoding the human milk protein K-casein is disclosed in PCT Ppublication No. WO 93/15196 (Hansson et al., "DNA Encoding Kappa- Casein, Process for Obtaining the Protein and Use Thereof") which is being incorporated herein by reference for the purpose of teaching a method of constructing an expression system for a DNA sequence encoding a polypeptide having an amino acid sequence which has the biological activity of human ~-casein. The DNA sequence is shown in Fig. 8 (SEQ ID
NO: 1:) and the amino acid sequence of the polypeptide which it encodes is shown in Fig. 9 (SEQ ID NO: 2:). The encoded polypeptide was determined to have the antimicrobial activity or opioid activity of human K-casein. The DNA sequence shown can be used in the production of recombinant human K-casein using either a procaryotic or a eukaryotic system, or can be used to produce transgenic non-human mammals. Recombinant human K-casein can be used as a constituent of infant formula to improve the nutritional and biological value of the formula or, as in the case of the present invention, to provide protection against enteric infection by human rotavirus.
The DNA sequence disclosed by Hansson et al. was determined from a cDNA clone isolated from a human mammary gland cDNA
library. Construction of the expression system and its molecular biological characterization employed standard recombinant DNA
methods. The procedure used was as follows: E. coli Y1090 bacteria were grown on LB (Luria-Bertoni) plates containing 50 ~g/ml of carbenicillin. A single colony was isolated and grown overnight in a LB containing 0.2% maltose and 10 mM MgSO4. 0.4 ml of the culture was then mixed with diluted library phages and W096/08269 PCT~S95/05676 adsorption was allowed for 15 minutes at 37OC. The infected culture was mixed with 7 ml soft agarose (0.75% agarose in LB and 10 mM MgS04). The soft agarose mixture was poured on 150 mm LB
plates. The plates were incubated at 420C for about 3.5 hours until plaques were visible. Thereafter, each plate was overlaid with a membrane (DuPont NENX, Colony Plaque Screen) previously saturated in lO mM IPTG (Isopropyl-B-D-thiogalactoside)~ and incubated overnight at 370C. The positions of the membranes were indicated before the membranes were removed. The membranes were washed in TTBS (tris buffered saline (TBS) containing 0.05% Tween 20), and incubated in TTBS containing 20% FCS and ~-casein antisera which had been raised in rabbits, purified, and diluted 1:25 for 2 hours at room temperature. The membranes were washed two times for 5 minutes in TTBS at room temperature.
Biotinylated goat-antirabbit IgG in TBS (50mM Tris-HCL pH 7.9, 150 mM NaCl) was added and the membranes incubated for 1 hour at room temperature. The membranes were washed again with TTBS two times for 5 minutes at room temperature. The conjugate of streptavidin and biotinylated alkaline phosphatase in TTBS was added followed by an incubation for l hour at room temperature.
The membranes were then washed four times in TTBS for 5 minutes and rinsed three times in a buffer containing 50 mM Tris-HCl pH
9.8, 3 mM MgCl2, 50 ~g/ml XP (5-bromo-4-chloro-3-indoylphosphate) and lO0 ~g/ml NBT (Nitroblue tetrasolium grade III).
Approximately lO0 positive plaques were identified.
The isolated plaques were purified by dilution and repeated screening. Phage DNA was prepared and the DNA preparations were digested with EcoRI. The digested DNA was separated by agarose electrophoresis and a number of EcoRI fragments were cloned into EcoRI digested and alkaline phosphatase treated pUC 18 plasmids and subsequently transformed into E. coli TG2. Transformants were selected on plates containing 50 ~g/ml of carbenicillin, 40 ~g/ml of X-gal (5-bromo-4-chloro-3-indoyl-~-D-galactoside) and l mM IPTC (Isopropyl-~-D-thiogalactoside). Plasmid DNA was analyzed from a number of transformants. A transformant W096/08269 2 1 99707 PCT~S95/05676 harboring a plasmid containing the full-length cDNA fragment encoding human K-casein was designated pS 270. Plasmid pS 70 DNA
was subjected to restriction endonuclease analysis. The complete nucleotide sequence of both strands of the region encoding K-casein was determined using a T7 sequencing kit (Pharmacia, Uppsala, Sweden) on double stranded templates as described by the vendor. Specific oligonucleotides complementary to pUC 18 or K-casein sequences were used as primers for sequencing reactions.
The nucleotide sequence contained an open reading frame sufficient to encode the entire amino acid sequence of a K-casein precursor protein consisting of 162 amino acids and a signal peptide of 20 amino acids.
Plasmid DNA, designated pS 270 was deposited in the collection of Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D.3300 Braunschweig, Germany and is identified by accession #DSM 6878.
Purified human or bovine K-casein or their derivatives can be administered enterally in the form of an oral mucosal dosage unit or with a feeding tube, eg. nasojejunal or jejunal. In oral use, K-casein should be between 2% and 50~ by weight of the preparation. In pharmaceutical preparations, K-casein should be mixed with a solid pulverulent carrier such as lactose, sorbitol, starch, or gelatin and pressed into tablet form. Multiple-unit dosages can also be prepared. Tablets and granules can be coated with substances such as polymers which change the dissolution rate in the gastrointestinal tract. Examples of coatings include anionic polymers having a pKa of above 5.5. Liquid preparations of human or bovine K-casein can be prepared as 0.2~ by weight of the active compound and glycerol. The daily dose of the active compound will vary with the administrative route.
The discovery disclosed herein of the antiviral activity of the secretory milk protein, K-casein, makes feasible the use of the agent in infant formula, in pharmaceutical substances useful for the prevention and treatment of rotaviral infection in all groups at risk, as a nutritional supplement for patients W096/08269 PCT~S95/05676 undergoing antibiotic therapy, and as an ingredient in other bioactive enteral nutritional products. This list is meant to be suggestive and should not be interpreted as limiting the potential uses of ~-casein.

WO 96/08269 . - PCTIUS95/05676 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: Abbott Laboratories (ii) TITLE OF INVENTION: Inhibition of Human Rotavirus Infection (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Donald O. Nickey ROSS Products Division Abbott Laboratories (B) STREET: 625 Cleveland Avenue (C) CITY: Columbus (D) STATE: Ohio (E) COUNTRY: United States of America (F) ZIP: 43215 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage (B) COMPUTER: IBM Compatible (C) OPERATING SYSTEM: MS-DOS Version 6.21 (D) SOFTWARE: WordPerfect Version 6.0a (vi) CURRENT APPLICATION DATA:
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(B) FILING DATE:
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(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/308,882 (B) FILING DATE: 16-SEP-1994 (A) APPLICATION NUMBER: US 08/308,883 (B) FILING DATE: 16-SEP-1994 (ix3 TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (614) 624-3774 (B) TELEFAX: (614) 624-3074 (C) TELEX: None (2) INFORMATION FOR SEQ ID NO: 1 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 857 base pairs (B) TYPE: Nucleic acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: cDNA
(A) DESCRIPTION: Hurnan milk kappa-casein (iii) HYPOTHETICAL: No (iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: Hurnan (A) ORGANISM: Homo sapiens (B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE: Adult (E) HAPLOTYPE:
(F) TISSUE TYPE: Mamrnar,v gland (G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE: Hurnan Mamrnary Gland (A) LIBRARY:
(B) CLONE:

(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 45...593 (C) IDENTIFICATION METHOD: DNA sequPnring and restriction analysis (D) OTHER INFORMATION: The encoded product of nucleotide SEQ ID
NO: 1 is the human milk protein, kappa-casein, having the amino acid sequence shown in SEQ ID NO: 2.
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. Hansson, et al.
(B) TITLE: DNA Encoding Kappa-Casein, Process for Obtaining the Protein and Use Thereof (C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER: PCT/W093/15196 (I) FILING DATE: 25-JAN- 1993 (J) PUBLICATION DATE: 05-AUG-1993 (K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 GAATTCCGAG AGAAGACCTG ACTGGCACGA GGAAAGGTGC AATA ATG AAG AGT

Met Lys Ser Phe Leu Leu Val Val Asn Ala Leu Ala Leu Thr Leu Pro Phe Leu Ala Val GAG GTT CAA AAC CAG AAA CAA CCA GCA TGC CAT GAG AAT GAT GAA AGA
152lu Val Gln Asn Gln Lys Gln Pro Ala Cys His Glu Asn Asp Glu Arg CCA TTC TAT CAG AAA ACA GCT CCA TAT GTC CCA ATG TAT TAT GTG CCA

Pro Phe Tyr Gln Lys Thr Ala Pro Tyr Val Pro Met Tyr Tyr Val Pro AAT AGC TAT CCT TAT TAT GGA ACC AAT TTG TAC CAA CGT AGA CCA GCT

Asn Ser Tyr Pro Tyr Tyr Gly Thr Asn Leu Tyr Gln Arg Arg Pro Ala ATA GCA ATT AAT AAT CCA TAT GTG CCT CGC ACA TAT TAT GCA AAC CCA

Ile Ala Ile Asn Asn Pro Tyr Val Pro Arg Thr Tyr Tyr Ala Asn Pro GCT GTA GTT AGG CCA CAT GCC CAA ATT CCT CAG CGG CAA TAC CTG CCA

Ala Val Val Arg Pro His Ala Gln Ile Pro Gln Arg Gln Tyr Leu Pro AAT AGC CAC CCA CCC ACT GTG GTA CGT CGC CCA AAC CTG CAT CCA TCA

Asn Ser His Pro Pro Thr Val Val Arg Arg Pro Asn Leu His Pro Ser TTT ATT GCC ATC CCC CCA AAG AAA ATT CAG GAT AAA ATA ATC ATC CCT

Phe Ile Ala Ile Pro Pro Lys Lys Ile Gln Asp Lys Ile Ile Ile Pro ACC ATC AAT ACC ATT GCT ACT GTT GAA CCT ACA CCA GCT CCT GCC ACT

Thr Ile Asn Thr Ile Ala Thr Val Glu Pro Thr Pro Ala Pro Ala Thr GAA CCA ACG GTG GAC AGT GTA GTC ACT CCA GAA GCT TTT TCA GAG TCC

Glu Pro Thr Val Asp Ser Val Val Thr Pro Glu Ala Phe Ser Glu Ser ATC ATC ACG AGC ACC CCT GAG ACA ACC ACA GTT GCA GTT ACT CCA CCT

Ile Ile Thr Ser Thr Pro Glu Thr Thr Thr Val Ala Val Thr Pro Pro ACG GCA TAAAAACACC AAGGAAATAT CAAAGAACAC AACGCAGGAC

Thr Ala CAAATTACTA CTTCACACTC TCCTTCAGCC A l-l-l GTCTGC CTTCAGTCAA

GATTTTCACA GATTCAGCTC TTCTCTCCTT ACATTTTACA TTCATGCCAC

TTGATTCTTG CACAATAAAG CCAACTGATT GCAAAAAA~ AAAAAA~
A A A A A A A A A A Q'~n AA~ U ' V
AAA~AA~ AAAAAA~ AAAAAA~ CGAATTC

(2) INFORMATION FOR SEQ ID NO: 2 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 182 arnino acids (B) TYPE: Arnino acid (C) STRANDEDNESS:
(D) TOPOLOGY: Linear (ii) MOLECULE TYPE: Protein (A) DESCRIPTION:

W O 96/08269 PCTrUS95/05676 (iii) HYPOTHETICAL:
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(A) ORGANISM:
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(A) LIBRARY:
(B) CLONE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME/SEGMENT:
(B) MAP POSITION:
(C) UNITS:
(ix) FEATURE:
(A) NAMEtKEy:
(B) LOCATION:

wo 96/08269 Pcr/usss/os676 (C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBUBLICATION INFORMATION:
(A) AUTHORS: L. ~n~o~, et al.
(B) TITLE: DNA Encoding Kappa-Casein, Process for Obtaining the Protein and Use Thereof (C) JOURNAL:
(D) VOLUME:
(E) ISSUE:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER: PCT/W093/15196 (I) FILING DATE: 25-JAN-1993 (J) PUBLICATION DATE: 05-AUG-1993 (K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 et Lys Ser Phe Leu Leu Val Val Asn Ala Leu Ala Leu Thr Leu Pro Phe Leu Ala Val Glu Val Gln Asn Gln Lys Gln Pro Ala Cys His Gin Asn Asp Glu Arg Pro Phe Tyr Gln Lys Thr Ala Pro Tyr Val Pro Met Tyr Tyr Val Pro Asn Ser Tyr Pro Tyr Tyr Gly Thr Asn Leu Tyr Gln Arg Arg Pro Ala Ile Ala Ile Asn Asn Pro Tyr Val Pro Arg Thr Tyr Tyr Ala Asn Pro Ala Val Val Arg Pro His Ala Gln Ile Pro Gln Arg - Gln Tyr Leu Pro Asn Ser His Pro Pro Thr Val Val Arg Arg Pro Asn Leu His Pro Ser Phe Ile Ala Ile Pro Pro Lys Lys Ile Gln Asp Lys Ile Ile Ile Pro Thr Ile Asn Thr Ile Ala Thr Val Glu Pro Thr Pro Ala Pro Ala Thr Glu Pro Thr Val Asp Ser Val Val Thr Pro Glu Ala Phe Ser Glu Ser Ile Ile Thr Ser THr Pro Glu Thr Thr Thr Val Ala Val Thr Pro Pro Thr Ala W096/08269 2 ~ 9 9 7 0 7 PCT~S95/05676 What is claimed is:

1. An enteral nutritional product comprising an anti-rotaviral infection effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein at a concentration exceeding that found in human or bovine milk.

2. The product of claim 1, wherein the enteral nutritional product is infant formula.

3. A pharmaceutical composition comprising a therapeutically effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein for the therapeutic or prophylactic treatment of infections of the gastrointestinal tract caused by human rotavirus.
4. A method of preventing or retarding the onset of or treating an infection of a mammalian cell caused by human rotavirus comprising contacting the cell with an effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein.

5. A method of preventing or retarding the onset of or treating infantile gastroenteritis comprising treating an infant or child at risk or afflicted with the disease according to the method of claim 4.

6. The method of claim 4, wherein the subject is an immunodeficient patient.

Claims (6)

What is claimed is:

1. An enteral nutritional product comprising an anti-rotaviral infection effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein at a concentration exceeding that found in human or bovine milk.

2. The product of claim 1, wherein the enteral nutritional product is infant formula.

3. A pharmaceutical composition comprising a therapeutically effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein for the therapeutic or prophylactic treatment of infections of the gastrointestinal tract caused by human rotavirus.

4. A method of preventing or retarding the onset of or treating an infection of a mammalian cell caused by human rotavirus comprising contacting the cell with an effective amount of an agent selected from the group consisting of native or recombinant unhydrolyzed human kappa-casein and native or recombinant unhydrolyzed bovine kappa-casein.

5. A method of preventing or retarding the onset of or treating infantile gastroenteritis comprising treating an infant or child at risk or afflicted with the disease according to the method of claim 4.

6. The method of claim 4, wherein the subject is an immunodeficient patient.