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EP0760674A1 - Inhibition de l'infection de cellules de mammiferes par le virus respiratoire syncytial - Google Patents

Inhibition de l'infection de cellules de mammiferes par le virus respiratoire syncytial

Info

Publication number
EP0760674A1
EP0760674A1 EP95914824A EP95914824A EP0760674A1 EP 0760674 A1 EP0760674 A1 EP 0760674A1 EP 95914824 A EP95914824 A EP 95914824A EP 95914824 A EP95914824 A EP 95914824A EP 0760674 A1 EP0760674 A1 EP 0760674A1
Authority
EP
European Patent Office
Prior art keywords
casein
human milk
human
infection
rsv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95914824A
Other languages
German (de)
English (en)
Inventor
Pradip Mukerji
Amanda Eun-Yeong Seo
Steven Neal Anderson
Joseph Paul Schaller
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Abbott Laboratories
Original Assignee
Abbott Laboratories
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/249,554 external-priority patent/US5506209A/en
Priority claimed from US08/249,555 external-priority patent/US5538952A/en
Application filed by Abbott Laboratories filed Critical Abbott Laboratories
Publication of EP0760674A1 publication Critical patent/EP0760674A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
    • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • 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
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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

Definitions

  • the present invention relates generally to inhibiting the infection of mammalian cells by Respiratory Syncytial Virus, and more specifically to the use of native or recombinant human ⁇ -casein and hydrolysates thereof for inhibiting the infection of mammalian cells by Respiratory Syncytial Vi us.
  • Respiratory Syncytial Virus is the single most frequent cause of acute respiratory tract infection in infants and children. Infants less than six months of age are most frequently and seriously affected. In most immunologically normal subjects, infection with RSV is limited to the respiratory mucosa, and is associated with the development of bronchiolitis, pneumonia and reactive airway disease. RSV infection in immunocompromised subjects has until recently been associated with increased mortality in infants and increased morbidity in other age groups. It has recently been reported in PEDIATRIC NOTES. Vol. 18, No. 14, January 27. 1994. that periods of high incidence of acute respiratory disease and numbers of deaths in elderly people were followed within 2-3 weeks by reports of high numbers of RSV or influenza virus isolates. The analyses indicate that RSV is as important as influenza viruses in causing morbidity and deaths among the elderly.
  • RSV-neutralizing components from breast milk may reach an infant ' s respiratory tract di ectly as a result of regurgitation and inhalation of milk during and after feeding.
  • the mucosa of the respiratory tract may gain direct protection in this way.
  • WO 91/06308 filed byAndersson et al . for “ANTIBACTERIAL COMPOSITION", and a published article by the same authors (Aniansson et al . , "Anti- adhesive activity of human casein against Streptococcus pneumonia and Haemophilus influenzae", MICROBIAL PATHOGENESIS.8:315-323 (1990) disclose the use of a milk fraction having a molecular weight of at least 5,000 da!tons for "therapeutic prophylactic, and/or diagnostic use in infections caused by S. pneumonae and/or H. influenzae” . but it is suggested in these publications that the beneficial effect is provided by kappa-casein. However, the present invention relates to the use of native or recombinant human ⁇ -casein and hydrolysates of both to inhibit RSV infections.
  • W093/04172 relates to a DNA sequence encoding human ⁇ -casein, but does not disclose the capacity of either native or recombinant human ⁇ -casein to inhibit the attachment of RSV to human cells.
  • W091/08675 discloses an infant formula which contains recombinant forms of both human alpha-lactalbumin and human -casein.
  • 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 ?-casein to inhibit the attachment of RSV to human cells is not taught or suggested in said publication.
  • the two assays (a HEp-2 cell assay and a LLC-MK2 cell assay) which were used for determining the bioactivity of /?-casein are described below. These assays have not been published heretofore, although the HEp-2 cell assay was based upon established methodology.
  • ⁇ -casein cDNA and the expression system from Symbicom AB, P.O. Box 1451, S-90124 Umea, Sweden.
  • the human ⁇ -casein cDNA used had been previously cloned and sequenced by Lonnerdal et al . , Cloning and sequencing of a cDNA encoding human milk ⁇ -casein. (SEQ.ID NO: 1:) Federation of European Biochemical Societies Letters 269, 153-156 (1990).
  • the recombinant human ⁇ -casein was obtained from E. coli and purified according to the method of Hansson et al .
  • Human ⁇ -casein cDNA was isolated by Hansson et al . as a 1.1-kb fcoRI fragment from a human lambda gt mammary gland library, and was subcloned into pUC19, which was designated pS21.
  • the cDNA was modified by introduction of synthetic oligonucleotides in the 5 ' and 3' termini. To introduce a suitable cloning site in the 5' end, Ndel , a translational start, was inserted in front of the sequence encoding mature human ⁇ -casein. To adapt the initial part of the translated sequence to E.coli codon usage, six synthetic oligonucleotides were constructed and ligated.
  • a 303-bp Accl/Bgl II fragment was isolated and cloned into a pUC18 derivative and designated plasmid pS22.
  • Four synthetic oligonucleotides containing the sequence encoding the carboxy-terminal end and translation stop followed by BamH and £c ⁇ RI sites were constructed resulting in the sequence 5 ' AGATCTACCCTGTGA CTCAGCCAC ⁇ GCCCCAG ⁇ CATAACCCCA ⁇ AGTGTCTAATAAGGATCCGAA ⁇ C-3' . (SEQ ID NO: 3:) where the protein encoding sequence is underlined.
  • the synthetic fragment was cloned into Bglll/EcoRI digested pS22, resulting in plasmid pS23.
  • three fragments were ligated: an 89-bp Pst /Avail fragment from pS24; a 197-bp Avail /AccI fragment from pS21; and Pstl/AccI digested pS23.
  • the resulting plasmid pS25 was digested with Ndel/BamWI and a 641-bp fragment was isolated and cloned into the vector pET-3a.
  • the resulting expression vector was designated pS26.
  • the E . coli signal sequence of the enterotoxin STII gene was introduced in front of the ⁇ -casein encoding sequence.
  • a modified STII sequence with Ncol- and Ndel-compatible ends and an internal Clal site was obtained by using a synthetic oligonucleotide, 5 ' -CATGAAAAAGAATATCGCA ⁇ TC ⁇ c ⁇ GCATCGATG ⁇ CGT ⁇ TTTCTATTGCTACAAATGCATATG-3' (SEQ ID NO: 4:).
  • pS25 was digested with I_I/£CORI and a 619-bp fragment was isolated.
  • This fragment was ligated with a syntheti c o 1 i g o n u c 1 e ot i d e fragment , 5'CATATGCACGTGAAACCATCGAATCCCTGAGCTCGAG-3' (SEQ ID NO: 5:), and Ndel/EcoRI- digested pUC19.
  • the resulting plasmid was designated pS27.
  • the final expression vector,pS28 was constructed by ligating three fragments: a 700- bp Ndel/Hindlll ⁇ -casein fragment isolated from pS27, the STII signal sequence, and a Ncol/ Hindi 11-digested pACAT7 vector.
  • the expression vectors pS26 and pS28 were used to transform E.coli strains BL2KDE3), BL21(DE3)pLysS. and BL21(DE3)pLysE.
  • the bacteria were grown in Luria Broth medium containing 50 ⁇ g/ml carbenicillin, and when B121(DE3)pLysS and BL21(DE3)pLysE were used the mediumwas supplemented with 25 ⁇ g/ml chloramphenicol .
  • the cultures were grown to a density yielding an optical density (OD) of 0.6 at a wavelength of 600 nanometers (OD 600 ), then 0.4 mM IPTG was added to induce the T7 system.
  • the cells were harvested about 90 minutes after induction.
  • Recombinant ⁇ -casein was isolated using standard procedures. The inducible T7-based expression system resulted in high-level expression of recombinant ?-casein. Bacteria were harvested and the cells pelletted by centrifugation. The supernatant contained the periplasmic proteins and the pellet the cytoplasmic fraction. The recombinant proteins obtained were compared with native ⁇ -casein, which had been purified by standard methods including either ion-exchange chromatography followed by reversed-phase HPLC or gel filtration. Recombinant and native ⁇ -casein were compared by standard biochemical techniques comprising SDS-PAGE, Western blotting, amino acid analysis,peptide mapping, phosphate analysis, and mass spectrometry. Recombinant ⁇ -casein expressed in E.coli was found to comigrate with full- length, nonphosphorylated native human -casein, which is one of seven native isoforms.
  • Recombinant human ⁇ -casein has also been expressed in S. cerevisiae using the pYES 2.0 vector (Invitrogen Corp.. San Diego, CA). but the expression level was approximately 10% of that obtained in E.coli . However, Hansson et al . found that S. cerevisiae appeared to express phosphorylated human milk ⁇ -casein.
  • the human ⁇ -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 37°C in 0.1 M NH 4 HC0 3 , pH 7.8. These digests were dried and resuspended in appropriate buffers prior to use in the assays discussed above.
  • the Long strain of RSV was grown in HEp-2 cells under Eagle minimal essential medium (MEM) with 2% fetal bovine serum (FBS).
  • the virus was harvested at a cytopathic effect (CPE) of 3 to 4+, sonicated for 10 seconds at 50% power with a Microson ultrasonic bell disrupter (Heat Systems - Ultrasonics, Inc., Farmingdale, N.Y.), divided into portions and stored at -70°C.
  • CPE cytopathic effect
  • Microson ultrasonic bell disrupter Heat Systems - Ultrasonics, Inc., Farmingdale, N.Y.
  • the neutralization tests were performed in 96-well, flat-bottomed, tissue culture, microtiter plates (catalog no. 3596; Costar, Cambridge, Mass.). Serum or a monoclonal antibody (MAb) in the form of ascites fluid, which had been heat inactivated at 56°C for 30 min. , was added to duplicate wells and serial fourfold dilutions were performed in the microtiter plates. All dilutions were in MEM-2% FBS. and the final volume was 75 ⁇ l per well. Approximately 6050% tissue culture infective doses of virus in 25 ⁇ l of MEM-2% FBS then were added to each well, and the mixture was incubated for 2 h at 4°C.
  • Serum or a monoclonal antibody (MAb) in the form of ascites fluid which had been heat inactivated at 56°C for 30 min.
  • MAb monoclonal antibody
  • PBS phosphate- buffered saline
  • the Enzyme Linked I muno Sorbent Assay was performed on the same day as the fixation, or the plates were stored overnight at 4°C and the ELISA was performed on the next day.
  • the wells were precoated with 200 ⁇ l of PBS with 0.5% gelatin for 30 min at 35°C, the contents were aspirated, the wells were washed with PBS (pH 7.2)-0.5% Tween 20 and 75 ⁇ l of bovine anti-RSV serum (BaRSV) Burroughs Wellcome Co., Research Triangle Park, N.C.) diluted in PBS 0.5% gelatin plus 0.5% Tween 20 and 2% normal goat serum was added and incubated for 1 hour at 35.5°C.
  • human and bovine ?-casein solutions were prepared first in 20 mM ethanol ami ne, 6 M urea, pH 9.5 and then washed twice in PBS by ultrafiltration using Centricon membrane filters (Ami con, MA) with a cut-off of 3,000 daltons. After resuspending in appropriate buffer for the HEp-2 cell assay described above, these samples were tested in the assay. Experiments with different designated numbers were performed in different days. As shown in Table 1, human ⁇ -casein caused an inhibition of infection/ virus replication of 50% or more at concentrations of 0.4 mg/ml or greater.
  • the RSV inhibition assay quantitatively determines the ability of a test reagent (antibody or other bioactive compound) to inhibit the infection of monkey kidney cells (LLC-MK2) (ATCC CCL 7) in microtiter plates. Infected cells were identified using an immunoperoxidase method. The method is described briefly below.
  • LLC-MK2 cells were seeded into fibronectin treated Costar microtiter plates (5.0 X 10 3 cells per well) and incubated for 3-4 days prior to use in the infectivity reduction assay.
  • the Long strain of RSV was diluted in MEM to 10-20,000 infected cell units (ICU/mL). and added to an equal volume (200 ⁇ L) of serially diluted sample preparations at suitable concentration (ex. 0.5, 1.0, and 2.0 mg casein/mL). Mixtures of diluted test samples and virus were then incubated for 2 hours at 4°C prior to adding to LLC-MK2 cells.
  • culture medium Prior to addition of the diluted sample- virus mixtures to microtiter plates, culture medium was removed and the monolayers rinsed one time with MEM. All diluted sample-virus mixtures were tested in triplicate wells. The diluted sample-virus mixtures were allowed to absorb to LLC-MK2 monolayers for 2 hours at 37°C in a humidified C0 2 incubator. Following incubation, 150 ⁇ l of MEM was added to all wells and the plates incubated at 37°C for 16 hours in the C0 2 incubator. After overnight incubation, the culture medium was removed and the monolayers fixed with cold ethanol.
  • microtiter plates were rinsed once with 200 ⁇ l Dulbecco ' s PBS per well, and bovine anti-RSV antibody (200 ⁇ l) was added to all wells. Following a 30 minute incubation at room temperature and three rinses with PBS/0.5% chick albumen (PBS/CEA), peroxidase labeled rabbit anti-bovine IgG was added to all wells and incubated at room temperature for 30 minutes. Microtiter plates were then rinsed 3 times with PBS/CEA and diaminobenzadine substrate added and incubated for 20 minutes. Plates were then rinsed as above with PBS/CEA, and the number of stained RSV-infected cells per well determined using an inverted microscope. RESULTS FROM LLC-MK2 CELL ASSAY
  • an enteral liquid nutritional product such as infant formula, comprising one or more proteins not contained in human milk in combination with a therapeutically effective amount of at least one of the forms of human -casein described in the preceding paragraph.
  • the infection of mammalian cells by RSV may be inhibited by administering via a nasal passageway, or as a throat spray, a formulation containing a therapeutically effective amount of at least one of the forms of human ⁇ -casein identified in the preceding paragraph.
  • a nasally administered formulation may be in the form of either drops or a spray.
  • enteral nutritional, throat spray and nasal products and methods are believed to be effective in inhibiting the infection of mammalian cells by RSV because the interaction of the human ⁇ -casein and RSV is believed to occur via direct contact rather than following digestion and absorption of the ⁇ -casein.
  • 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.
  • 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.
  • 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 respiratory tract infection in human infants.
  • MOLECULE TYPE Cloned cDNA representing the product of a human genomic DNA segment
  • nucleotide SEQ ID NO: 1 is the human milk protein, ⁇ -casein.
  • GCA AGG GAG ACC ATA GAA AGC CTT TCA AGC AGT GAG GAA TCT ATT 90
  • MOLECULE TYPE Synthetic ohgonucleotide
  • MOLECULE TYPE Synthetic ohgonucleotide
  • MOLECULE TYPE Synthetic ohgonucleotide
  • MOLECULE TYPE Synthetic ohgonucleotide

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Abstract

L'infection de cellules de mammifères par la virus respiratoire syncytial RSV peut être empêchée par la β-caséine humaine native, par une forme recombinée de β-caséine humaine et par des hydrolysats de l'une et l'autre. La β-caséine humaine ou son hydrolysat peuvent être contenus dans un produit d'alimentation entérale liquide, tel que du lait maternisé. Ce produit d'alimentation entérale peut être utilisé par exemple pour prévenir et traiter les infections des voies respiratoires chez les nourrissons. La β-caséine humaine ou son hydrolysat peuvent également être administrés sous la forme d'un spray pour la gorge ou par voie nasale en gouttes ou en spray.
EP95914824A 1994-05-26 1995-03-27 Inhibition de l'infection de cellules de mammiferes par le virus respiratoire syncytial Withdrawn EP0760674A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US249555 1981-03-31
US08/249,554 US5506209A (en) 1994-05-26 1994-05-26 Product for inhibition of infection of mammalian cells by respiratory syncytial virus
US08/249,555 US5538952A (en) 1994-05-26 1994-05-26 Inhibition of infection of mammalian cells by respiratory syncytial virus
US249554 1994-05-26
PCT/US1995/003628 WO1995032727A1 (fr) 1994-05-26 1995-03-27 Inhibition de l'infection de cellules de mammiferes par le virus respiratoire syncytial

Publications (1)

Publication Number Publication Date
EP0760674A1 true EP0760674A1 (fr) 1997-03-12

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ID=26940164

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95914824A Withdrawn EP0760674A1 (fr) 1994-05-26 1995-03-27 Inhibition de l'infection de cellules de mammiferes par le virus respiratoire syncytial

Country Status (6)

Country Link
EP (1) EP0760674A1 (fr)
JP (1) JPH10500100A (fr)
AU (1) AU697616B2 (fr)
CA (1) CA2190609A1 (fr)
NZ (1) NZ283578A (fr)
WO (1) WO1995032727A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7982008B2 (en) 2002-11-27 2011-07-19 David Bar-Or Treatment of diseases and conditions mediated by increased phosphorylation

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ283281A (en) * 1994-05-26 2000-07-28 Abbott Lab Liquid Nutritional Product or throat spray containing beta-casein from Human milk for inhibiting attachment of Haemophilus influenzae to Human cells
US5707968A (en) * 1994-05-26 1998-01-13 Abbott Laboratories Inhibition of attachment of H.influenzae to human cells
US5942254A (en) * 1995-02-27 1999-08-24 Abbott Laboratories Phosphorylated recombinant human β-casein expressed in a bacterial system
JP2000500141A (ja) * 1995-11-06 2000-01-11 アボツト・ラボラトリーズ リン酸化された組換えヒトベータ−カゼインを用いるヒト細胞に対するインフルエンザ菌の付着阻止方法
WO2004050118A1 (fr) * 2002-11-29 2004-06-17 Morinaga Milk Industry Co., Ltd. Inhibiteur de la cysteine protease
WO2011138489A1 (fr) * 2010-05-06 2011-11-10 Consejo Superior De Investigaciones Científicas (Csic) Utilisation d'hydrolysats de caséines en tant qu'antiviraux

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2631470B2 (ja) * 1987-05-15 1997-07-16 雪印乳業株式会社 感染防御剤
SE465109B (sv) * 1989-10-30 1991-07-29 Bengt Andersson Antibakteriell komposition
US5795611A (en) * 1989-12-20 1998-08-18 Slattery; Charles W. Human infant formulas containing recombinant human alpha-lactalbumin and beta-casein
WO1993004171A1 (fr) * 1991-08-19 1993-03-04 Symbicom Aktiebolag Beta-caseine humaine, procede de preparation et utilisation
JPH08501688A (ja) * 1992-09-22 1996-02-27 ニュージーランド デアリー リサーチ インスティテュート ベータ・カゼイン強化製品の製造方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9532727A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7982008B2 (en) 2002-11-27 2011-07-19 David Bar-Or Treatment of diseases and conditions mediated by increased phosphorylation
US8507651B2 (en) 2002-11-27 2013-08-13 Dmi Acquisition Corp. Treatment of diseases and conditions mediated by increased phosphorylation

Also Published As

Publication number Publication date
WO1995032727A1 (fr) 1995-12-07
AU2192095A (en) 1995-12-21
NZ283578A (en) 1998-05-27
MX9605830A (es) 1998-06-30
AU697616B2 (en) 1998-10-15
JPH10500100A (ja) 1998-01-06
CA2190609A1 (fr) 1995-12-07

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