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WO2007122517A2 - Virus h5 pseudotypés et leurs utilisations - Google Patents

Virus h5 pseudotypés et leurs utilisations Download PDF

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Publication number
WO2007122517A2
WO2007122517A2 PCT/IB2007/002494 IB2007002494W WO2007122517A2 WO 2007122517 A2 WO2007122517 A2 WO 2007122517A2 IB 2007002494 W IB2007002494 W IB 2007002494W WO 2007122517 A2 WO2007122517 A2 WO 2007122517A2
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Prior art keywords
hemagglutinin
virus
entry
cell
pseudotyped
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WO2007122517A3 (fr
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Isabelle Nefkens
Ralf Altmeyer
Jean-Michel Garcia
Philippe Buchy
Malik Peiris
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HKU-PASTEUR RESEARCH CENTRE Ltd
Institut Pasteur
Institut Pasteur du Cambodge
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HKU-PASTEUR RESEARCH CENTRE Ltd
Institut Pasteur
Institut Pasteur du Cambodge
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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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/60—Vectors comprising as targeting moiety peptide derived from defined protein from viruses
    • C12N2810/6072—Vectors comprising as targeting moiety peptide derived from defined protein from viruses negative strand RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/11—Orthomyxoviridae, e.g. influenza virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to the field of Influenza in the presence of Influenza antibodies and, more particularly, the present invention relates to the use of hemagglutinin pseudotyped viruses in methods for detecting the presence or absence of Influenza antibodies in a sample.
  • the present invention also relates to the use of the hemagglutinin pseudotyped viruses in methods for the detection of modulators of influenza virus entry in a cell.
  • HPAI highly pathogenic avian influenza
  • Avian influenza viruses preferentially bind ⁇ 2-3-linked sialic acids (SA) (Skehel et al., 1982; Skehel et al., 1983, Russell et al., 2006) while human influenza viruses preferentially recognize ⁇ 2- 6-linked SA. Subsequent virus entry and uncoating is dependent on low pH (Skehel and Wiley, 2000). During the final stage of the virus life cycle the HA binds to the SA receptor requiring the enzymatic activity of the neuraminidase for the release of the viruses from the cell surface (Dong et al. 1992).
  • SA sialic acids
  • H5N1 microneutralization tests confirmed by western-blot assays are the gold-standard for detection of anti-H5 specific antibodies in humans (Rowe et al., 1999).
  • H5N1 microneutralization tests require BSL-3 containment which precludes such studies in many affected countries.
  • the conventional haemagglutination inhibition (HI) test is not suitable for serodiagnosis of avian H5N1 infections in humans (Rowe et al 1999).
  • the modified HI test using horse erythrocytes (Stephenson et al., 2003) is currently under evaluation but is not a functional assay for inhibition of entry and is not amenable to high throughput testing.
  • An aspect of the invention is to provide new serodiagnostic tools and new inhibiting or promoting compounds in the field of Influenza.
  • Such an aspect is particularly achieved by providing a method for detecting the presence or absence of Influenza antibodies in a sample, comprising the steps of: a) contacting the sample with an hemagglutinin pseudotyped virus under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a). Said method is especially performed in vitro on a sample previously obtained from a subject to be diagnosed.
  • Another aspect of the invention concerns isolated and purified hemagglutinin pseudotyped virus, wherein said hemagglutinin is encoded by : - a polynucleotide encoding an H1 hemagglutinin having a nucleotide sequence as set forth in SEQ ID NO 2;
  • polynucleotide encoding an H5 hemagglutinin having a nucleotide sequence as set forth in SEQ ID NO1 or 5.
  • kits for the detection of the presence or absence of antibodies indicative of Influenza virus comprising:
  • a reagent to detect an hemagglutinin-antibody immune complex - optionally a biological reference sample lacking antibodies that immunologically bind with said hemagglutinin;
  • a comparison sample comprising antibodies which can specifically bind to said hemagglutinin; wherein said hemagglutinin pseudotyped virus, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • FIG. 1A Production of lentiviral particles pseudotypes with a synthetic H5 envelope protein.
  • HEK293 T cells (“producer cells”) are transfected with provirus expressing the luciferase or GFP reporter gene driven by the LTR promoter and a plasmid expressing the synthetic H5 gene. The cells will subsequently secrete pseudotyped viruses containing the proviral RNA and surrounded by the synthetic H5 gene.
  • the supernatant containing the secreted pseudotyped viruses is harvested, filtered and incubated with the target cells.
  • the synH5 pp will interact with their receptors and the HA2 will fuse with the plasma membrane. 5. After endocytosis, the nucleopcapsid is released into the cytoplasm. 6. The nucleocapsid will then brake down and the viral mRNA is reverse transcribed into DNA. Finally, the viral DNA is transported into the nucleus where it will be integrated into the DNA of the host cell. There it will express the reporter gene which can be detected by a lucifease assay or FACS analysis for the luciferase or GFP gene respectively.
  • FIG. 1B Expression of H5 in the producer cells.
  • FIG. 1 A western assay was performed on cell lysates from the HEK293T producer cells. The blot was then stained with a human serum against H5N1. Lane 1 : HEK293T cells, lane 2: HEK293T cells transfected with pNL Luc E- R- and pCDNA-synH5.
  • Figure 2A The yield of H5 pp in the supernatant is increased in the presence of sNA. sNA was added to the HEK293T producer cell line after transfection. 24 h later, the supernatant was harvested, filtered and incubated with Huh7 cells. The yield is expressed in relative luciferase untis (RLU).
  • RLU relative luciferase untis
  • Figure 2B and C:B FACS analysis of MDCK cells infected with H5pp expressing the eGFP reporter gene.
  • Figure 3 Infectious particle containing secreted H5 protein and p24 proteins can be purified on a sucrose gradient.
  • Figure 4A Different cell types are permissive for H5pp and the entry is dependent on sialic acid.
  • Huh7 cells were pre-incubated for various time periods with sNA (0.025 U/ml) prior to infection with H5pp. At the time of infection, SNA and MAA staining was performed to determine ⁇ 2-6- linked and ⁇ 2-3- linked sialic acid (SA) expression respectively. All data are expressed in percentage compared to the no treatment control, which is considered 100 %.
  • H5 pp entry is pH-dependent.
  • Cells were treated with medium containing the indicated concentrations of NH4CI as described in materials and methods prior to infection with H5pp or AMLVpp. Infectivity was measured by luciferase expression and are expressed as % of control (untreated cells).
  • FIG. 6A Infection of H5pp, but not VSV-G are neutralized by pre-incubation of H5pp with sera from H5N1 infected patients.
  • Figure 6B Sera from H5N1 infected persons and vaccinated persons neutralize the infection of H5pp.
  • NIH #1-#5 are sera from H5N1 vaccinated persons.
  • Figure 6C Sera from H5N1 infected and vaccinated poultry neutralize the infection of H5pp.
  • Figures 7A shows a preferred nucleotide sequence of an hemagglutinin used according to the present invention and set forth as SEQ ID NO 1.
  • H5 P0408008 (A/Cambodia/2005) from a H5N1 infected patient from Cambodia. Negative cis-acting motifs have been removed and codon as well as signal peptide optimised.
  • Figures 7B shows a preferred nucleotide sequence of an hemagglutinin used according to the present invention and set forth as SEQ ID NO 2.
  • Optimised sequence CAC86622 (A/New Caledonia/20/99(H1 N1 ). Negative cis-acting motifs have been removed and codon as well as signal peptide optimised.
  • Figures 7C shows a preferred nucleotide sequence of an hemagglutinin used according to the present invention and set forth as SEQ ID NO 3.
  • Negative cis-acting motifs have been removed and codon as well as signal peptide optimised.
  • Figures 7D shows a preferred nucleotide sequence of an hemagglutinin used according to the present invention and set forth as SEQ ID NO 4. Optimised sequence of H7N7 Netherlands. Negative cis-acting motifs have been removed and codon as well as signal peptide optimised.
  • Figures 7E shows a preferred nucleotide sequence of an hemagglutinin used according to the present invention and set forth as SEQ ID NO 5.
  • H5 pseudotyped particles preferably contemplated by the present invention are capable only of a single-round infection and do not produce progeny virus, and can therefore be produced under lower biosafety requirements than the wild-type virus.
  • the inventors have thus developed a system to produce lentiviral particles pseudotyped with an hemagglutinin, preferably H5 (H5pp) isolated from a patient in
  • H5pp show similar entry characteristics with respect to receptor usage, pH requirement and neutralization compared to the wild type H5N1.
  • the system described herein finds a particular use in serodiagnostic assays and analysis of cellular pathways of influenza entry, and more particularly for H5N1 subtype.
  • the present invention provides a method for detecting the presence or absence in a sample of Influenza antibodies.
  • the detecting method of the present invention comprises the following steps: a) contacting the sample with an hemagglutinin pseudotyped virus under conditions sufficient to form an immune complex; and b) detecting the presence or absence of the immune complex formed in a).
  • the method of the invention may detect non- neutralizing and/or neutralizing antibodies with respect to Influenza.
  • the present method of the invention may consist of a neutralizing assay, such as the one described in the Examples Section.
  • the present method of the invention may take the form of an ELISA for instance.
  • the present method of the invention may also consist of any other suitable type of assay for the detection of anti-Influenza antibodies.
  • immuno complex it is meant that the antibodies in the sample bound, in a specific manner, to the hemagglutinin.
  • sample refers to a variety of sample types obtained from an individual and can be used in the method of the invention.
  • the definition encompasses blood and any other liquid samples of biological origin which may be suspected of containing anti-Influenza antibodies.
  • the sample consists of a blood sample from a subject selected from the group consisting of a human, an avian and any animal subject of being infected by an influenza virus, and preferably of a H5N1 subtype.
  • under condition sufficient to form an immune complex refers to the conditions in terms of time and temperature for instance that are used to allow antibodies in the sample to specifically bind to the hemagglutinin of the pseudotyped virus.
  • conditions that are preferably contemplated in a neutralizing assay are shown in the Examples Section.
  • a preferred hemagglutinin pseudotyped virus contemplated by the present invention consists of a viral expressing vector.
  • a contemplated hemagglutinin pseudotyped virus consists of an isolated and purified hemagglutinin pseudotyped virus, such as an hemagglutinin pseudotyped lentivirus.
  • the isolated and purified hemagglutinin pseudotyped virus comprises an hemagglutinin which is encoded by :
  • polynucleotide encoding an H5 hemagglutinin having a nucleotide sequence as set forth in SEQ ID NO 1 or 5.
  • the hemagglutinin sequences preferably contemplated by the present invention have been advantageously optimised in order to eliminate splice site, cryptic splice sites, RNA instability motifs and in order to optimise the codon and signal peptide (Fig 7A-7E).
  • the hemagglutinin is H5 and has a nucleotide sequence as set forth in SEQ ID NO 1 or 5.
  • the hemagglutinin pseudotyped virus contemplated by the present invention advantageously comprises a reporter protein, such as luciferase. Any other reporter gene, such as GFP, that is suitable according to one skilled in the art is within the scope of the present invention.
  • the method of the present invention finds particular advantageous applications in the serodiagnosis of H5N1 exposed or infected humans and animals in outbreak or epidemic situations, and in the serodiagnosis in large scale seroprevalence in humans and animals to determine the degree of protective immunity against H5N1 in the general population.
  • the methods of the invention such as the H5pp technology requires only BSL2 containment and therefore renders seroneutralisation techniques for H5N1 for instance accessible to countries and institutions which do not have BSL3 laboratories required for microneutralisation.
  • the method of the present invention further finds a particular advantageous application in the serodiagnosis of large number of sample in HTS format in BSL laboratories.
  • Yet another advantageous application of the present method consists in the detection of protective H5N1 antibody levels in H5N1 exposed or vaccinated humans, animals and avians.
  • a further embodiment of the invention consists of the use of an hemagglutinin pseudotyped virus as defined above for the detection of modulators of Influenza virus entry in a cell such as a compound that inhibits or promotes such entry.
  • the contemplated hemagglutinin pseudotyped virus can be advantageously used in order to identify Influenza entry factors and inhibitors.
  • the H5pp specifically reproduces the entry step of H5N1 and it can therefore be used to identify cellular factors (receptors, etc.) that play a role in H5N1 entry.
  • H5pp can be used to identify H5N1 entry inhibitors, e.g., small molecule inhibitors.
  • the present invention provides methods for identifying a compound that inhibits or promotes the entry of an Influenza virus into a cell; the method comprises the steps of : a) providing a mixture containing said cells and the compound to be tested; b) contacting said mixture with an hemagglutinin pseudotyped virus as define above under conditions to allow entry of said virus into the cell; and c) evaluating the capacity of the compound to inhibit or to promote the entry of the hemagglutinin pseudotyped virus into said cell.
  • the expression "compound that inhibits” refers to a regulatory compound that inhibits, interferes with the entry into a cell of an Influenza virus, such as one of the H5N1 type.
  • a "compound that promotes” as used herein is a regulatory compound that promotes, induces, or facilitates the entry into a cell of an Influenza virus, such as one of the H5N1 type.
  • the present invention further provides a kit for use within the detection method of the present invention.
  • a kit typically comprises two or more components necessary for performing an antibody detection assay.
  • Components may be compounds, reagents, containers and/or equipment.
  • one container within a kit may contain an hemagglutinin pseudotyped virus, such as H5pp, that will allow the binding onto it of a specific antibody such as an anti-H5 antibody.
  • One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
  • a further embodiment of the present invention concerns a serodiagnostic kit for the detection of the presence or absence of antibodies indicative of Influenza virus, comprising: - an hemagglutinin pseudotyped virus as defined above;
  • comparison sample comprising antibodies which can specifically bind to said hemagglutinin; wherein said hemagglutinin pseudotyped virus, reagent, biological reference sample, and comparison sample are present in an amount sufficient to perform said detection.
  • the expression “specifically binds to” refers to antibodies that bind to one or more epitopes of an hemagglutinin of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • H5N1 Highly pathogenic avian influenza (HPAI) H5N1 has spread globally in birds and infected over 270 humans with an apparently high mortality rate.
  • Study Design We have generated and evaluated H5 hemagglutinin pseudotyped lentiviral particles encoding the luciferase reporter (H5pp).
  • H5pp entry into target cells depends on ⁇ 2-3 cell surface sialic acids and requires low pH for membrane fusion. H5pp infectivity is specifically neutralized by sera from patients and animals infected with H5N1 and correlates well with conventional microneutralization test. Conclusions: H5pp reproduce H5N1 influenza virus entry into target cells and potentially provides a high-throughput and safe method for sero-epidemiology.
  • H5pp The pseudotyped particles production and sucrose gradient purification were performed as described previously (Lozach et al., 2004). Briefly, HEK293T cells were transfected with pNL Luc E- R- and pCDNA-synH5 (for luciferase) or pcHMWS- EGFP, pCDNA-synH5 and pCMV-dR8.91 (for eGFP) and grown in the presence of soluble Vibrio cholerae neuraminidase (sNA) (6,2 mU/ml; Roche).
  • sNA Vibrio cholerae neuraminidase
  • MDCK cells (4000 cells/well) were seeded in 96-well plates in 100 uL of DMEM. 104 RLU of H5pp were incubated with twofold serial dilutions of serum (starting dilution 1 :20) for 1 hour at 37°C (CO2 incubator) in 60 uL total DMEM. Subsequently, 100 uL of fresh medium was added and 140 uL of the virus-antibody mixture was transferred to the cells. The luciferase assay was performed 48h later by direct addition of Steady-Glow Luciferase substrate (Promega). Sera were scored positive when they inhibited the H5pp infectivity >80% at serum dilutions >1/20.
  • Huh7 cells were treated with 0.025 U/ml sNA (Roche) for the indicated periods of times. They were harvested by Accutase (Sigma) treatment, washed and distributed over a 96-well plate. Cells were incubated with Fluorescein SNA (Vector laboratories, 10 ⁇ g/ml), Biotinylated MAA Il (Vector laboratories, 20 ⁇ g/ml) or PBS containing 1 % BSA (1 h, 4°C) and washed three times. When needed, secondary staining was performed with Streptavidin-FITC (BD, 25 ⁇ g/ml) prior to analysis by FACScan.
  • BD Streptavidin-FITC
  • Sera Human sera from confirmed H5N1 patients were provided by lnstitut Pasteur Cambodia and the University of Hong Kong.
  • Sera from H5N1 vaccinated volunteers, collected at the 28 day post second dose of the vaccine were provided by Linda Lambert (National Institute of Allergy and Infectious Diseases, Rockville, MD).
  • Avian sera were provided by lnstitut Pasteur Cambodia and by Robert Webster (St. Jude Children's Research Hospital, Memphis, TN).
  • Serostatus for mammalian sera was confirmed by microneutralization tests and for avian sera by haemagglutination inhibition tests (WHO 2002; OIE 2004).
  • the HA of A/Cambodia/408008/05 (H5N1 ) virus was used. Sequence analysis confirmed that this is a clade 1 H5N1 virus with no known mutations relevant for receptor binding including E190 and G225, suggesting that the H5 protein retained the binding characteristics for ⁇ 2,3-linked SA.
  • the HA sequence was codon- optimized (Geneart, Germany), cloned into a eukaryotic expression vector and protein expression verified after transfection into HEK293T cells by Western (Figure 1 B). Three main bands were seen which is consistent with the expected molecular weight for the uncleaved HAO and the cleaved subunits HA1 and HA2.
  • H5pp were generated as described in Figure 1A.
  • Figure 2A shows that H5 pseudotyped but not non-enveloped particles yielded a luciferase signal in MDCK cells.
  • Addition of sNA during the production of H5pp increased the infectivity by more than 1 log (Figure 2A).
  • FACS and microscopy analysis show that H5pp infectivity could also be observed when using a different reporter gene (eGFP) ( Figure 2B).
  • Electron microscopy confirmed that viral particles with an average size of 130 nm were generated in transfected cells and could be visualized at the plasma membrane (Figure 2C).
  • Example 2 H5pp reproduce key steps of H5N1 entry mechanism Removal of the sialic acids from the cell surface, by pre-incubation of cells with sNA treatment, decreased infection (Figure 4A).
  • SNA Sambucus Nigra lectin
  • MAA Maackia amurensis lectin Il
  • sNA treatment decreased the expression of both ⁇ 2-3-linked and ⁇ 2-6- linked SA, but with different kinetics.
  • Treatment for 30 min diminished the expression of ⁇ 2-3-linked SA ( Figure 4B grey bars), while the ⁇ 2-6-linked SA were not affected until more prolonged incubation times (Figure 4B black bars).
  • Example 3 H5pp are neutralized by patient sera
  • Initial neutralization experiments showed that a convalescent serum from a H5N1 infected patient (TH001 ) reduced H5pp infectivity to background levels while infectivity of VSV-Gpp was not affected (Figure 6A).
  • a serum taken from a another H5N1 patient (p0302295) at day 10 after the onset of disease was less potent in neutralizing H5pp but still inhibited infectivity of H5pp.
  • H5pp H5 hemagglutinin
  • H5pp entry requires ⁇ 2,3-linked SA and low endosomal pH and can be abrogated by sera containing H5N1 -specific antibodies.
  • Selective removal of cell surface ⁇ 2,3-linked SA (Ada, et al., 1961 ; Tomlinson et al., 1992) downregulates H5pp infection highlighting that H5pp are an ideal tool to study influenza cell tropism such as the role of critical HA residues in receptor usage.
  • H5pp encode a reporter gene which allows for high throughput analyses under lower biosafety requirements than wild type H5N1.
  • H5pp neutralization assay Using a novel H5pp neutralization assay, the inventors detected neutralizing antibodies in sera from previously confirmed avian and human H5N1 cases. The inventors have observed a tight correlation of the H5pp neutralization assay with characterized positive and negative human and avian (Table 1AB). The two false positive sera were detected in the H5pp assay came from elderly persons and it was previously reported that false positive results in the H5 microneutraliztion test are also more common in the elderly (Rowe et al., 1999). While a more comprehensive clinical and epidemiological evaluation is needed, the results presented herein indicate the H5pp neutralization assay is a novel approach that can be used for large-scale H5 seroprevalence studies on human and animal sera.
  • the advantages over existing techniques for detection of H5N1 neutralizing antibodies include easy and flexible read-out, handling under BSL2 condition and the requirement of small quantities of serum.
  • the system is suited for automated high throughput screening in a 96-well plate or 384-well plate format.
  • the H5pp particles can be produced from synthetic codon optimized DNA without access to the live H5N1 virus or cloned viral genes.
  • the flexibility of the system encourages the development of a multiplex assay where different subtypes or clades of influenza viruses can be screened simultaneously.
  • the method of detecting anti-Influenza antibodies of the present invention is clearly a valuable tool in global efforts to increase the surveillance of influenza viruses in avians, animals and humans.
  • Table 1A Overview of the human sera tested in the H5pp neutralization test.
  • the criterion for H5pp positive is ⁇ 80% reduction of infectivity at a serum dilution of ⁇ 1/20
  • Table 1B Overview of the avian sera tested in the H5pp neutralization test.
  • the criterion for H5pp positive is > 80% reduction of infectivity at a serum dilution of > 1/20
  • HI hemagglutination inhibition
  • HA hemagglutinin
  • H5 hemagglutinin of H5N1
  • H5pp H5 pseudotyped viral particles
  • AMLV amphotrophic envelope of the murine leukemia virus sNA: soluble recombinant neuraminidase from Vibrio cholerae
  • MAA Maackia amurensis lectin Il
  • HPAI highly pathogenic avian influenza
  • Mink lung epithelial cells unique cell line that supports influenza A and B virus replication. J CHn Microbiol 36(12), 3718-20.
  • Trypanosoma cruzi trans-sialidase restoration of complement resistance of desialylated sheep erythrocytes. Glycobiology 2(6), 549-51.

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Abstract

La présente invention se rapporte au domaine de la grippe, en présence d'anticorps de la grippe. Elle se rapporte plus particulièrement à l'utilisation de virus pseudotypés d'hémagglutinine dans des méthodes destinées à détecter la présence ou l'absence d'anticorps de la grippe dans un prélèvement. L'invention se rapporte en outre à l'utilisation des virus pseudotypés d'hémagglutinine et à des méthodes de détection de modulateurs de la pénétration du virus de la grippe dans une cellule.
PCT/IB2007/002494 2006-04-26 2007-04-26 Virus h5 pseudotypés et leurs utilisations Ceased WO2007122517A2 (fr)

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CA2541872 2006-04-26
CA 2541872 CA2541872A1 (fr) 2006-04-26 2006-04-26 Pseudotypes du virus h5 et utilisations connexes

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WO2007122517A3 WO2007122517A3 (fr) 2008-05-08

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008087563A3 (fr) * 2006-12-29 2008-11-27 Inst Pasteur Of Shanghai Lentivirus pseudotypé avec la grippe hémagglutinine et procédés d'utilisation
WO2009036063A1 (fr) * 2007-09-11 2009-03-19 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs viraux pseudotypés et leurs procédés de fabrication et d'utilisation
WO2011107439A1 (fr) * 2010-03-01 2011-09-09 Epixis Méthode permettant de mesurer l'infectivité virale
WO2015023461A3 (fr) * 2013-08-06 2015-09-03 The Trustees Of The University Of Pennsylvania Molécules d'acide nucléique de grippe et vaccins fabriqués a partir de celles-ci
JP2016052333A (ja) * 2007-11-12 2016-04-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア インフルエンザウイルスの複数のサブタイプに対する新規ワクチン

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999013905A1 (fr) * 1997-09-18 1999-03-25 The Trustees Of The University Of Pennsylvania Mutants de poche de fixation de recepteur d'hemagglutinine de virus de grippe a
CN100531802C (zh) * 2000-06-01 2009-08-26 株式会社载体研究所 含有血凝素活性膜蛋白质的假型逆转录病毒载体

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008087563A3 (fr) * 2006-12-29 2008-11-27 Inst Pasteur Of Shanghai Lentivirus pseudotypé avec la grippe hémagglutinine et procédés d'utilisation
WO2009036063A1 (fr) * 2007-09-11 2009-03-19 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Vecteurs viraux pseudotypés et leurs procédés de fabrication et d'utilisation
JP2016052333A (ja) * 2007-11-12 2016-04-14 ザ トラスティーズ オブ ザ ユニバーシティ オブ ペンシルバニア インフルエンザウイルスの複数のサブタイプに対する新規ワクチン
US10076565B2 (en) 2007-11-12 2018-09-18 The Trustees Of The University Of Pennsylvania Vaccines against multiple subtypes of influenza virus
WO2011107439A1 (fr) * 2010-03-01 2011-09-09 Epixis Méthode permettant de mesurer l'infectivité virale
EP2366776A1 (fr) * 2010-03-01 2011-09-21 Epixis Procédé de mesure de l'infectiosité virale
WO2015023461A3 (fr) * 2013-08-06 2015-09-03 The Trustees Of The University Of Pennsylvania Molécules d'acide nucléique de grippe et vaccins fabriqués a partir de celles-ci
US10398769B2 (en) 2013-08-06 2019-09-03 The Trustees Of The University Of Pennsylvania Influenza nucleic acid molecules and vaccines made therefrom

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CA2541872A1 (fr) 2007-10-26

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