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WO1992018616A1 - Virus de la grippe d'une nouvelle reference et methodes de determination de la sensibilite aux drogues antivirales - Google Patents

Virus de la grippe d'une nouvelle reference et methodes de determination de la sensibilite aux drogues antivirales Download PDF

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Publication number
WO1992018616A1
WO1992018616A1 PCT/US1992/003030 US9203030W WO9218616A1 WO 1992018616 A1 WO1992018616 A1 WO 1992018616A1 US 9203030 W US9203030 W US 9203030W WO 9218616 A1 WO9218616 A1 WO 9218616A1
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Prior art keywords
influenza
virus
amantadine
antiviral
cells
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PCT/US1992/003030
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English (en)
Inventor
Alan P. Kendal
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United States Department of Commerce
United States Department of the Army
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United States Department of Commerce
United States Department of the Army
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Publication of WO1992018616A1 publication Critical patent/WO1992018616A1/fr
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    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16121—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • the present invention relates to novel reference standards and methods that can facilitate consistent results in the screening of influenza viruses for susceptibility or resistance to the antiviral drug amantadine or any other drug that inhibits viral replication in a way that reduces the amount of type specific viral antigens in infected cells.
  • the present invention relates to methods for screening drugs and drug development for antiviral activity against influenza A viruses and methods for measuring virus isolates from patients treated or considered for treatment with an anti-influenza drug.
  • Influenza viruses remain an important cause of morbidity and mortality, causing outbreaks in most years.
  • Virus-specific substances other than vaccines which are available to reduce the disease impact include particularly the antiviral agent, amantadine (J. S. Oxford, J. Antimicrobial. Chemotherapy 1:7-23 (1975) and G.G. Jackson, In Kendal AP, Patriacrca
  • PA eds. Options for the Control of Influenza. New York: Alan R. Liss, 1986:293-315), or in some countries the derivative substance rimantadine.
  • vaccination before the influenza season is recommended to prevent infection or reduce its complications, whereas amantadine is recommended to be used once influenza has begun to occur.
  • Amantadine can be effective in stopping outbreaks of influenza A viruses in closed population settings, and has been reported in some cases to speed
  • amantadine-resistant mutants can be selected in vitro by growth of influenza A viruses in the presence of the drug (J.S. Oxford et al., J. Hyg.
  • rimantadine can be detected occasionally in the respiratory secretions of children or adults
  • a genetic and biochemical marker for amantadine-resistance exists, namely a mutation within a short region of the influenza virus M2 protein (A.J. Hay et al., EMBO J.4:3021-3024 (1985), R.B. Belshe et al., J. Virol.62:1508-1512 (1988), F.G. Hayden et al., (1989) and Centers for Disease
  • M2 protein is demonstrated to have the properties of a transmembrane spanning protein, including a section of about 20 hydrophobic amino acid residues towards the N-terminal end of the molecule (R.A. Lamb et al., Cell 40:627-633 (1985)).
  • biological assays rather than molecular/genetic assays are likely to be required to first detect the presence of resistant viruses in patients and contacts.
  • test procedures and reagents should be readily available, that can be applied in different regions of the world.
  • rimantadine is widely available for use (D.M. Zlydnikov et al., Rev. Infect. Dis.3:408-421 (1981)), or in China, where many new strains of influenza seem to first appear.
  • Viruses usually used for this purpose in research laboratories include the highly amantadine sensitive Asian influenza strains (H2N2),
  • the present invention addresses the above concerns by providing a novel reference strain of influenza A virus sensitive to amantadine and a resistant mutant derived from it. These viruses are believed to be safer for use as internal controls in the laboratory than previously used reference strains which are potential human pathogens.
  • the present invention also provides novel and simple standardized methods to measure inhibition of influenza virus without the requirement of multiple cycles of virus replication.
  • the present invention elates to an influenza A mutant viral strain designated A/Turkey/Oregon/71 mutant ARl (AR-T/ORE/71).
  • A/Turkey/Oregon/71 mutant ARl influenza strain is embodied in the deposited strain given accession # VR 2321 deposited on April 16, 1991, American Type Culture Collection.
  • the present invention relates to a method of detecting
  • influenza replication inhibition in a sample comprising the steps of culturing cells that need support no more than one round of replication of influenza virus, incubating these cells with either antiviral drug or medium alone followed by infecting the cell cultures with an antiviral drug resistent reference influenza strain, an antiviral drug resistent drug sensitive reference influenza strain or a sample, and incubating these infected cells at an elevated temperature such that in one round of viral replication faster than normal levels of protein synthesis occur in the stages prior to virus maturation. After this procedure, the incubated cells are contacted with a monoclonal antibody specific to influenza nucleoprotein or matrix protein and the complex formed between influenza nucleoprotein or matrix protein on the infected cells and the antibodies are detected.
  • An example of such an embodiment in the present invention relates to the method of detecting inhibition of influenza virus replication in a sample comprising the same steps described above using primary chicken embryo fibroblasts as the cultured cells.
  • the present invention relates to the method of detecting
  • the antiviral drug resistant reference influenza virus strain is AR-T/ORE/71 and the
  • antiviral drug sensitive reference influenza virus strain is WT-T/ORE/71.
  • the antiviral drug, amantadine is required and/or another related drug with a similar mode of action as amantadine.
  • the present invention relates to a method of screening for antiviral compounds comprising the steps and modifications of the steps used in the previous method to detect inhibition of influenza virus replication in a sample.
  • modification is to incubate unknown antiviral testing compounds in cell cultures in parallel with amantadine and amantadine related drugs under the same conditions.
  • the present invention relates to a method of detecting virus isolates previously recovered from a patient's sample before or after treatment with antiviral drugs comprising the steps described above which differs only that in addition to infecting the cell cultures with control reference sensitive and resistant influenza strains, the cell culture are infected in parallel with the virus previously recovered from the patient's sample.
  • the present invention relates to a diagnostic kit for detecting influenza A virus comprising in combination, an insoluble surface or support containing primary chicken fibroblast cells, amantadine free medium, amantadine containing medium or antiviral drug containing medium; control resistant and sensitive reference strains AR-T/ORE/71 and WT-T/ORE/71, monoclonal antibodies against influenza A nucleoproteins and a means for detecting the amount of antibodies in a test sample which binds to the influenza A nucleoprotein.
  • Figure 1 demonstrates amantadine inhibition of replication of reference viruses and representative epidemic isolates.
  • the present invention relates to novel reference internal standards and novel methods that provide both safe and consistent results in the screening of influenza viruses for susceptibility or resistance to the antiviral drug, amantadine or any other related drug with a similar mode of action.
  • the methodology may also be applicable to the general task of screening drugs for antiviral activity against influenza viruses or testing of virus isolates from patients before and after treatment with an anti-influenza drug.
  • the present invention relates to reference strains of influenza virus sensitive and resistant to amantadine.
  • A/Turkey/Argon/71 H7N3 a non-human, non-pathogenic avian influenza has been utilized as a sensitive reference virus control.
  • transmembrane protein of the M2 gene has been developed as a resistant reference virus control.
  • These reference strains are utilized as internal quality controls in an any drug sensitivity method that has a general requirement for internal test controls to demonstrate the validity and
  • the normal antiviral sensitive control viruses that have been used in the past are strains such as A/Singapore/57 (H2N2), a prototype of Asian influenza. It is undesirable to distribute such a virus for routine laboratory use as this has the potential to permit reintroduction of Asian
  • influenza into the human population. Similarly, it is undesirable to distribute a resistant strain of influenza that has the potential to infect
  • the reference strains are not infectious to man, non-pathogenic to animals, they have readily
  • H7N3 viruses appears to eliminate the need to use potential human pathogens for control purposes in amantadine- resistance screening studies in diagnostic or research laboratories in replication inhibition assays.
  • the present invention relates to a method to measure inhibition of virus by an immunoassay procedure without requiring multiple cycles of virus replication.
  • the method of the present invention also has the advantages of giving results in one working day, if needed. It also permits specific identification of drugs that inhibit virus replication prior to the stage of virus particle assembly.
  • primary chicken embryo fibroblast cells are infected with reference strains described above and human virus at 36 to 37o for about 5 to 6 hours. Under such conditions human influenza viruses only abortively infect these cells. However, even an abortive cycle of virus replication can produce enough antigen to quantitate in an enzyme immunoassay. Thus, when primary chicken embryo fibroblasts are infected with a multiplicity of greater than one infectious unit per cell, there is adequate amounts of antigen synthesized in a single cycle of infection to measure inhibition in the presence of amantadine or related drugs with a similar mode of action. Inhibition of viral replication is detected by an immunoassay, for example, performed with monoclonal antibodies to conserved epitopes, such as, for example, the monoclonal antibodies developed against the
  • nucleoproteins and matrix (M1) protein of influenza A and/or B virus (Walls et al., (1986)).
  • Other types of immunoassay can also be used for detection as one skilled in the art will appreciate, for example, time-resolved fluroimmunoassay or any other immunoassay-based procedure.
  • the antibody-viral protein complexes formed can be detected using standard methodologies known in the art. For even greater viral antigen protein detection, the
  • immunoassay can be performed with avidin-biotin based test kits. Such reagents are commercially available.
  • Another aspect of the present invention relates to the method above performed in other cell types, for example, primary monkey kidney cells or MDCK cells.
  • the combination of safe control viruses and an assay which is not dependent on availability of cell lines should facilitate the performance of reliable amantadine-sensitivity screening in
  • the assay described above can, if
  • MDCK cells which are commonly used in virus laboratories for influenza work.
  • Another aspect of the method of the present invention includes performing the method with lower virus input concentration and longer periods of incubation to permit multiple cycles of virus replication.
  • permissive cell types such as MDCK, for example, may be used.
  • the present invention further relates to diagnostic kits.
  • the diagnostic kits comprise the non-hazard sensitive and resistant reference viruses of the present invention and reproducible
  • Monoclonal antibodies that are commercially available (Walls et al., (1986)) are used to detect influenza virus strains in these kits. Such antibodies provide sensitive and
  • the antigen detection sensitivity can be increased further by means of enhancing the immunoassay with an avidin-biotin based test kit.
  • avidin-biotin based test kit Such reagents are commercially available from
  • concentration (or higher) is normally present in any clinical isolate, and corresponds to a multiplicity of infection of about 1 median infectious unit/cell. However, a range of virus dilutions should be used to ensure the correct dilution is obtained.
  • the cells infected with reference and human viruses are incubated at 36-37 degrees C for about 5-6 hours in a standard tissue culture
  • a temperature of 37 degrees C is used to increase the rate of virus antigen production over that obtained at 33-34 degrees C, the temperature normally used in amantadine sensitivity testing with human influenza viruses.
  • the cells are next fixed and an enzyme immunoassay is performed using first, monoclonal antibody to the nucleoprotein of influenza A virus (Walls et al., (1986)), and second, horseradish peroxidase conjugated to staphylococcal protein A (a standard commercial reagent). All steps for the washing, color development, and measurement are standard laboratory procedures and well-known in the art.
  • the resistant virus should show less than about 40% inhibition by amantadine at a
  • Wild- type (WT) virus (WT-/0RE/71) has a hemagglutinin that is not cleaved by endogenous cell proteases, and thus the virus does not cause multi-organ highly lethal infections in avian species (M. Orlich et al., Virology 176:531-538 (1990)).
  • Apathogenic avian influenza H7 subtype viruses have also never been reported to infect man.
  • An amantadine-resistant mutant (AR-T/ORE/71) was prepared from WT-T/ORE/71 by passage in primary chick embryo cell culture using methods similar to those described by Hay et al., (1985), including two sequential plaque
  • TPCK-trypsin was included at a concentration of 0.5 or 1 microorgan/ml in all multiple cycle growth passages. Two nucleotide differences were found between the parent and mutant A/T/ORE/71 viruses that encode changes in adjacent amino acids within the region of M2 protein
  • A/T/ORE PRO LEU VAL ILE ALA ALA SER ILE ILE GLY ILE LEU HIS LEU ILE LEU A/T/ORE (R) PRO LEU VAL ILE ALA ALA SER ILE ILE GLU MET LEU HIS LEU ILE LEU
  • This mutant designated A/Turkey/Oregon/71 mutant ARl, or AR-T/ORE/71, is the subject of one part of the invention.
  • the gene encoding the protein which is the target for amantadine was sequenced, and two adjacent amino acid substitutions identified in the appropriate portion of the protein, as markers for amantadine resistance (see Table 1).
  • the existence of two mutations increases the genetic stability of the mutant over previous amantadine resistant mutants that carry only a single change. They also provide a laboratory marker of the origin of the virus should it be knowingly or unknowingly
  • the mutant virus has been deposited at the
  • an in vitro replication inhibition assay was used modified from procedures previously used to measure neutralizing antibody titers in sera (Harmon et al., J. Clin. Microbiol. 26:333-337 (1988)) or the occurrence of amantadine-resistant viruses in nursing home outbreaks of influenza A virus where amantadine was used for control purposed.
  • H2N2 amantadine-sensitive Asian influenza strain A/Ann Arbor/6/60
  • H3N2 amantadine-resistant mutant derived from it.
  • This resistant mutant contains one sequence change in the transmembrane spanning region of M2 protein.
  • Representative type A (H3N2) viruses isolated from patients in Kassel, Germany, in the winter of 1989/90 were also tested. These isolates had low passage levels, (a single egg passage after isolation in MDCK cells), and consequently low hemagglutinin titers, of about 32-64.
  • an amantadine-resistant H3N2 virus isolated from a child treated with rimantadine was included (Belshe et al., 1988).
  • H3N2 resistant strain also has one sequence change in the transmembrane spanning region of M2 protein.
  • the replication inhibition assay which relates to the subject matter of the present invention was performed with the above reference strains as follows. Primary chick embryo cells were grown to confluence in 96 well microplates. Cells were washed with serum free Dulbecco's medium, residual medium carefully removed by vacuum
  • Virus hemagglutinin titers in the egg fluids were about 640-1280
  • H2N2 and H7N3 viruses reference H2N2 and H7N3 viruses
  • 32-64 H3N2 human isolates
  • Cells were immediately returned to the 37°C incubator, and after 6 hours medium was removed, and cells fixed for about 5 min with 100 ⁇ l of cold acetone:phosphate buffered saline (4:1), and then dried with a current of air.
  • Levels of type- specific influenza A antigens in the fixed cells were determined by an ELISA method using a pool of two ascitic fluids containing monoclonal antibodies to influenza A NP and Ml proteins (Walls et al., (1986)), and peroxidase-conjugated Staphylococcal A protein.
  • H2N2 and WT-T/ORE/71 (H7N3) reference viruses As shown in Figure 1, very high levels of inhibition were observed for the Asian (H2N2) and WT-T/ORE/71 (H7N3) reference viruses, as well as for a current epidemic strain of H3N2 influenza, at a range of input virus dilutions that allowed for the large difference in titer between the laboratory adapted reference strains (HA about 1,000/ml) and the recent isolates (HA about 32/ml). Similar results were obtained for the other recent H3N2 virus isolates tested.
  • the resistant mutant AR-T/ORE/71 resembled the H2N2 resistant mutant in the very low levels of inhibition by amantadine at even the lowest inputs of virus tested in this assay.
  • the previously reported H3N2 resistant isolate was also only slightly inhibited by amantadine. * * * * * * While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the

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Abstract

Nouvelles normes et nouvelles méthodes de référence qui peuvent produire des résultats cohérents dans le dépistage des virus de la grippe en vue de la détermination de leur sensibilité ou de leur résistance à l'amantadine antivirale ou à toute autre drogue stoppant la réplication virale dans des conditions de nature à réduire le nombre d'antigènes viraux spécifiques d'un type dans les cellules infectées. L'invention porte en particulier sur des méthodes de sélection de drogues et sur l'élaboration de drogues ayant une activité antivirale dirigée contre les virus influenza A et sur des méthodes de mesure des isolats de virus de patients traités ou suceptibles d'être traités avec un médicament anti-grippe.
PCT/US1992/003030 1991-04-16 1992-04-16 Virus de la grippe d'une nouvelle reference et methodes de determination de la sensibilite aux drogues antivirales Ceased WO1992018616A1 (fr)

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US685,408 1991-04-16

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010339A1 (fr) * 1992-11-05 1994-05-11 Evotec Biosystems Gmbh Vaccination evolutive
US7893108B2 (en) 2004-07-14 2011-02-22 President And Fellows Of Harvard College Antiviral methods and compositions

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CLINICAL MICROBIOLOGY, Volume 23, No. 2, issued February 1986, H.H. WALLS et al., "Characterization and Evaluation of Monoclonal Antibodies Developed for Typing Influenza A and Influenza B Viruses", pages 240-245. *
JOURNAL OF CLINICAL MICROBIOLOGY, Volume 26, No. 2, issued February 1988, M.W. HARMON et al., "Antibody Response in Humans to Influenza Virus Type B Host-Cell-Derived Variants after Vaccination with Standard (Egg-Derived) Vaccine or Natural Infection", pages 333-337. *
JOURNAL OF GENERAL VIROLOGY, Volume 42, issued 1979, J. LOHMEYER et al., "Biosynthesis of the Influenza Virus Envelope in Abortive Infection", pages 73-88. *
JOURNAL OF MEDICAL VIROLOGY, Volume 27, issued 1989, M.W. HARMON et al., "Immunoassay for Serologic Diagnosis of Influenza Type A Using Recombinant DNA Produced Nucleoprotein Antigen and Monoclonal Antibody to Human IgG", pages 25-30. *
VIRUS RESEARCH, Volume 5, issued 1986, B. GIESENDORF et al., "Studies on the Temperature Sensitivity of Influenza A Virus Reassortants Nonpahtogenic for Chicken", pages 27-42. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994010339A1 (fr) * 1992-11-05 1994-05-11 Evotec Biosystems Gmbh Vaccination evolutive
US7893108B2 (en) 2004-07-14 2011-02-22 President And Fellows Of Harvard College Antiviral methods and compositions

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