WO2005018538A2 - Polypeptides du sras (syndrome respiratoire aigu severe), et anticorps desdits polypeptides et leur utilisation a des fins de diagnostics, vaccination, et applications therapeutiques - Google Patents

Polypeptides du sras (syndrome respiratoire aigu severe), et anticorps desdits polypeptides et leur utilisation a des fins de diagnostics, vaccination, et applications therapeutiques Download PDF

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Publication number
WO2005018538A2
WO2005018538A2 PCT/US2004/017715 US2004017715W WO2005018538A2 WO 2005018538 A2 WO2005018538 A2 WO 2005018538A2 US 2004017715 W US2004017715 W US 2004017715W WO 2005018538 A2 WO2005018538 A2 WO 2005018538A2
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Prior art keywords
sars
polypeptide
bioconjugate
antibodies
set forth
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PCT/US2004/017715
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English (en)
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Frank Q. Li
Wan-Ching Lai
Yong Liang Chu
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Vaxim, Inc.
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Publication of WO2005018538A2 publication Critical patent/WO2005018538A2/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates generally to polypeptides and, in particular, to SARS polypeptides and fragments thereof and to the use of these polypeptides in diagnostic and therapeutic applications.
  • Severe Acute Respiratory Syndrome is a respiratory disorder of humans where patients exhibits both flu-like and cold-like symptoms. Although the disease was originally identified in China and various other portions of South East Asia, it has been exported by human travel and contact to North and South
  • PCR analysis While reliable, PCR is a laboratory assay, that cannot be conducted in the clinic, at home, on the street, etc. It requires substantial investment in apparatus and infrastructure. Further, notwithstanding the availability of the nuclear and amino acid structure of the virus responsible, reliable treatments have yet to emerge.
  • the virus-induced condition is frequently lethal, j although there are survivors.
  • an isolated polypeptide which binds antibodies to the SARS virus.
  • the polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NOS. 1-32.
  • the polypeptide can comprise up to 25, 50, or 100 amino-acid residues.
  • the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NOS. 1-32.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 1.
  • a bioconjugate comprising a hyaluronic acid polymer conjugated to a polypeptide as set forth above and an article of manufacture which comprises a solid support and the bioconjugate immobilized on a surface of the support are also provided.
  • a method of detecting the presence of anti-SARS antibodies in a sample is also provided which comprises: contacting the sample with a bioconjugate as set forth above wherein the bioconjugate is immobilized on a surface of a solid support; washing the surface of the solid support; and detecting anti-SARS antibodies on the surface of the support.
  • a pharmaceutical composition comprising an isolated polypeptide or a bioconjugate as set forth above and a method of immunizing a mammal against the SARS virus which comprises administering the pharmaceutical composition to the mammal are also provided.
  • a method of isolating anti-SARS antibodies is also provided which comprises: contacting serum from a recovered SARS patient with a bioconjugate as set forth above, wherein the bioconjugate is immobilized on a surface of a solid support wherein SARS antibodies in the serum bind to the bioconjugate; releasing the bound antibodies from the solid support; and collecting the antibodies released from the solid support.
  • a pharmaceutical composition comprising anti-SARS antibodies isolated by the above method and a method of immunizing a mammal against the SARS virus which comprises administering the pharmaceutical composition to the mammal are also provided.
  • an isolated polypeptide which binds to angiotensin-converting enzyme 2 (ACE2), a SARS coronavirus receptor.
  • ACE2 angiotensin-converting enzyme 2
  • the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ JO NOS: 39, 109, 110, 111, 112 and 113.
  • the polypeptide can comprise up to 25, 50, or 100 amino-acid residues.
  • the polypeptide can have an amino acid sequence selected from the group consisting of SEQ JJD NOS: 39, 109, 110, 111, 112 and 113.
  • the polypeptide has the amino acid sequence of SEQ ID NO:39.
  • a bioconjugate comprising a hyaluronic acid polymer conjugated to a polypeptide as set forth above and an article of manufacture which comprises a solid support and the bioconjugate immobilized on a surface of the support are also provided.
  • a pharmaceutical composition comprising an isolated polypeptide or a bioconjugate as set forth above is also provided.
  • a method of treating SARS is also provided which comprises administering a pharmaceutical composition as set forth above to a patient infected with the SARS virus.
  • FIG. 1 is a photograph of a rectangular membrane array which has been exposed to the sample of an individual who had recovered from SARS and was confirmed positive by PCR.
  • FIG. 2 is a photograph of a rectangular membrane array which has been exposed to the sample of an individual who was without either SARS symptoms or infection as confirmed by PCR.
  • FIG. 3 is a photograph of a rectangular membrane array which has been exposed to the sample of a first patient exhibiting SARS symptoms but confirmed negative by PCR.
  • FIG. 4 is a photograph of a rectangular membrane array which has been exposed to the sample of a second patient exhibiting SARS symptoms but confirmed negative by PCR.
  • FIG. 1 is a photograph of a rectangular membrane array which has been exposed to the sample of an individual who had recovered from SARS and was confirmed positive by PCR.
  • FIG. 2 is a photograph of a rectangular membrane array which has been exposed to the sample of an individual who was without either SARS symptoms or infection as confirmed by PCR.
  • FIG. 3 is a photograph of a rectangular membrane array which has been exposed to
  • FIG. 5 is a photograph of a rectangular membrane array which has been exposed to a sample of a second individual with SARS as confirmed by PCR.
  • FIG. 6 is a photograph of a rectangular membrane array which has been exposed to the sample of a third patient exhibiting SARS symptoms but confirmed negative by PCR.
  • FIG. 7 is a bar chart showing the immunoreactivity of ACE2 to its antibodies as a function of ACE2 concentration.
  • FIGS 8A-8D are bar charts showing the binding activities of HA-peptide conjugates to ACE2 protein.
  • FIG. 9 is a bar chart showing the binding of ACE2 protein to a conjugate of
  • FIG. 10 is a bar chart showing the competitive binding of ACE2 protein to free peptide 25 (P25) and to a conjugate of HA and peptide 25 (HA-P25).
  • Peptide 21 an antigenic determinant of the SARS virus, a seventeen amino acid sequence, designated herein "Peptide 21", which is bound by antibodies to the SARS virus. This isolated peptide has the following sequence: FERDISNVPFSPDGKPC SEQ ID NO: 1
  • polypeptide fragments include the following: Pept #78 PDGKPC SEQ ID NO:2 Pept #79 SPDGKPC SEQ ID NO:3 Pept #80 FSPDGKPC SEQ JJ3 NO:4 Pept #81 PFSPDGKPC SEQ ID NO:5 Pept #82 VPFSPDGKPC SEQ JJD NO:6 Pept #83 NVPFSPDGKPC SEQ JJD NO:7 Pept #84 SNVPFSPDGKPC SEQ JD NO:8 Pept #85 ISNVPFSPDGKPC SEQ ID NO:9 Pept #86 DISNVPFSPDGKPC SEQ ID NO: 10 Pept #87 RDISNVPFSPDGKPC SEQ ID NO: 11 Pept #88 ERDISNVPFSPDGKPC SEQ ID NO: 10
  • All of the above polypeptides including Peptide 21 (SEQ ID NO:l) and the fragments thereof identified as Peptides 78 - 88 (SEQ TD NOs:2-12), are aspects of the invention.
  • SARS virus genome was first submitted to a standard computer blast program to identify antigenic determinants. Of 138 potential antigenic determinants, the forty (40) most antigenic polypeptides were selected by the computer program and the remaining ninety eight (98) polypeptides were selected by the inventors as potential determinants.
  • the polypeptides were plated onto a membrane by attaching a linker arm to a hyaluronic acid (HA) moiety, and affixing the HA moiety to the membrane surface.
  • HA hyaluronic acid
  • the suspect polypeptide is exposed, spatially, to potential antibodies.
  • All the selected peptides were plotted onto a rectangular membrane array, such as that illustrated by rows A-H and columns 1-12 of FIGS. 1-6.
  • the membrane was blocked with five (5 %) percent dry milk in 20 mL PBS for one hour at room temperature.
  • the resulting affixed membranes in A2 - A7 were exposed to serum of patients either exhibiting SARS symptoms, exposed to SARS, or normal (as a control). As shown in FIG.
  • FIG. 5 is included as a reflection of a failed array, which was also exposed to a sample of a patient with SARS confirmed by PCR. As can be seen, no positive dot was shown which would have reflected binding to an antigenic peptide (the placement of the peptides was random, and changed for each membrane) but neither was a control dot blot formed, indicating that the exposure had been handled improperly. As a result of these double blind studies, a specific polypeptide bound by antibodies to the SARS virus has been identified. This polypeptide can be used diagnostically, therapeutically or as a vaccine. Additional SARS Antigenic Polypeptides In addition to the aforementioned polypeptides, twenty (20) additional
  • SARS antigenic polypeptides originated from S and N proteins in SARS corona virus have also been identified. These twenty polypeptide sequences are listed as follows:
  • SARS antigenic polypeptides All of the aforementioned polypeptides are referred to collectively herein as "SARS antigenic polypeptides".
  • SARS antigenic polypeptides can be used for diagnosis.
  • a support bearing a SARS antigenic polypeptide e.g., SEQ ID NO:l
  • a membrane on which a conjugate of hyaluronic acid and a SARS antigenic polypeptide are placed is combined with a sample of the patients blood.
  • Patients with active SARS infections should have anti-SARS antibodies in the blood which will bind to the active polypeptide.
  • Binding can be detected by a wide variety of modalities, such as providing a color indicator which is occluded upon binding, or by providing a migrating or changing color indicator or chemiluminescent indicator or the like.
  • a wide variety of reporting agents are also available.
  • a patient unaffected with SARS should not show antibody that binds to the peptide, and accordingly, a negative result should be obtained.
  • Devices of this type are simple to calibrate with a collection with antibody.
  • antibodies raised against a SARS antigenic polypeptide as described herein including straight forward murine monoclonal antibodies, produced through well known hybridoma techniques or polyclonal antibodies generated from rabbit, goat, or other species, may be used as detection reagents, h this case, the antibody is the stationary detection reagent, and again the blood sample is introduced. Where SARS virus is present in the blood, binding should occur, detected either colorimetrically, or through another modality which is detected visually (e.g., chemiluminescence) or through instruments (fluorescence or wavelengths other than those normally seen). Again, such diagnostics can be calibrated and confirmed relatively easily by exposure to the virus itself.
  • the SARS antigenic polypeptides identified above can be conjugated to Hyaluronic Acid (HA) or some other biopolymer which is water soluble and biodegradable may be used as a therapeutic which, when injected in the patient, will stimulate an immune response.
  • HA Hyaluronic Acid
  • patients suspected of having SARS that is, patients exhibiting SARS symptoms but not confirmed through PCR or other methods, may benefit from prompt inoculation with the therapeutic, to assist the body in rapid generation of therapeutic titers of the SARS antibody raised against the SARS antigenic polypeptide.
  • polypeptide fragment which has been implicated in the region critical to the ability of the corona virus that produces SARS to bind to the cell surface receptor of the target cell or host, which permits release of the nuclear material of the virus into the host and self- replication.
  • This polypeptide comprises an amino acid sequence as set forth below: NYKYRYLRHGKLRP (SEQ ID NO:39).
  • an inhalant can be prepared comprising a polypeptide having the amino acid sequence of SEQ ID NO:39.
  • the inhalant for example, can have concentrations of polypeptide of from 1 microgram/mL up to 1 g/mL.
  • the inhalant can be sprayed directly and can compete for receptor sites, thereby preventing or slowing infection.
  • human or humanized antibodies to the SARS antigenic polypeptides or peptide fragments can be administered, as passive immunization therapeutic agents, passive immunization, antibodies are provided to the individual by administration of exogenous antibodies.
  • Human antibodies prepared from genetically altered hosts such as mice which are better than 95% humanized and humanized antibodies produced by mice and other hosts which are subsequently altered by switching out CDR regions to preserve binding ability while reducing immunogenicity are well known in the art and can be used.
  • Such antibodies raised against SARS antigenic polypeptides and peptide fragments again using a conjugates of the SARS polypeptide and HA or a similar biopolymer, can be used as an antigen for generation of the antibodies.
  • SARS antigenic polypeptide as described herein can be used to raise titers in individuals exposed to SARS but not yet positively identified as infected.
  • administration of conjugated peptide to human individuals who are likely SARS targets, either by reason of geography, age, immune condition or the like, should be an effective way of raising antibody titers in the individual so immunized that will bind SARS and defeat infection, upon exposure, hi contrast to the passive immunization using the administration of significant titers of exogenous antibodies, this process relies on the patients own immune system to generate antibodies to Peptide 21 (SEQ ID NO: 1).
  • the SARS antigenic polypeptide can be conjugated or otherwise derivitized so that it will be "recognized” as immunogenic by the host.
  • a seventeen (17) amino acid residue of the polypeptide itself is unlikely to be sufficiently immunogenic to generate significant antibody titers without conjugation.
  • the SARS antigenic polypeptides also provide a method for improving short-term therapeutics for patients infected with SARS.
  • the only effective medication currently available is serum from recovered patients. Recovered patients are few, the amount of blood that can be recovered is limited, and the number of patients that can be treated with that recovered blood (anti- serum) is even more limited. Production of therapeutic antibodies, either human or humanized, will take time.
  • a conventional column packed with a SARS antigenic polypeptide as disclosed herein can be used.
  • the column, with immobilized SARS antigenic polypeptide will "pull" antibodies present in the serum of recovered patients out of solution.
  • the highly purified antibodies can be subsequently released from the column, collected, and administered on a broader basis. Isolation of the antibodies using this method also permits wider patient selection, long-term storage of the antibody and transportation of the antibody to distant sites.
  • the SARS antigenic polypeptides described herein can be used both to augment short term solutions, and provide long term solutions that include diagnosis of SARS infection, therapeutic treatment of SARS-infected patients, and vaccination of patients. Therapy can include active immunization, provided the patient does not have a compromised immune system, passive immunization using "chimeric" and/or "humanized” antibodies, and human antibodies collected from surviving
  • Peptide Fragments of SARS-CoV S Protein that Bind ACE2 The SARS coronavirus spike (S) protein mediates infection of receptor- expressing host cells.
  • Angiotensin-converting enzyme 2 (ACE2) is a functional receptor for the SARS coronavirus. Dimitrov, Cell Previews, pp. 652-653; Li et al, Nature, Vol. 26, pp. 450-454 (January 27, 2003).
  • ACE2 Angiotensin-converting enzyme 2
  • a 193 amino acid fragment of the S protein i.e., residues 318-510 has been found to bind ACE2 more efficiently than the full SI domain of the protein. Wong et al.. J. Biol. Chem., Vol. 279, Issue 5, pp. 3197-3201 (January 30, 2004).
  • the published amino acid sequence of the SARS CoV S protein is shown below (SEQ ID NO: 107).
  • ELISA for Testing ACE2 Immunoreactivitv A stock solution of recombinant human ACE2 (R&D Systems, 0.138 mg/ml in 12.5 mM tris, 75 mM NaCl, 0.5 ⁇ m ZnCl 2 , 50% glycerol, pH 7.5) was serially diluted as 1.38 ⁇ g/ml, 138 ng/ml, 13.8 ng/ml, 1.38 ng/ml and 138 pg/ml in coating buffer (NaHCO 3 /Na 2 CO 3 , 563/215 mM, pH 9.6). 100 ⁇ l of each dilution of
  • ACE2 was added into a well of 96-well microtiter plate (Nunc) and incubated at 37° C for 90 min. For each concentration of ACE2, duplicated wells were coated. After washing with Tris buffered saline supplemented with 0.2% Tween-20 (TBST) 3 times, the plate was blocked with 150 ⁇ l blocking buffer (1 % fetal bovine serum albumin, 1 % BSA, 1 % dry milk, 0.1 % NaN 3 in PBS) for 2 hr and washed for 5 times with TBS-Tween.
  • TBS-Tween 150 ⁇ l blocking buffer
  • a stock solution of biotinylated anti-human ACE2 ectodomain antibody [R&D Systems, 50 ⁇ g/ml in assay diluent (0.1% BSA, 0.1% NaN 3 , pH 7.2, PBS)] was diluted in 250 ng/ml with assay diluent, 100 ⁇ l of diluted antibody was added to each well of plate and incubated at 37° C for 90 min. After washing 5 times, to each well was added 100 ⁇ l of 1 : 1000 diluted streptavidin alkaline phosphatase conjugate in assay diluent, incubated at 37° C for 30 min.
  • the plate was blocked with 150 ⁇ l blocking buffer (1 % fetal bovine serum albumin, 1 % BSA, 1 % dry milk, 0.1 % NaN 3 in PBS) for 90 min and washed 5 times with TBST.
  • blocking buffer (1 % fetal bovine serum albumin, 1 % BSA, 1 % dry milk, 0.1 % NaN 3 in PBS
  • a stock solution of recombinant human ACE2 (0.138 mg/ml in 12.5 mM tris, 75 mM NaCl, 0.5 ⁇ m ZnCl 2 , 50% glycerol, pH 7.5) was diluted in 0.28 ⁇ g/ml with assay diluent, 100 ⁇ l of diluted ACE2 (28 ng/well) was added to each well of the plate and incubated at 37° C for 2 hrs, then kept at 4° C for overnight.
  • FIGS. 8A-8D The time of substrate color development for FIGS. 8 A and 8B was 12 hours at 4° C, whereas for FIGS. 8C and 8D was 24 hr at 4° C.
  • FIGS. 8A-8D The time of substrate color development for FIGS. 8 A and 8B was 12 hours at 4° C, whereas for FIGS. 8C and 8D was 24 hr at 4° C.
  • FIGS. 8A and 8C represent a concentration of HA-Peptide of 1.07 mg/ml in the coating whereas FIGS. 8B and 8D represent a concentration of HA-Peptide 0.74 mg/ml in coating.
  • Titration of ACE2 Binding to HA-P25 100 ⁇ l of diluted HA-peptide #25 (HA-P25, stock concentration of 1.1 mg/ml) in coating buffer (30 ⁇ g/ml, 3 ⁇ g/well) was added into each well of 96-well microtiter plate. The plate was incubated at 37° C for 2 hrs.
  • a stock solution of recombinant human ACE2 (0.138 mg/ml in 12.5 mM tris, 75 mM NaCl, 0.5 ⁇ m ZnCl 2 , 50% glycerol, pH 7.5) was diluted in 1.38 ⁇ g/ml with assay diluent.
  • N, N-dimethylfonnimide DMF
  • 10 ⁇ l of 10 mg/ml free peptide 100 ⁇ g/ml was added.
  • 10 ⁇ l of DMF was added to another 1 ml of diluted ACE2 (1.38 ⁇ g/ml in assay diluent).
  • ACE2 with free P25 (ACE2,138 ng/well; free P25 10 ng/well) or ACE2 alone (138 ng/well), was added to each well of plate and incubated at 37 °C for 2 hrs, then kept at 4° C overnight. After washing 3 times, 100 ⁇ l of diluted biotinylated anti-human ACE2 ectodomain antibody (50 ng ml in assay diluent, 5 ng/well) was added to each well of the plate and incubated at 37 °C for 2 hours.
  • biotinylated anti-human ACE2 ectodomain antibody 50 ng ml in assay diluent, 5 ng/well
  • polypeptides identified above as binding the ACE2 receptor can be used to treat patients infected with the SARS virus.
  • Membrane Assays Sera from 63 SARS patients were tested on membranes coated with 94 different polypeptides. These polypeptides were selected from the S (spike) and N (nucleocapsid) proteins of the SARS virus. The published amino acid sequence of the SARS CoV N protein is shown below (SEQ ID NO: 108).
  • Table 2 shows the number of samples that interacted with a given polypeptide on the membrane.
  • the peptide UD numbers listed in Table 2 correspond to those listed in Table 1.
  • Dot-Blot Strip Assays Uing Polypeptide Mixtures Tests were also conducted on strips having dots of different polypeptide mixtures. The polypeptide mixtures used are set forth below. The polypeptide identification numbers used in this study are the same as those set forth above in Table 1.
  • Polypeptide mixture 1 contains PEPT 1, PEPT 2, PEPT 3, PEPT 4, PEPT 6, PEPT 8, PEPT 10, PEPT 15, PEPT 16, PEPT 17, PEPT 18 and PEPT 21.
  • Polypeptide mixture 2 contains PEPT 1, PEPT 2, PEPT 4, PEPT 6, PEPT 15, PEPT 16, PEPT 18 and PEPT 21.
  • the strips used included three dots of polypeptide mixture 2 and one dot of polypeptide mixture 1.
  • the strips also included a dot of normal Human IgG as a control. The results are shown below in Table 3.
  • Table 3 Table 3

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Abstract

L'invention porte sur des polypeptides du syndrome respiratoire aigu sévère (SRAS) et leurs bioconjugués, et sur des polymères d'acide hyaluronique. Lesdits bioconjugués une fois immobilisés sur un support solide peuvent servir au diagnostic de patients infectés par le virus du SRAS, et à isoler les anticorps du SRAS présents dans le sérum de patients guéris. L'invention porte également sur des préparations pharmaceutiques comprenant lesdits bioconjugués et pouvant servir à traiter les patients atteints du SARS ou à immuniser les personnes présumées avoir été exposées au virus du SRAS.
PCT/US2004/017715 2003-06-04 2004-06-04 Polypeptides du sras (syndrome respiratoire aigu severe), et anticorps desdits polypeptides et leur utilisation a des fins de diagnostics, vaccination, et applications therapeutiques WO2005018538A2 (fr)

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Cited By (6)

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WO2008060331A2 (fr) * 2006-05-19 2008-05-22 Amgen Inc. Anticorps au coronavirus sras
WO2014134439A1 (fr) * 2013-03-01 2014-09-04 New York Blood Center, Inc. Composition immunogène pour infection de coronavirus de syndrome respiratoire du moyen orient (mers-cov)
CN112341526A (zh) * 2020-09-24 2021-02-09 杭州医学院 新型冠状病毒核衣壳蛋白特异性抗原多肽及其应用
US11103575B2 (en) 2014-02-28 2021-08-31 The New York Blood Center, Inc. Immunogenic composition for MERS coronavirus infection
WO2021198999A1 (fr) * 2020-04-03 2021-10-07 Axon Neuroscience Se Vaccins à base d'épitope pour le traitement de maladies associées au coronavirus
WO2021229077A1 (fr) * 2020-05-15 2021-11-18 Biomvis S.R.L. Vésicules de membrane externe bactérienne portant des polypeptides de coronavirus, procédé de préparation, compositions et utilisation de celles-ci

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WO2008060331A2 (fr) * 2006-05-19 2008-05-22 Amgen Inc. Anticorps au coronavirus sras
WO2008060331A3 (fr) * 2006-05-19 2008-10-02 Amgen Inc Anticorps au coronavirus sras
US7728110B2 (en) 2006-05-19 2010-06-01 Amgen, Inc. Antibodies to SARS coronavirus
WO2014134439A1 (fr) * 2013-03-01 2014-09-04 New York Blood Center, Inc. Composition immunogène pour infection de coronavirus de syndrome respiratoire du moyen orient (mers-cov)
US9889194B2 (en) 2013-03-01 2018-02-13 New York Blood Center, Inc. Immunogenic composition for MERS coronavirus infection
US11103575B2 (en) 2014-02-28 2021-08-31 The New York Blood Center, Inc. Immunogenic composition for MERS coronavirus infection
WO2021198999A1 (fr) * 2020-04-03 2021-10-07 Axon Neuroscience Se Vaccins à base d'épitope pour le traitement de maladies associées au coronavirus
WO2021229077A1 (fr) * 2020-05-15 2021-11-18 Biomvis S.R.L. Vésicules de membrane externe bactérienne portant des polypeptides de coronavirus, procédé de préparation, compositions et utilisation de celles-ci
CN112341526A (zh) * 2020-09-24 2021-02-09 杭州医学院 新型冠状病毒核衣壳蛋白特异性抗原多肽及其应用

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