WO1996008269A1 - Inhibition de l'infection a rotavirus humain - Google Patents

Inhibition de l'infection a rotavirus humain Download PDF

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Publication number
WO1996008269A1
WO1996008269A1 PCT/US1995/005676 US9505676W WO9608269A1 WO 1996008269 A1 WO1996008269 A1 WO 1996008269A1 US 9505676 W US9505676 W US 9505676W WO 9608269 A1 WO9608269 A1 WO 9608269A1
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WIPO (PCT)
Prior art keywords
casein
human
bovine
cells
rotavirus
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PCT/US1995/005676
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English (en)
Inventor
Pradip Mukerji
Pedro Antonio Prieto
Amando Eug-Yong Seo
Jeffrey Harris Baxter
Richard Dale Cummings
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Abbott Laboratories
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Publication date
Priority claimed from US08/308,883 external-priority patent/US5576300A/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Priority to NZ285593A priority Critical patent/NZ285593A/xx
Priority to MX9701979A priority patent/MX9701979A/es
Priority to JP8510160A priority patent/JPH10505828A/ja
Priority to EP95919035A priority patent/EP0784479A1/fr
Priority to AU24744/95A priority patent/AU711437B2/en
Publication of WO1996008269A1 publication Critical patent/WO1996008269A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4732—Casein
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to a secretory protein, kappa-casein ( ⁇ -casein) , which inhibits the attachment of human rotavirus (HRV) to mammalian cells.
  • ⁇ -casein kappa-casein
  • 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, 1984-1991, 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 protection against infection and rotavirus infection has been observed in breast-fed infants.
  • Yolken et al. report a nonimmunoglobulin natural substance, milk mucin, that inhibits the replication of rotavirus.
  • Mucin is a sialic acid-containing glycoprotein.
  • Yolken et al. demonstrate that mucin and mucinous components found in human milk show anti-rotaviral activity.
  • Yolken et al. first showed mucin binding to African Green Monkey Kidney (MA-104) cells infected with human and simian strains of rotavirus.
  • tissue culture techniques they next showed that a fraction of human milk, subsequently identified as human milk mucin complex, inhibited rotavirus replication.
  • Yolken et al. also demonstrated the efficacy of mucin- containing milk components in preventing experimental rotavirus gastroenteritis in suckling mice.
  • WO 94/09651 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 discloses a method for preventing adhesion of E. coll to human epithelial cells by treating them with ⁇ -casein, a sialic acid-conjugated protein derived from cow's milk.
  • ⁇ -casein which can be purified from human or bovine milk, or manufactured in reco binant form using cDNA, inhibits the attachment of human rotavirus to mammalian cells.
  • 60-70% inhibition of human rotavirus binding was obtained when the cells were treated with either human or bovine ⁇ -casein.
  • ⁇ -casein which is a mammary product believed to be without adverse side effects, can be used for the prevention and treatment of rotavirus infection.
  • 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 ⁇ -casein.
  • Fig. 4 shows that human ⁇ -casein retained its ability to inhibit binding by human rotavirus to MA-104 cells after being treated with either neura inidase, fucosidase or both.
  • Fig. 5 shows that bovine ⁇ -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 Pronase* on the ability of bovine ⁇ -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 ⁇ -casein to inhibit infection of MA-104 cells.
  • Fig. 8 shows the DNA sequence and the amino acid sequence of human ⁇ -casein.
  • Fig. 9 shows the amino acid sequence which has the biological activity of human ⁇ -casein.
  • 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 ⁇ -casein in inhibiting the attachment of human rotavirus (HRV) to simian cells, and thereby preventing the virus from entering the cells and causing infection.
  • HRV human rotavirus
  • the agent of this invention, ⁇ -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 ⁇ -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.
  • 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 K- casein was detected either by comparison with human ⁇ -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 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 4°C 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.
  • Bovine ⁇ -casein was purchased from Sigma (catalog #CO406) and had a purity of greater than 80%.
  • Example 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 (1991) .
  • 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 125 I-labeled human rotavirus (HRV) suspensions. Radiolabeled virus was prepared as described in Fig.l. The plates were incubated for 90 minutes at 37"C and washed three times with TBS. The cells were then lysed by adding 0.2% sodium hydroxide and 2% SDS solution.
  • BSA bovine serum albumin
  • 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 ⁇ -casein was shown to be dose- dependent in the same experiment.
  • bovine ⁇ -casein When bovine ⁇ -casein was treated with neuraminidase to 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 ⁇ -casein was digested in 100-fold excess with a protease, Pronase* (Calbiochem, La Jolla, CA) , for 16 hours at 37°C in 0.1M tris buffer at pH 8.0.
  • 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 ⁇ -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.
  • 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) .
  • BME Basal Medium Eagle
  • FBS Fetal Bovine Serum
  • BioWhittaker 2 mM L-glutamine
  • 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 37°C in a 5% C0 2 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.
  • HRV was activated with 10 IU/ml trypsin per 1 ml HRV and incubated for 30 minutes at 36°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.
  • plates containing MA-104 cells were washed 3 times with 15 ⁇ l diluent medium (150 ⁇ l/well/wash) after which another 50 ⁇ l of diluent medium was added to each well.
  • MA-104 plates were then incubated at 37°C in 5% C0 2 until the preincubation period for the HRV+test agent was completed.
  • a 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 37°C 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.
  • 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.
  • PBS-CEA chick egg albumin
  • 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 dia inobenzidine (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.
  • Kappa-Casein - Second Assay Method - Counting Infected Cells 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 examination. 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 ⁇ -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.
  • Example 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 37°C 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 ⁇ -casein with the protease eliminated inhibition of infection of MA-104 cells by HRV.
  • bovine ⁇ -casein Undigested bovine ⁇ -casein showed between 86% and 93% inhibition of viral attachment to Wa cells. Hydrolyzed bovine ⁇ -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.
  • Human ⁇ -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 ⁇ -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 ⁇ -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 ⁇ -casein.
  • the DNA sequence shown can be used in the production of recombinant human ⁇ -casein using either a procaryotic or a eukaryotic system, or can be used to produce transgenic non-human mammals.
  • Recombinant human ⁇ -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 MgS0 4 .
  • TTBS tris buffered saline (TBS) containing 0.05% Tween 20
  • TTBS tris buffered saline
  • FCS FCS
  • ⁇ -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
  • the membranes were washed again with TTBS two times for 5 minutes at room temperature.
  • TTBS Tris-HCl pH 9.8, 3 mM MgCl 2 , 50 ⁇ g/ml XP (5-bromo-4-chloro-3-indoylphosphate) and 100 ⁇ g/ml NBT (Nitroblue tetrasolium grade III) . Approximately 100 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-j3-D-galactoside) and 1 mM IPTC (Isopropyl-0-D-thiogalactoside) .
  • Plas id DNA was analyzed from a number of transformants.
  • a transformant harboring a plasmid containing the full-length cDNA fragment encoding human ⁇ -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 ⁇ -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 Sam lung 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.
  • K-casein should be between 2% and 50% by weight of the preparation.
  • ⁇ -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.
  • ⁇ -casein The discovery disclosed herein of the antiviral activity of the secretory milk protein, ⁇ -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 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.
  • nucleotide SEQ ID NO: 1 is the human milk protein, kappa-casein, having the amino acid sequence shown in SEQ ID NO: 2.

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Abstract

Produit nutritionnel entérique contenant soit de la caséine λ humaine, soit de la caséine λ bovine en une concentration supérieure à celle que l'on trouve dans le lait humain ou bovin et suffisante pour inhiber l'infection de cellules mammaliennes par le rotavirus humain.
PCT/US1995/005676 1994-09-16 1995-05-05 Inhibition de l'infection a rotavirus humain WO1996008269A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
NZ285593A NZ285593A (en) 1994-09-16 1995-05-05 An enteral nutritional product containing casein that is sufficient to inhibit infection of mammalian cells by human rotavirus
MX9701979A MX9701979A (es) 1994-09-16 1995-05-05 Inhibicion de infeccion por rotavirus humano.
JP8510160A JPH10505828A (ja) 1994-09-16 1995-05-05 ヒトロタウイルス感染の阻害
EP95919035A EP0784479A1 (fr) 1994-09-16 1995-05-05 Inhibition de l'infection a rotavirus humain
AU24744/95A AU711437B2 (en) 1994-09-16 1995-05-05 Inhibition of human rotavirus infection

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US30888294A 1994-09-16 1994-09-16
US308,883 1994-09-16
US08/308,883 US5576300A (en) 1994-09-16 1994-09-16 Method for inhibition of human rotavirus infection
US308,882 1994-09-16

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CA (1) CA2199707A1 (fr)
MX (1) MX9701979A (fr)
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6849593B1 (en) * 1997-09-16 2005-02-01 Pharis Biotec Gmbh Bifidogenic peptides
US8211476B2 (en) 2003-03-14 2012-07-03 Meiji Co., Ltd. Compositions against rotavirus infection and processes for producing the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4180645B2 (ja) 2005-09-30 2008-11-12 森永乳業株式会社 グルカゴン様ペプチド−１分泌促進剤及び食後血糖値上昇抑制剤

Citations (5)

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WO1993015196A1 (fr) * 1992-01-23 1993-08-05 Symbicom Aktiebolag And codant la kappa-caseine, procede permettant d'obtenir cette proteine et son utilisation
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US8211476B2 (en) 2003-03-14 2012-07-03 Meiji Co., Ltd. Compositions against rotavirus infection and processes for producing the same
US8440233B2 (en) 2003-03-14 2013-05-14 Meiji Co., Ltd Compositions against rotavirus infection and processes for producing the same

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EP0784479A1 (fr) 1997-07-23
NZ285593A (en) 1999-03-29
JPH10505828A (ja) 1998-06-09
AU2474495A (en) 1996-03-29
MX9701979A (es) 1997-06-28
AU711437B2 (en) 1999-10-14
CA2199707A1 (fr) 1996-03-21

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