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WO2008138120A1 - Système de vecteur lentiviral pseudotypé de l'hémagglutinine h5n1 du virus de la grippe aviaire pour une identification rapide d'antiviraux et une neutralisation de polypeptides - Google Patents

Système de vecteur lentiviral pseudotypé de l'hémagglutinine h5n1 du virus de la grippe aviaire pour une identification rapide d'antiviraux et une neutralisation de polypeptides Download PDF

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WO2008138120A1
WO2008138120A1 PCT/CA2008/000886 CA2008000886W WO2008138120A1 WO 2008138120 A1 WO2008138120 A1 WO 2008138120A1 CA 2008000886 W CA2008000886 W CA 2008000886W WO 2008138120 A1 WO2008138120 A1 WO 2008138120A1
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influenza
virus
cells
particles
infection
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Xiojian Yao
Zhujun Ao
Gary Kobinger
Darwin Kobasa
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Canada Minister of National Health and Welfare
University of Manitoba
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University of Manitoba
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Definitions

  • the avian influenza H5N1virus is a subtype of the Influenza A virus that can cause illness in humans and many other animal species.
  • the earliest infections of humans by H5N1 coincided with an epizootic of H5N1 influenza in Hong Kong's poultry population, infecting 18 people with a fatality rate of 33% (Subbarao, Klimov et al. 1998; Chan 2002). Since then, the H5N1 virus has spread to over 60 countries or regions and infected several million wild and domestic birds. Between 2003 and Feb 2007, there were a total of 272 confirmed human cases of H5N1 viral infection, 166 of which were fatal, according to the World Health Organization (World Health Organization 2007).
  • H5N1 viruses are not readily transmissible between humans, it is quite possible that they can acquire such transmissibility via mutations and/or gene reassortment from circulating human influenza A viruses. Due to the high virulence of this virus and its endemic presence as well as its significant ongoing mutations, the H5N1 virus is the world's largest current pandemic threat and preparations are being made for a potential epidemic.
  • antivirals provide an additional line of defense, particularly important for controlling a rapidly spreading pandemic (Ferguson, Cummings et al. 2005; Longini, Nizam et al. 2005).
  • two classes of influenza antivirals are available: inhibitors for viral M2 ion channel proteins (amantadine and rimantadine) and neuraminidase inhibitors (zanamavir and oseltamivir).
  • amantadine and rimantadine neuraminidase inhibitors
  • zanamavir and oseltamivir neuraminidase inhibitors
  • their extensive use coupled with the recombination potential of the influenza genome has promoted the emergence of drug resistant strains (Bright, Shay et al. 2006), (Kiso, Mitamura et al. 2004).
  • influenza HA-pseudotyped lentiviral/MLV vectors have been successfully used to study gene transfer into lung epithelial cells or vaccination against H5N1 virus (Neumann, Watanabe et al. 2000; Kobinger, Weiner et al. 2001 ; McKay, Patel et al. 2006; Szecsi, Boson et al. 2006).
  • a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA, M2 and NA and the additional viral proteins would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.
  • a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins under conditions suitable for assembly into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles in the presence of an agent of interest where in the absence of the agent the virus-like particles would infect the cells capable of infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.
  • a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA 1 M2 and NA and the additional viral proteins would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.
  • a method of treating an H5N1 influenza infection comprising administering to an individual in need of such treatment an effective amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8.
  • a method of immunizing an individual in need of such treatment against H5N1 infection comprising administering an immunizing amount of at least one of the peptides as set forth in any one of SEQ ID No. 1-8 to said individual.
  • FIG. 1 Biochemical and function analysis of H5N1-VLP harboring HA cleavage site modification.
  • A. HA, NA and M2 proteins from H5N1 (A/Hanoi/30408/2005) influenza viruses or VSV-G protein were cotransfected with lentiviral vector containing GFP gene and Gag-Pol expressing plasmids, as indicated in material and methods.
  • cells were labeled with [ 35 S] methionine for 12 hours.
  • H5N1-VLP was pelleted through 20% sucrose. Purified H5N1-VLPs were divided into two equal parts, and one of them was treated with 3 ⁇ g of trypsin/ml for 1 h.
  • H5N1-VLPs and VSV- VLPs were dissolved in RIPA buffer, and viral proteins were precipitated by human anti- influenza serum, rabbit anti-NA and mouse anti-P24 antibodies and analysyzed by PAGE followed by autoradiography of gel.
  • HAO HA precursor protein
  • HA1 HA surface subunit
  • HA2 HA transmembrane subunit
  • NA neuraminidase
  • CA HIV capsid protein.
  • B Dose titration of trypsin for H5N1-pseudotyped lentiviral vectors transduction. Purified H5N1 - VLPs were used to infect 293T cells in the absence or presence of various concentration of trypsin (different).
  • Viral vector titers were determined by P24 ( pg/0.5x10 5 ). Upper panel: The transduction efficiency, determinated as the GFP-positive cells, was measured by fluorescence-activated cell sorter (FACS) analysis at 48-72hr after infection. Lower panel: The transduction efficiency in 293T cells was also analyzed by PAGE-WB using anti-GFP antibody.
  • FACS fluorescence-activated cell sorter
  • FIG. 2 Both NA and M2 proteins are required for an efficient transduction.
  • A. The lentiviral vectors were pseudotyped with H5N1 HA alone, or with HA+NA, or HA+M2, or HA+NA+M2. The purified vectors titers were quantified by HIV-1 p24 antigen.
  • B. The same amount of viral vectors from A. were used to transduce 293T cells, and 48-72 h post-transduction GFP-positive cells was measured by FACS analysis.
  • C The lentiviral vector was pseudotyped with H5N1 HA, NA and increasing amount of M2 expressing plasmids. Equal amounts of VLPs were used to infect 293T cells and the expression of GFP protein in transduced cells was analyzed by PAGE-WB using anti- GFP antibody.
  • FIG. 3 The efficiency of H5N1 -pseudotyped lentiviral vectors transduction differed among cell types.
  • A The equal amount of p24 antigen H5N1 or VSV-G -pseudotyped lentiviral vectors were used to transduced human embryonic kidney 293T cells, A549 lung carcinoma cell and CD4+ C8166 T cells. The transduction efficiency, determined as the GFP-positive cells, was measured by FACS analysis at 48-72h after infection.
  • B Fluorescence microscopic images of GFP expression at 48h by H5N1-VLPs applied to 293T and A549 cells.
  • FIG. 4 NA inhibitor Oseltamivir interfered with H5N1 -pseudotyped lentiviral vectors' transduction in the late and early entry steps.
  • A. HA, NA and M2 proteins from H5N1 influenza viruses or VSV-G protein were cotransfected with an HIV packaging plasmid and transfer vector in 293T cells. 16 hrs later, transfected cells were washed with serum free medium fresh DMEM was added without or with 10OnM Oseltamivir. Pseudotyped VLPs were concentrated from cell free supematants at 48h post- transfection by ultracentrifugation and quantified by HIV-1 p24 antigen. B.
  • pseudotyped VLPs (p24) from Oseltamivir treated or non-treated vector producing cells were used to infect 293T cells and the GFP-positive cells were measured by FACS analysis.
  • C The same amount of H5N1 or VSV-G pseudotyped VLPs (p24) from non- Oseltamivir treated producing cells were used to infect 293T cells in the absence or presence of 10OnM Oseltamivir. The GFP-positive cells were measured by FACS analysis.
  • FIG. 5 Evaluation of the effects of Oseltamivir on the H5N1 pseudotyped vector transduction by measure the ⁇ -gal expression using 96 well plates.
  • Vector pHx'LacZWP encoding for ⁇ -gal was co-transfected with H5N1 envelope /VSV-G expression plasmids and HIV packaging plasmid in the absence or presence of oseltamivir.
  • a 2x fold dilution of the cultured viral vector stocks were used to transduce 293T cells in 96well plates. 48h post-transduction, the ⁇ - galactosidase activity in cell lysates was measured.
  • Oseltamivir (10OnM) was added to A549 cells in 96wells, followed by adding serial dilution of viral vectors. 48 later, /3-gal positive cells were showed by MAGI assay and counted using Elisapot Reader.
  • FIG. 6. Bar graph showing inhibition of VLP formation by HA 15 mer peptides.
  • FIG. 7. HA sequence of influenza A/Hanoi/30408/2005 virus.
  • the glycoproteins HA and NA were efficiently incorporated onto HIV vector particles which could transduce different cell lines in a trypsin-dependent manner. Simultaneous expression of NA and M2 maximized vector production and transduction.
  • the H5N1-pseudotyped HIV vector was sensitive to oseltamivir (tamiflu, an NA inhibitor), amantadine (an M2 inhibitor) or HA peptides independently.
  • This H5N1-pseudotyped HIV vector has been adapted for high-throughput screening of drug candidates such as small molecules, peptides or antibodies which can be safely performed in a biosafety level 2 environment.
  • the H5N1-pseudotyped HIV vector system mimics the influenza virus entry process and can greatly accelerate drug discovery to prepare against the next pandemic.
  • Described herein is a trypsin-dependent H5N1 HA-pseudotyped lentiviral vector system, which is comprised of plasmids expressing H5N1 HA, NA and M2 membrane proteins and an HIV-based vector expressing a recombinant green fluorescent protein (GFP) or ⁇ -Gal.
  • GFP green fluorescent protein
  • Transduction efficiency was measured quantitatively by FACS analysis or ⁇ -Gal activity. This system was evaluated on its transduction efficiency, antiviral sensitivity, and its adaptability for screening antivirals against H5N1 -avian influenza virus surface proteins.
  • H5N1 HA-pseudotyped lentiviral vectors system in which the cleavage site of H5 was modified to render a "high-pathogenic" H5 to be a "low- pathogenic" form, as being described previously by Li et a/., (Li, Liu et al.
  • M2 play key roles to initiate virus infection.
  • HA protein has been known to be responsible for binding to sialic acid receptors on the cell surface and also mediates fusion of viral membrane and endosomal membrane after endocytosis (Klenk and Rott 1988; Skehel and Wiley 2000).
  • M2 is a small transmembrane protein with an ion channel activity which regulates the pH inside the virion during viral entry cells (Bron, Kendal et al. 1993).
  • the NA has initially been shown to play an essential role in facilitating virus release from infected cells (Palese, Schulman et al. 1974; Palese and Compans 1976; Air and Laver 1989).
  • a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA in the presence of an agent of interest under conditions where in the absence of the agent the influenza HA, M2 and NA would assemble into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles under conditions suitable for infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles wherein an infection rate in the presence of the agent lower than the control infection rate indicates that the agent is a potential anti- influenza agent.
  • cells 'capable of infection refers to any cells known in the art which are capable of or suitable for infection by influenza virus.
  • control does not necessarily need to be repeated each time.
  • additional viral proteins are provided for assembly of the viral particles.
  • the HA peptide has been modified such that exogenous trypsin is required is required for HA to be cleaved into HA1 and HA2.
  • trypsin is added during the assembly phase. As discussed herein, this requirement for the addition of trypsin greatly enhances the safety of the recombinant particles.
  • a method of assessing the anti-influenza effect of an agent of interest comprising: providing influenza HA, M2 and NA and additional viral proteins under conditions suitable for assembly into infectious virus-like particles; incubating the particles with cells capable of infection by assembled virus- like particles in the presence of an agent of interest where in the absence of the agent the virus-like particles would infect the cells capable of infection; and comparing the infection rate of the virus-like particles to the infection rate of control virus-like particles.
  • control virus-like particles are for example particles grown under similar conditions in the absence of the agent of interest and used to infect cells of the same or similar type, for providing a rate of infection for comparison purposes.
  • the rate of infection may be determined by any suitable means known in the art.
  • a suitable reporter is used wherein the levels of the reporter are determined or measured.
  • this system has several advantages. Specifically, HA, M2 and NA of new viral strains, emerging viral strains or viral strains of concern can easily be substituted for the H5N1 proteins, meaning that potential anti-virals can quickly and easily be screened for effectiveness against a large number of strains. Furthermore, as discussed below, the effectiveness of the anti-virals at the assembly and infection stages can be measured using this system.
  • HIV-based system was used to form the core particles into which HA, M2 and NA were inserted. It is of note that other systems for expressing proteins of interest on the surface of a viral particle known in the art may be used.
  • H5N1-pseudotyped HIV vector As discussed below, the sensitivity of H5N1-pseudotyped HIV vector to an anti-influenza compound was also assessed.
  • the NA inhibitor oseltamivir efficiently inhibited HA-VLPs production (Fig.4A). Results indicated that H5N1- VLPs derived from oseltamivir-treated cells almost completely lost its infectivity, even through equal amounts of VLPs were used to compare the transduction efficiency with VLPs from the non-oseltamivir treated samples (Fig.4B and 5A). In addition, in the absence of NA expression in producer cells, the H5-pseudotyped VLPs exhibited extremely low transduction (Fig. 2B and C).
  • NA not only facilitated the release of the VLPs, but also directly affected their transduction ability. It is possible that the NA inhibitor oseltamivir could promote virus aggregation during and/or after virus release, and consequently inhibit viral infectivity (Tai, Escarpe et al. 1998). Moreover, in the H5N1 HA- psedutyped lentiviral vector system, oseltamivir was also found to be capable of interfering with the viral entry step directly (Fig.4C, Fig.5B and 5C).
  • trypsin-dependent HA-pseudotyped lentiviral vectors system can be used not only for studying viral envelope glycoprotein interactions with their corresponding cellular receptors, but also for identifying compounds having activity against the functions of these avian influenza membrane proteins and the mechanism involved.
  • this system can be rapidly adapted for monitoring the potential impact of membrane proteins of other highly pathogenic influenza viruses for possible influenza pandemics.
  • H5N1-pseudotyped vector infection in 293T cells we have tested HA 15-mer combinatorial peptide library (108 peptides) for their inhibition effect on H5N1-pseudotyped vector infection in 293T cells. These peptides encompass the full-length heammaglutinin sequence of H5N1 influenza A/Hanoi/30408/2005 virus strain as shown in Figure 7. H5N1-pseudotyped vector was incubated with 293T cells in the presence of
  • the transduced 293T cells were measured by vector-induced ⁇ -Gal activity assay. As can be seen in Figure 6, 8 peptides were found to cause more than a 50% reduction in H5N1 -vector induced ⁇ -Gal activity.
  • the inhibitory peptides are:
  • TRSKVNGQSGRMEFF SEQ ID NO. 6
  • peptides can be used as direct peptide inhibitors to prevent H5N1 HA infection.
  • an effective amount of any one of a peptide comprising or consisting of or consisting essentially of an amino acid sequence as set forth in any one of SEQ ID Nos. 1-8 or combinations thereof can be administered to an individual in need of such treatment, for example, an individual infected with, suspected of being infected with, at risk of infection with or showing symptoms consistent with infection with H5N1 HA or another influenza strain.
  • these peptides may be used as vaccines for immunizing individuals in need of such treatment against H5N1 or other influenza strain infection. It is of note that the peptides may be used in the formulation of a vaccine using means known in the art, for example, the HBcAg system in which capsid-like particles are formed which express the peptide of interest on the outer surface of the particles. Similarly, other methods known in the art for increasing antigenicity of small peptides that is recognition of small peptides by a host as foreign may be used.
  • H5N1 HA proteins harboring cleavage site modification can be incorporated into HIV-based vector particles and lead to a trypsin-dependent transduction.
  • H5N1 avian influenza virus entry system we first constructed three H5N1 membrane protein expressors by synthesizing HA, NA and M2 cDNAs based on H5N1 influenza A virus (A/Hanoi/30408) sequence from database and cloned into a pcGAalpha vector.
  • H5N1 HA gene we modified HA cDNA by deleting a five-amino acid sequence in the cleavage site of the HA protein (Fig. 1A).
  • VLPs virus-like particles
  • H5N1 pseudotyped VLPs but not in VSV-G pseudotyped VLPs (Fig. 1 B, middle panel). Both H5N1 and VSV-G pseudotyped VLP show similar level of HIV capsid (p24) protein (Fig. 1 B, lower panel).
  • H5N1 NA and M2 are required for an efficient HA-pseudotyped lentiviral vector particle production and transduction.
  • H5N1 HA-pseudotyped VLPs were transduced with similar amounts (as adjusted by VLP-associated p24 levels) of HA-VLPs in the presence of trypsin (4 ⁇ g/ml) or VSV-G-VLPs. After 48 hours, each cell population was collected, fixed with 4% paraformaldyhyde and analyzed by FACS analysis. In contrast to VSV-G-VLPs, which transduce all three kinds of cells efficiently, the H5N1 HA-VLPS showed variable transduction ability.
  • H5N1 HA-VLPs displayed a cell type-specific transduction ability and the 293T cell is a sensitive cell line for our H5N1 -mediated virus entry system.
  • this trypsin-dependent HA-pseudotyped lentiviral vectors system is not only valuable for studying the mechanism involved in viral entry, but also can be used for screening the anti-viral agents against influenza A virus that target to HA, NA or M2 proteins.
  • NA inhibitor oseltamivir interfered H5N1-pseudotyped lentiviral vectors' transduction in the late and early entry steps
  • the direct effect of oseltamivir on the early stage of HA-VLP's transduction was also tested.
  • the H5N1 HA-VLPs and VSV-G-VLPs were produced in 293T cells without the treatment of oseltamivir and then, similar amounts of VLPs were incubated with 293T cells in the absence or presence of oseltamivir for 2h at 37 0 C.
  • the cell culture was washed with DMEM to remove VLPs and oseltamivir, and cultured in fresh DMEM for 48h.
  • the GFP-positive cells were measured by FACS analysis. As shown in Fig.
  • oseltamivir reduced the HA-VLPs transduction to 50%, while there was no effect on VSV-G-VLP mediated transduction.
  • All of these results indicate that the NA- inhibitor oseltamivir effectively inhibited H5N1 HA-pseudotyped VLP's activity and meanwhile, proved that this trypsin-dependent HA-pseudotyped lentiviral vector system can be a safe and sensitive system for screening and testing different antivirals against H5N1 HA, NA and M2 proteins as well as against HA, NA and M2 from other influenza strains using the methods described herein.
  • H5N1-pseudotyped lentivial vectors system for large scale testing of anti-H5N1 agents.
  • this H5N1 HA-pseudotyped vector system may provide a safe and sensitive way for testing antivirals against H5N1 membrane proteins.
  • a quantitative 96-well plate method as a potential screening assay.
  • a HIV-1 vector pHx'LacZWP containing a LacZ reporter gene, instead of pHx'EGFPWP was used to produce HA-VLPs expressing ⁇ -galactosidase ( ⁇ -Gal). These HA-VLPs were then used to transduce 293T or A549 cells in 96 well plates (Fig.
  • the MAGI assay was performed with 96 well plates after A549 cells in each well were transduced by HA-VLPs, and the ⁇ -Gal positive cells were scanned and counted (Fig.5B and C).
  • the results indicate that the presence of oseltamivir during transduction reduced the transduction efficiency by 30 to 50% in different viral vector dilutions (Fig. 5B and C).
  • this trypsin-dependent HA-pseudotyped lentiviral vectors system can provide a safe and sensitive experimental platform for rapid screening of compounds and/or polypeptides against the H5N1 -avian influenza virus entry step.
  • Plasmid constructs The avian influenza H5N1 HA, NA and M2 cDNAs were synthesized based on the sequences of Influenza A/Hanoi/30408/2005 virus from the Influenza Sequence Database. Also, a two-step PCR technique was used to generate a trypsin-dependent H5N1 HA containing a five basic amino acid (RRRKK) deletion and the addition of a threonine residue at proximal to the cleavage site of the protein, as described before (Li, Liu et al. 1999) (shown in Fig 1A).
  • RRRKK five basic amino acid
  • Each of the synthesized HA, NA and M2 cDNA was cloned into a pCAGalpha vector (Watson, Kobinger et al. 2002).
  • the HIV-based vectors encoding for GFP gene (pHxEGFPwp) or encoding for lacZ gene (pHxLacZwp) and the helper packaging construct pCMV ⁇ R8.2 encoding for the HIV helper function, were described previously (Watson, Kobinger et al. 2002).
  • a vesicular stomatitis virus G (VSV-G) glycoprotein expressor was previously described (Yao, Mouland et al. 1998).
  • DMEM Dulbecco's Modified Eagles Medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • the primary human bronchial epithelial cells were collected from a normal transplant donor and grown in a bronchial epithelial growth media from Cambrex. DNA transfection in 293T cells was performed with CaPO4 precipitation methods as previously described (Yao, Kobinger et al. 1999).
  • Antibodies used in immunoprecipitation are as follows.
  • the rabbit polyclonal to Avian influenza NA protein was purchased from Cedarlane Lab (Hornby, Ontario, Canada).
  • the anti-HIVp24 monoclonal antibody used in this study was previously described (Yao, Mouland et al. 1998).
  • the rabbit anti-GFP was obtained from Molecular Probes Inc.
  • the NA inhibitor Oseltamivir (tamiflu) was obtained from Roch Inc. Trypsin (TPCK-treated) was purchased from Sigma Inc.
  • Enhanced ⁇ - galactosidase assay kit (CPRG) was purchased from Genlantis Inc. (San Diego, CA).
  • HIV- 1 p24 ELISA Kit was obtained from the AIDS Vaccine Program of the Frederick Cancer Research and Development Center.
  • the pseudotyped HIV-based vector was produced by triple transfection of 293T cells using CaPO4 precipitation methods. Briefly, 293T cells were transfected with the appropriate envelope expression plasmids, the HIV packaging plasmid pCMV ⁇ R8.2 and the HIV-based vector PxEGFPwp or pHxLacZWP (Kobinger, Weiner et al. 2001). After 48 hours of transfection, viral vectors were concentrated from the supernatant through ultracentrifugation, and virus titers were quantified by using RT activity Assay (Yao, Kobinger et al. 1999) or HIV-1 p24 Antigen Capture Assay Kit.
  • VLPs H5N1 or VSV-G- pseudotyped virus-like particles (VLPs) (adjusted by P24 level) or serial dilution (2xfold) of VLPs were incubated with target cells in the 0.5% BSA growth medium containing 4 ⁇ g/ml trypsin. After an overnight incubation, transduced cells were replaced with fresh medium. 48h-72h later, the percentage of transduced (GFP-positive) cells was determined using fluorescence-activated cell sorter (FACS) analysis (Becton Dickenson FACS Calibur).
  • FACS fluorescence-activated cell sorter
  • LacZ-containing vector The relative transduction efficiency of LacZ-containing vector was evaluated by measuring ⁇ -galactosidase in transduced cell lysates using ⁇ -galactosidase assay kit (CPRG) by Spectra max plus (Molecular Devices Coporation) or by detecting the GaI positive cells using the MAGI assay as described previously (Kimpton and Emerman 1992). The blue positive cells were counted by Elisapot Reader (AID Autoimmun Diagnostika GmbH). Inhibitor treatment of 293T producer and target cells: 293T cells were plated and transfected with HA, NA and M2 expressors and HIV-based vector as described above.
  • Oseltamivir 100 ⁇ g/ml was added to the medium and incubated for additional 24 hours prior to HA-VLPs harvest.
  • Oseltamivir 100 ⁇ g/ml was added to the medium and incubated for additional 24 hours prior to HA-VLPs harvest.
  • A549 or 293T cell monolayer cultures in 24-well or 96-well plate were transduced with VLPs in the absence or presence of oseltamivir for
  • Transduced (GFP-positive) cells were analyzed by FACS analysis or the induced ⁇ - galactosidase activity in the cells was measured as described above. lmmunoprecipitation analyses.
  • HA-VLP-producing 293T cells were starved in methionine-free DMEM for 30 min at

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Abstract

L'invention porte sur des glycoprotéines de la grippe HA et NA qui ont été efficacement incorporées sur des particules du vecteur du VIH qui ont pu effectuer, de façon dépendante de la trypsine, une transduction de différentes lignées cellulaires. Une expression simultanée de NA et M2 a rendu maximale la transduction et la production du vecteur. De façon intéressante, le vecteur du VIH pseudotypé par H5N1 s'est montré indépendamment sensible à l'oséltamivir (tamiflu, un inhibiteur de NA), à l'amantadine (un inhibiteur de M2) ou à des peptides HA. Ce vecteur du VIH pseudotypé par H5N1 a été adapté pour un criblage haut débit de candidats de médicament, tels que de petites molécules, des peptides ou des anticorps qui peuvent fonctionner de façon sûre dans un environnement de niveau 2 de biosécurité. Le système de vecteur du VIH pseudotypé par H5N1 imite le processus d'entrée du virus de la grippe et peut grandement accélérer la découverte de médicaments pour se préparer contre la prochaine pandémie.
PCT/CA2008/000886 2007-05-11 2008-05-12 Système de vecteur lentiviral pseudotypé de l'hémagglutinine h5n1 du virus de la grippe aviaire pour une identification rapide d'antiviraux et une neutralisation de polypeptides Ceased WO2008138120A1 (fr)

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WO2009053535A3 (fr) * 2007-10-26 2009-07-23 Glykos Finland Oy Vaccin peptidique contre le virus de la grippe

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Publication number Priority date Publication date Assignee Title
WO2009053535A3 (fr) * 2007-10-26 2009-07-23 Glykos Finland Oy Vaccin peptidique contre le virus de la grippe

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