WO2011115583A1 - A novel platform for retrovirus-like particle (vlp)-display of vaccine antigens - Google Patents

A novel platform for retrovirus-like particle (vlp)-display of vaccine antigens Download PDF

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WO2011115583A1
WO2011115583A1 PCT/SG2011/000109 SG2011000109W WO2011115583A1 WO 2011115583 A1 WO2011115583 A1 WO 2011115583A1 SG 2011000109 W SG2011000109 W SG 2011000109W WO 2011115583 A1 WO2011115583 A1 WO 2011115583A1
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virus
vlp
diii
rige
domain
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PCT/SG2011/000109
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Mah Lee Mary Ng
Pierre Boulanger
Saw-See Hong
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National University Of Singapore
Centre National De La Recherche Scientifique
Université Claude Bernard Lyon I
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Definitions

  • VLP VIRUS-LIKE PARTICLE
  • the present invention relates generally to a platform for retrovirus-like particle (VLP)-display of vaccine antigens.
  • VLP retrovirus-like particle
  • Flaviviruses like West Nile and dengue viruses have established a stronghold in many parts of the tropical and sub-tropical countries world wide. They are one of the most important re-emerging diseases. These viruses are transmitted by mosquito vectors and with global warming, the potential territorial expansion for the spread of these diseases are real. Dengue viruses affects hundreds of millions people each year. To date there are still no vaccines nor anti-virals for human use for either virus. In previous studies, we have demonstrated that the flavivirus envelope (E) domain III (E :DIII) is highly immunogenic and is able to induce potent neutralising antibodies [1-5]. Efficient vaccines are those inducing a robust neutralizing immune response.
  • VLPs Virus-like particles mimicking the natural virions are an advantageous alternative since they are not infectious [6]. This is best illustrated by the VLP -based strategy against the human papilloma virus (HPV), recently approved for human vaccination against HPV infections and HPV-associated cervical lesions which can be cancerous in nature [7,8].
  • HPV human papilloma virus
  • VLPs are highly organised particulate structures which differ in nature and composition with the virus type, but shared the common feature of being devoid of viral genome, (i) VLPs solely made up of viral capsid proteins can be produced with e.g. HPV, as evoked above, or with hepatitis E virus (HEV) [9]. (ii) On the other hand, VLPs solely consisting of viral envelope glycoproteins have been isolated, and their use as vaccines is envisaged. This is the case for the subviral particles formed by hepatitis B virus (HBV) envelope glycoprotein S, or by chimeric HBV and hepatitis C virus (HCV) envelope proteins [10,11], and influenza virus [12].
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • VLPs based on retroviruses or lentiviruses represent another type of subviral structure. They are membrane-enveloped particles made up of a backbone of viral polyprotein Gag (or Gag precursor), wrapped by an envelope derived from the host cell plasma membrane.
  • the HIV-1 Gag precursor spontaneously assembles into VLPs which bud at the plasma membrane of Gag-expressing cells, e.g. recombinant baculovirus-infected cells, and which are structurally similar to immature virions except that they do not contain any viral genetic material [13-19].
  • Gag-expressing cells e.g. recombinant baculovirus-infected cells
  • membrane-enveloped viruses bring with them part of the plasma membrane of the producing cells, and some host cell membrane glycoproteins foreign to the virus [20].
  • deletion of the gene encoding the viral envelope protein and substitution by another type of membrane-targeted protein lead to virus pseudotypes.
  • Such pseudotypes have been described in various enveloped viruses, e.g. retroviruses, lentiviruses, vesicular stomatitis virus (VSV), HCV and baculovirus [21- 26].
  • CD16-RIgE clone One publication describing the construction of the CD16-RIgE clone is by Clemenceau et al. [41]. In this paper, the group made a lentivirus to express the CD16-RIgE protein in B-cells.
  • the present invention relates to a fusion protein
  • CD 16 signal peptide comprising (i) a CD 16 signal peptide; (ii) an antigenic peptide region downstream of the CD 16 signal peptide; and (iii) an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
  • the present invention relates to a nucleic acid molecule encoding a promoter operably linked to (i) a sequence encoding the fusion protein as described herein; or (ii) a sequence encoding a CD 16 signal peptide upstream of a cloning site and a sequence encoding an IgE receptor transmembrane domain and cytoplasmic tail downstream of the cloning site.
  • the present invention relates to a method of forming a pseudotype virus-like particle, the method comprising coexpressing in a cell (i) a fusion protein as described herein; and (ii) a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein.
  • the present invention relates to a pseudotype virus-like particle comprising (i) protein core comprising a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein; (ii) a membrane envelope surrounding the protein core; and (iii) a fusion protein incorporated into the membrane envelope, the fusion protein comprising an antigenic peptide region and an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
  • the present invention relates to a vaccine comprising a pseudotype virus-like particle as described herein.
  • the present invention relates to use of a vaccine of the fifth aspect for vaccination of a human or animal.
  • FIG. 1 is a depiction of a VLP-display of the flavivirus antigenic E :DIII domain.
  • A Amino acid sequence of the CD16-RIgE cloning platform.
  • the CD16-RIgE is a chimeric molecule composed of the extracellular domain of CD 16 and the transmembrane domain (TM) and cytoplasmic tail (CT) of the gamma chain of the human high affinity IgE receptor (FceRIy), abbreviated RIgE in the present study.
  • the symbols for the different domains of the amino acid (aa) sequence are the following : signal peptide (amino acid residues 1-22); boxed, ectodomain of CD 16 (aa 23-209); underlined with dotted line, TM domain (aa 210-231); underlined with solid line, CT domain (aa 232-273); shaded residues, deletion (aa 23-206) of the CD 16 ectodomain, replaced by the flavivirus envelope Dili domain.
  • Figure 2 shows coexpression of HIV-1 Pr55Gag and CD16-RIgE in Sf9 cells.
  • Sf9 cells were infected with AcMNPV-Gag alone (left lane) or with AcMNPV- Gag and AcMPV-CD16-RIgE (right lane), at equal MOI each (10 pfu/cell), and harvested at 48 h pi.
  • FIG. 3 shows the pseudotyping of VLP.
  • A Western blot analysis. HIV-1 Gag VLPs were produced by Sf9 insect cells and pseudotyped with CD 16- RlgE alone (i), VSV-G alone (ii) or double VSV-G- and CD16-RIgE-pseudotyped VLPs (iii; rightmost lane).
  • B Electron microscopy, (i) double VSV-G- and CD 16- RlgE-pseudotyped VLPs, and (inset, ii) nonpseudotyped VLPs. Note the difference between the 'naked' envelope (ii) and 'furry' envelope (i) of VLPs.
  • FIG. 4 shows the confocal IF microscopy of human cells expressing the E:DIII-RIgE fusion proteins.
  • HEK-293 cells were transfected with pcDNA3 expressing the fusion DIII-WNWe-RIgE, and analysed by fluorescence imaging at 48 h posttransfection without cell permeabilization.
  • Plasma membrane was stained with red-fluorescent WGA, nuclei with blue-fluorescent Hoechst 3342, and DIII-WNKu- RlgE was immunodetected with monoclonal anti-His 6 tag antibody and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody.
  • A Control cells transduced with empty pcDNA3 plasmid.
  • FIG. 1 Cells transduced with pcDN A3 -DIII-WNWe-RIgE.
  • Panels (i) show views from the apical pole; panels (ii) show the corresponding sagittal cell plane reconstructed from the Z-stack images. Note that the DIII-WNWe-RIgE proteins were accessible at the surface of the nonpermeabilized cells, and that they localized in membrane microdomains different from the WGA-reacting regions.
  • FIG. 5 shows the expression of E:DIII-RIgE fusion proteins in insect cells.
  • A Western blot analysis. Sf9 were mock-infected (control lane 1), infected with parental baculovirus vector (control lane 2), or recombinant AcMNPV expressing CD16-RIgE (control lane 3), DIII-DENj-RIgE (lane 4), DIII-WNKu-RIgE (lane 5), and DIII-WNWe-RIgE (lane 6), respectively, and harvested at 48 h pi.
  • the 22-23 kDa band of the envelope E:DIII-RIgE fusion proteins was detected using monoclonal anti-oligoHis tag antibody and complementary phosphatase-labeled antibody.
  • Nonpermeabilized Sf9 cells were infected with AcMNPV expressing (i) CD16-RIgE (control, non-tagged fusion protein), (ii) DIII-DENrRIgE, (iii) DIII-WNWe-RIgE, (iv), DIII-WNKu-RIgE. Cells were harvested at 48 h pi and reacted with monoclonal anti-His 6 tag antibody and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody.
  • C Confocal IF image of DIII-WNWe-RIgE-expressing Sf9 cells showing that the fluorescent signal of the oligoHis tag mainly localized at the cell surface and outlined the cell contour.
  • FIG. 6 shows the protein analysis of E:DIII-pseudotyped VLPs.
  • VLPs were isolated from Sf9 cells expressing HIV-1 Pr55Gag alone (control VLPs, left side of the blots) or co-expressing Pr55Gag and flavivirus DIII-RIgE fusion construct (right side of the blots).
  • A WNKu
  • B DENi .
  • VLPs were purified by .
  • the Gag polyprotein which constituted the structural backbone of the VLPs, migrated as a major protein species with an apparent MW of 55 kDa (Pr55Gag), the DIII-DEN r RIgE and DIII-WNKu-RIgE as a 23 kDa and 22 kDa proteins, respectively.
  • the 23-22 kDa bands were absent from control VLPs.
  • Gag was detected using a polyclonal antibody, whereas DIII-DENrRIgE and DIII-WNKu-RIgE were detected with monoclonal anti-His 6 tag antibody;
  • in retroviral particles 4,000 to 5,000 copies of Gag constitute the protein core, versus only 60 copies of envelope glycoproteins are inserted in the viral envelope.
  • C The fractions from sucrose-D 2 0 gradient corresponding to density 1.10-1.20 and containing the VLPs were pooled, further purified and concentrated by pelleting through a sucrose cushion, and analyzed as above. The dual color blot was first reacted with polyclonal antibody against Pr55Gag and secondary peroxidase-labeled anti-rabbit IgG, which revealed the Gag
  • FIG. 7 shows the immuno-EM analysis of E :DIII-pseudotyped VLPs.
  • Figure 8 shows immunological analysis of specificity and 3D
  • VLP-displayed E:DIII Conformation of VLP-displayed E:DIII.
  • Aliquots of control VLP samples (VLP- void), VLP pseudotyped with DIII-DENi-RIgE (VLP-DEN1), or with DIII-WNKu- RlgE (VLP -WNKu) were coated on plates and probed by ELISA for reactivity with patient sera positive for (A) Dengue virus serotype 1, or (B) WNV Kunjin strain virus infection.
  • Figure 9 is a graph that shows quantitative evaluation by ELISA of antibodies in sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) on their surface (VLP-DEN1), or with control, nonpseudotyped VLP (VLP-0).
  • E-DIII Dengue virus serotype 1 envelope domain III
  • VLP-0 control, nonpseudotyped VLP
  • Figure 10 is a graph that shows quantitative evaluation by ELISA of antibodies in sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) at their surface (VLP-Kun), or with control, nonpseudotyped VLP (VLP-0). The different dilutions of mouse sera were tested against WNKu virions adsorbed on the wells. The different symbols indicate the sera from individual animals.
  • Figure 11 is a graph that shows antibody specificity by ELISA.
  • Figure 12 is a graph that shows antibody specificity by ELISA.
  • Different dilutions of sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) on their surface (VLP-Kun) were tested against virions of West Nile virus strain Kunjin, Dengue virus serotypes 1, 2, 3 and 4 adsorbed on the wells. Results presented are the mean of triplicate values obtained with sera from 5 different animals ⁇ standard deviation (SD).
  • SD standard deviation
  • Figure 13 is a graph that shows evaluation of antibodies against DENV serotype 1 by homologous neutralization (NA) assays.
  • Sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) at their surface (VLP-DEN1) were diluted to 1 :10 and tested against live virions of Dengue virus serotypes 1 in PRNT assays.
  • NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three
  • Figure 14 is a graph that shows evaluation of antibodies against West Nile virus Kunjin by homologous neutralization (NA) assays.
  • Sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) at their surface (VLP-Kun) were diluted to 1 : 10 and tested against live virions of WNKu in PRNT assays.
  • NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • Figure 15 is a graph that shows neutralization (NA) assays against heterologous DENV serotypes. Different dilutions of sera from mice immunised with
  • FIG. 16 is a graph that shows neutralization (NA) assays against heterologous DENV serotypes. Different dilutions of sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) on their surface (VLP-DEN1), were tested against live virions of Dengue virus serotype 3 in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • NA neutralization
  • Figure 17 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses.
  • NA neutralization
  • NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • Figure 18 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses.
  • NA neutralization
  • Different dilutions of sera from mice immunised with VLP displaying the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 1 in PRNT assays.
  • N A activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • Figure 19 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses.
  • NA neutralization
  • Different dilutions of sera from mice immunised with VLP the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 2 in PRNT assays.
  • NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • Figure 20 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses.
  • NA neutralization
  • Different dilutions of sera from mice immunised with VLP the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 3 in PRNT assays.
  • NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ⁇ SD).
  • VLPs The non-replicative nature of VLPs makes them highly attractive candidates for the design of subunit vaccines which carry the immunological structures of the virus in the absence of infectious genetic material.
  • VLPs can induce both humoral and cellular-mediated immune responses [27].
  • WNV West Nile virus
  • DENV dengue virus
  • the use of the baculovirus-insect cell expression system for production of VLP and WNV and DENV envelope glycoprotein domains represented a logical approach for the development of VLP vaccines against these flaviviruses.
  • the nonviral glycoprotein pseudotyping platform utilizes a chimeric human surface glycoprotein derived from the CD 16 molecule (abbreviated CD16-RIgE). It was surprisingly found by the present inventors that CD16-RIgE was incorporated into the envelope of extracellular VLPs with a high efficiency. It is shown herein that CD 16 ectodomain can be replaced by the envelope glycoprotein domain III of WNV and DENV, resulting in the exposure of the E:DIII epitopes at the surface of the VLP envelope.
  • CD16-RIgE chimeric human surface glycoprotein derived from the CD 16 molecule
  • Recombinant chimera CD16-RIgE can be used as a general VLP -pseudotyping platform, in coexpression with recombinant retroviral Gag polyprotein, for VLP-display of other viral envelope glycoproteins, or other pathogen proteins and their therapeutic application as potential vaccine vectors.
  • the present invention relates to a fusion protein comprising (i) a CD 16 signal peptide; (ii) an antigenic peptide region downstream of the CD 16 signal peptide; and (iii) an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
  • fusion protein refers to a protein comprising amino acid sequences that are in addition to, in place of, less than, and/or different from the amino acid sequences encoding the original or native full-length protein or subsequences thereof.
  • CD 16 signal peptide is intended to encompass an amino-terminal extension derived from immature CD 16 protein which directs the protein to be secreted.
  • CD 16 signal peptide directs the fusion proteins of the instant inventions to translocate across the cell membrane.
  • CD 16 protein signal peptide may be proteolytically removed by a signal peptidase during or immediately following membrane translocation of the fusion protein.
  • antigenic peptide refers to a peptide which elicits an immunological response in a subject.
  • immunological response includes, but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or gamma-delta T cells, and/or virus neutralizing antibodies directed specifically to an antigen or antigens included in the composition or vaccine of interest.
  • the host will display either a therapeutic or protective
  • IgE receptor transmembrane domain is intended to encompass that a portion of an immunoglobulin E (IgE) receptor protein that resides primarily in a membrane
  • IgE cytoplasmic tail is intended to encompass that portion of an IgE receptor protein that resides within the bounds of a cell, virus, VLP or other membrane-bound body.
  • the antigenic peptide region is not CD 16 ectodomain.
  • the term "ectodomain” is intended to encompass that portion of a protein which is located on the outer surface of a cell.
  • the ectodomain of the transmembrane protein CD 16 is that portion of the CD 16 protein which extends from a cell's outer surface into the extracellular space.
  • the fusion protein may be suitable for use in forming a pseudotype viruslike particle (VLP).
  • VLP pseudotype viruslike particle
  • the antigenic peptide region comprises an antigen useful for vaccination of a human or animal.
  • the antigenic peptide region comprises a viral, parasitic or bacterial antigen.
  • Viral antigens may be from RNA or DNA viruses.
  • RNA viruses from which a viral antigen may be obtained include, but are limited to, those in the following RNA virus families:
  • Arenaviridae such as lymphcytic choriomeningitis virus (LCM), Lassa virus, Junin, Tacaribe, Pichinde viruses, Machupo virus, and Guanito virus;
  • Bornaviridae such as Borna disease virus
  • Bunyaviradae such as Hanta virus, California encephalitis virus, Japanese encephalitis virus, LaCrosse virus, Rift Valley fever virus, Bunyavirus, Arbovirus, Kenya sheep disease virus, Phlebovirus, and Tospoviruses
  • Caliciviridae such as Human and animal calici viruses
  • Coronaviridae such as SARS Coronavirus
  • Filoviridae such as Ebola virus and Marburg virus
  • Flaviviridae such as Yellow Fever virus, Dengue Fever virus, West Nile virus, Hepatitis C virus, Pestiviruses, Bovine Viral Diarrhea virus, and Classical Swine Fever virus (and others as indicated below)
  • Nodaviridae such as Nodaviruses
  • Nodaviridae such as Nodaviruses
  • Orthomyxoviridae such as Influenza virus type A, Influenza virus type B, Influenza virus type C, Thogotovirus, and Fowl Plague disease virus; Paramyxoviridae, such as Parainfluenza viruses, Mumps virus, Measles virus, Subacute sclerosing
  • SSPE panencephalitis virus
  • RSV Respiratory syncytial virus
  • TPMV-like viruses Newcastle Disease virus
  • Rinderpest virus and Canine
  • Picornaviridae such as Human Enteroviruses, including Poliovirus
  • Coxsackie virus A Coxsackie virus B
  • Hepatitis A virus Hepatitis A virus
  • Rhinoviruses Foot and Mouth Disease virus
  • Enterovirus 70 Apthoviruses
  • Cardioviruses Reoviridae, such as Colorado Tick fever virus, Rotaviruses, Reoviruses, Coltivirus and
  • Retroviridae such as Human immunodefficiency virus (HIV), Human T- lymphotrophic virus (HTLV), Feline Leukemia virus (FeLV), Friend Leukemia virus (FLV), and MMTV (Mouse Mammary Tumor virus); Rhabdoviridae, such as Rabies virus, and Vesicular Stomatitis virus; and Togaviridae, such as Eastern Equine Encephalitis virus, Western Equine Encephalitis virus, Rubella virus (measles), Alphaviruses, Ross River virus, Astroviruses, Norwalk-like viruses, Hepatitis D and E viruses, Nipah virus, LR1 virus and Benyviruses.
  • HAV Human immunodefficiency virus
  • HTLV Human T- lymphotrophic virus
  • FeLV Feline Leukemia virus
  • FLV Friend Leukemia virus
  • MMTV Mammary Tumor virus
  • Rhabdoviridae such as Rabies virus, and Vesicular Stomatitis virus
  • viruses in the Flaviviridae from which a viral antigen may be obtained include, for example, those in the genera Flavivirus and Pestivirus, the "Hepatitis C-like viruses", and those in the Yellow fever virus group, Tick-borne encephalitis virus group, Rio Bravo group, Japanese encephalitis group, Tyuleniy group, Ntaya group, Kenya S group, Dengue group, and Modoc group.
  • viruses of the Flaviviridae which may be used in the present invention include, for example, but are not limited to, Gadgets Gully virus, Kyasanur Forest disease virus, Langat virus, including the British, Irish, Louping ill, Spanish and Vietnamese subtypes, Omsk hemorrhagic fever virus, Powassan virus, Karshi virus, Royal Farm virus, Tick-borne encephalitis virus, including the European, Far Eastern, and Siberian subtypes, Kadam virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, Bussuquara virus, Iguape virus, Naranjal virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4, Kedougou virus, Cacipacore virus, Japanese encephalitis virus, Koutango virus, Alfuy virus, Murray Valley encephalitis virus, St.
  • Gadgets Gully virus Kyasanur Forest disease virus, Langat virus, including the British, Irish, Louping ill,
  • leukoencephalitis virus Batu Cave virus, Phnom Penh bat virus, Rio Bravo virus, Cell fusing agent virus, Tamana bat virus, Border disease virus— BD31, Border disease virus— X818, Bovine viral diarrhea virus 1-CP7, Bovine viral diarrhea virus 1-NADL, Bovine viral diarrhea virus 1-Osloss, Bovine viral diarrhea virus 1-SDl, Bovine viral diarrhea virus 2-C413, Bovine viral diarrhea virus 2-New York '93, Bovine viral diarrhea virus 2-strain 890, Classical swine fever virus— Alfort/187, Classical swine fever virus— Alfort-Tubingen, Classical swine fever virus—Brescia, Classical swine fever virus— C, Pestivirus of giraffe, Hepatitis C virus, including genotype 10, genotype 11, genotype la, genotype lb, genotype 2a, genotype 2b, genotype 3a, genotype 4a, genotype 5 a, genotype 6a, and GB virus B, GB virus A
  • DNA viruses from which a viral antigen may be obtained include, but are limited to, those in the following DNA virus families:
  • Herpesviridae such as Herpes Simplex virus, cytomegalovirus, Epstein- Barr virus, Pseudorabies virus and Human Herpes virus 6; Adenovirus; Parvoviridae, such as Human parvovirus B19; Papillomaviridae, such as Human Papillomavirus; and Polyomaviridae, such as JC polyomavirus and BK polyomavirus; Poxviridae, such as Variola virus, Monkeypox virus and Myoma virus.
  • Examples of parasites from which a parasite antigen may be obtained include, but are limited to, those in the following parasite families:
  • Haemonchus Trichostrongylus, Ostertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris, Parascaris, Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, Wuchereria, Brugia and Onchocerca.
  • examples of parasites from which a parasite antigen may be obtained include, but are limited to, the following:
  • bacteria from which a bacterial antigen may be obtained include, but are limited to, the following:
  • Bacteria in general include but are not limited to: P. aeruginosa; E. coli, Klebsiella sp.; Serratia sp.; Pseudomanas sp.; P. cepacia; Acinetobacter sp.; S.
  • E. faecalis S. pneumonias; S. aureus; Haemophilus sp.; Neisseria Sp.; N. meningitidis; Bacteroides sp.; Citrobacter sp.; Branhamella sp.; Salmonella sp.;
  • Streptomyces sp. Trichomoras sp.; and P. mirabilis.
  • the antigenic peptide region comprises a Dili domain from a flavivirus envelope glycoprotein.
  • the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus or Dengue virus.
  • Domain III (Dili) of a flavivirus envelope glycoprotein is an
  • Ig-like domain Dili of Dengue virus envelope glycoprotein consists of 100 amino acids (residues 303-395) of the C-terminus. This domain has been suggested to be the receptor recognition and binding domain due to a number of reasons.
  • the Ig-like fold present in the Dili protein is commonly associated with structures that have an adhesion function. This domain extends perpendicularly to the surface of the virus with a tip projecting further from the virion surface than any part of the envelope glycoprotein.
  • studies have demonstrated that both recombinant Dill proteins and antibodies generated against Dili of E protein of flavivirus can inhibit entry of the flavivirus into target cells.
  • flavivirus with mutations in Dili of the envelope glycoprotein shows either attenuated virulence or the ability to escape immune neutralization [ 1 ] .
  • Exemplary strains of West Nile virus include, but are not limited to, West Nile B956, Madagascar 1978, Cyprus 1968, Kunjin 1960, Kunjin 1991, Wengler, goose Israel 1998, New York 1999, Kenya, Kenya, Senegal 1990, Philippine 1937, Kenya 1959, Central African Republicl 972a, Central African Republic 1972b, Central African Republic 1983, Madagascar 1986 and Madagascar 1988.
  • the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus strain Wengler or Kunjin or of Dengue virus serotype 1, 2, 3 or 4.
  • the present invention relates to a nucleic acid molecule encoding a promoter operably linked to (i) a sequence encoding the fusion protein as described herein; or (ii) a sequence encoding a CD 16 signal peptide upstream of a cloning site and a sequence encoding an IgE receptor transmembrane domain and cytoplasmic tail downstream of the cloning site.
  • nucleic acid molecule is intended to encompass DNA molecules and RNA molecules.
  • a nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • a nucleic acid is
  • operably linked when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • promoter is intended to encompass a
  • the promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity.
  • Non-limiting examples of promoters include, for example, constitutive, inducible, tissue-specific and tissue-preferred promoters. Conditions that may induce transcription from an inducible promoter include, for example, any chemicals or compounds, temperature, light wavelength or level.
  • the initiation complex can be modified by activating sequences termed “enhancers” or inhibitory sequences termed "silencers".
  • the term "cloning site" is intended to encompass a region which allows for the insertion of desired nucleic acid sequences. Typically, the cloning site comprises one or more restriction endonuclease recognition sites. Cloning sites include, but are not limited to, multiple cloning sites or polylinkers.
  • the nucleic acid molecule is an expression vector.
  • expression vector is intended to encompass a nucleic acid molecule, preferably a double-stranded DNA molecule, comprising a gene that is expressed in a host cell.
  • gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue- specific regulatory elements and enhancers.
  • These vectors can be designed, using known methods, to contain the elements necessary for directing transcription, translation, or both, of the nucleic acid in a cell to which it is delivered.
  • Known methodology can be used to generate expression constructs that have a protein-coding sequence operably linked with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques and synthetic techniques. For example, see Sambrook et al. ((2001) Molecular Cloning: a
  • a variety of expression vector/host systems may be utilized to contain and express the nucleic acid encoding the fusion protein of the present invention. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus,
  • Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS.cells (such as COS-7), W138, BHK, HepG2, 3T3, RTN, MDCK, A549, PC 12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the protein are described herein below.
  • the secreted peptide is purified from the growth medium by, e.g., the methods used to purify the fusion protein from bacterial and mammalian cell supernatants.
  • the nucleic acid is a baculoviral expression vector, wherein the promoter is a promoter that is expressed in insect cells.
  • the cDNA encoding the peptide may be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, Calif.). This vector is then used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant protein.
  • the fusion protein may be purified and concentrated from the media using a heparin- Sepharose column (Pharmacia, Piscataway, N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass.), and resuspended in PBS. SDS-PAGE analysis shows a single band and confirms the size of the fusion protein, and Edman sequencing on a Porton 2090 Peptide Sequencer confirms its N-terminal sequence.
  • the present invention relates to a method of forming a pseudotype virus-like particle, the method comprising coexpressing in a cell (i) a fusion protein as described herein; and (ii) a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein.
  • retroviral Gag polyprotein and “protein precursor of a retroviral Gag polyprotein” are intended to encompass retroviral group specific antigens that constitute retroviral structural proteins.
  • the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SrV), or a chimeric human-animal retroviral Gag polyprotein.
  • HIV- 1 Pr55Gag is intended to encompass a polyprotein complex comprising pi 7 (matrix) protein, p24 (capsid) protein, p2 (Spl) protein, p7 (nucleocapsid) protein, pi (Sp2) protein, and p6 protein of human immunodeficiency virus- 1 (HIV-1).
  • Proteins, polypeptides, and nucleic acids referred to herein are intended to encompass homologues, derivatives, variants, or fragments thereof.
  • a nucleotide sequence or polypeptide sequence is a "homologue" of, or is “homologous” to, another sequence if the two sequences have substantial identity over a specified region and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness).
  • Two polynucleotide sequences or polypeptide sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted), they share at least about 50% sequence identity, or if the sequences share defined functional motifs.
  • optimally aligned sequences may be considered to be substantially identical (i.e. to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%), 97%, 98%, 99 identity over a specified region.
  • An "unrelated" or “non-homologous” sequence shares less than 40% identity, though preferably less than about 25 % identity, with a polypeptide or polynucleotide of the invention over a specified region of homology.
  • identity and “identical” refer to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences.
  • a degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region.
  • Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at htt ://clustalw. genome.ad.jp, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.
  • a variant or derivative polypeptide refers to a polypeptide that has been mutated at one or more amino acids, including point, insertion or deletion mutation, but still retains the desired function(s), as well as non-peptides and peptide mimetics which possess the ability to mimic the desired function of the polypeptide.
  • a variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, including tagged polypeptides and fusion proteins; substitutions, for example conservative substitutions, site-directed mutants and allelic variants; and modifications, including peptoids having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications.
  • substitutions refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their , size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, for example, by reaction of a functional side group of an amino acid.
  • Variants and derivatives can be prepared, for example, by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of a polypeptide or fragment thereof, and screening for biological activity. Preferably, substitutions are made with conservative amino acid residues, i.e., residues having similar physical, biological or chemical properties.
  • conservative amino acid residues i.e., residues having similar physical, biological or chemical properties.
  • a skilled person will understand how to make such derivatives or variants, using standard molecular biology techniques and methods, described for example in Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3 rd ed., Cold Spring Harbour Laboratory Press), and how to test such derivatives or variants for their desired function, including using techniques as described in the Examples set out herein.
  • consists essentially of or “consisting essentially of' means that the sequence includes one or more amino acids at one or both ends of the described sequence, but that the additional amino acids do not materially affect the function of the polypeptide.
  • the polypeptide consisting essentially of a particular sequence may have one, two, three, five or ten amino acids at one or both ends of the described sequence, provided that such a protein still possesses the desired function.
  • the present invention relates to a pseudotype virus-like particle comprising (i) protein core comprising a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein; (ii) a membrane envelope surrounding the protein core; and (iii) a fusion protein incorporated into the membrane envelope, the fusion protein comprising an antigenic peptide region and an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
  • virus-like particle is intended to encompass a highly organised particulate structure which differs in nature and composition from the virus type, for example, by being devoid of viral genome and being non- replicative.
  • VLPs may be comprised of viral capsid proteins, for example from HPV or hepatitis E virus (HEV).
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • VLPs may be based on retroviruses or lentiviruses, for example, human immunodeficiency virus (HIV), and may be membrane-enveloped particles made up of a backbone of viral polyprotein Gag (or Gag precursor), wrapped by an envelope derived from the host cell plasma membrane.
  • HIV human immunodeficiency virus
  • the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SIV), or a chimeric human-animal retroviral Gag polyprotein.
  • the terms "pseudotype” or “pseudotyping”, are intending to encompass a VLP in which at least one viral envelope protein has been substituted with the fusion protein as described herein.
  • the antigenic peptide region comprises an antigen useful for vaccination of a human or animal.
  • the antigenic peptide region comprises a viral, parasitic or bacterial antigen, non-limiting examples of which are described herein.
  • the antigenic peptide region comprises a Dili domain from a flavivirus envelope glycoprotein.
  • the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus or Dengue virus.
  • the Dili domain is the Dill domain from the envelope glycoprotein of West Nile virus strains or of Dengue virus serotype 1, 2, 3 or 4.
  • the pseudotype virus-like particle is formed by the method of the third aspect.
  • the present invention relates to a vaccine comprising a pseudotype virus-like particle of the fourth aspect.
  • the vaccine of the present invention may be in the form of a
  • composition which may comprise one or more of the fusion protein or pseudotype virus-like particle, any additional active substance or substances or one or more additional therapeutic agents.
  • the pharmaceutical composition can be prepared by known methods for the preparation of pharmaceutically acceptable compositions suitable for vaccination of a human or animal, such that an effective quantity of the fusion protein or pseudotype virus-like particle and any additional active substance or substances is combined in a mixture with a pharmaceutically acceptable vehicle, carrier, excipient or diluent.
  • a pharmaceutically acceptable vehicle, carrier, excipient or diluent Suitable vehicles, carriers, excipients and diluents are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • compositions include, albeit not exclusively, solutions or suspensions of the fusion proteins or pseudotype virus-like particles, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffer solutions with a suitable pH and iso-osmotic with physiological fluids.
  • the pharmaceutical composition may be prepared in a physiologically and pharmacologically suitable buffer. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, and that will maintain the live state of the cells. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
  • the pharmaceutical composition may further comprise one or more adjuvants.
  • Suitable adjuvants include, but are not limited to, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,
  • GPI-0100 Galenica Pharmaceuticals, Inc., Birmingham, AL
  • non-metabolizable oil mineral and/or plant/vegetable and/or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in- water emulsion.
  • the emulsion can be based in particular on light liquid paraffin oil
  • isoprenoid oil such as squalane or squalene
  • esters of acids or of alcohols containing a linear alkyl group more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glyceryl tri- (caprylate/caprate) or propylene glycol dioleate
  • esters of branched fatty acids or alcohols in particular isostearic acid esters.
  • the oil is used in combination with emulsifers to form the emulsion.
  • the emulsifers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L 121.
  • mannide e.g. anhydromannitol oleate
  • glycol of polyglycerol
  • propylene glycol and of oleic isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L 121.
  • Pluronic products especially L 121.
  • the present invention relates to use of a vaccine of the fifth aspect for vaccination of a human or animal.
  • the fusion proteins, pseudotype virus-like particles and vaccines of the present invention are useful for vaccination of a human or animal.
  • animals include livestock such as cattle, horses, goats, sheep, and pigs; companion animals such as dogs and cats; and domesticated fowl such as chickens, ducks and geese.
  • livestock such as cattle, horses, goats, sheep, and pigs
  • companion animals such as dogs and cats
  • domesticated fowl such as chickens, ducks and geese.
  • "useful for vaccination of a human or animal” is intended to encompass eliciting an "immunological response", as defined herein.
  • Eliciting an immunological response is intended to encompass treating or preventing a disease or condition to obtain beneficial or desired results, including clinical results.
  • Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, amelioration or palliation of the disease state, and remission (whether partial or total).
  • Treating can also mean prolonging survival of a subject beyond that expected in the absence of treatment.
  • “Treating” can also mean inhibiting the progression of disease, slowing the progression of disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
  • the pharmaceutical composition of the present invention can be administered in any conventional manner.
  • administration methods include any that afford access by cells of the immune system to the pharmaceutical composition including parenteral, oral, transdermal, intradermal, intravenous, subcutaneous, intramuscular, intraocular, intraperitoneal, intrarectal, intravaginal, intranasal, intragastrical, intratracheal and intrapulmonarial, or any combination thereof.
  • an effective amount of the pharmaceutical composition is administered to the human or animal.
  • effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to induce protective immunity against a pathogen in the human or animal.
  • the dose and frequency of administration of the pharmaceutical composition that is to be used depends on the particular disease or condition being treated or prevented, the severity of the disease or condition, individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of
  • HEK-293 Human embryonic kidney cells (HEK-293) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown in Iscove's medium supplemented with 10 % FBS and 50 mg/ml gentamicin
  • VSV-G Recombinant baculovirus expressing VSV-G , HIV-1 Gag, and VLP production, (i) AcMNPV-VSV-G and AcMNPV-Gag. All foreign genes were inserted into the genome of Autographa californica MultiCapsid NucleoPolyhedrosis Virus (AcMNPV) under the control of a chimeric AcMNPV-GmNPV polyhedrin promoter, as described in previous studies [16-18,30,35].
  • AcMNPV- VSV-G expressed the attachment glycoprotein G of vesicular stomatitis virus (VSV).
  • VLP were first recovered by pelleting through a cushion of 20 % sucrose in TNE buffer (TNE : 100 mM NaCl, 10 mM Tris- HC1 pH 7.4, 1 mM Na 2 EDTA) at 30,000 rpm for 1 h at 15 °C in a Kontron TST55.5 rotor.
  • VLP pellets from step (a) were gently resuspended in PBS (0.20-0.25 ml), and (b) further purified by isopycnic ultracentrifugation in sucrose-D 2 0 gradients.
  • the HIV-1 mature capsid is a fullerene cone composed of approximately 250 hexamers and 12 pentamers of the viral CAp24 protein [36], which accounted for 1,560 copies of CAp24 per core, generated by the proteolytic cleavage of Pr55Gag molecules constituting the immature particle. These data are consistent with the value of 1,500 copies of CAp24 protein per mature core, determined by alternative methods [37,38]. However, less than half of the CAp24 proteins form mature cores, since cryoelectron microscopy and scanning electron microscopy showed that immature HIV-1 particle contains approximately 5,000 copies of closely packed Pr55Gag protein [37,39,40].
  • VLP titer was estimated by SDS-PAGE analysis of VLP and the intensity of the Pr55Gag signal in Coomassie blue-stained gels, in co- el ectrophoresis with a range of bovine serum albumin samples of known
  • a concentration of 1 mg/ml of Pr55Gag protein corresponded to a titer of ⁇ 2 x 10 E 12 VLP per ml.
  • CD16-RIgE consisted of the fusion of the ectodomain of FcyRIIIa (or CD 16), the human low-affinity receptor for IgG Fc (involved in antibody-dependent cell-mediated cytotoxicity; ADCC), to the transmembrane and cytoplasmic domains of the gamma polypeptide of the human high affinity receptor for IgE, FcsRIy.
  • the CD 16 ectodomain was responsible for the recognition of IgG Fc, whereas FceRIy transduced the intracellular signals [41].
  • the cDNA encoding the CD16-RIgE was then rescued using conventional overlapping PCR method, and inserted into the Nhel and Hindlll sites of the pBlueBac4.5 vector genome (Invitrogen, San Diego, CA). Recombinant AcMNPV-CD16-RIgE were isolated using beta-galactosidase staining of positive Sf9 cells and the blue plaque selection method.
  • VLP VLP were denatured in SDS/beta-mercaptoethanol-containing loading buffer at 100 °C for 2 min, and proteins were electrophoresed in SDS- denaturing, 10 %-polyacrylamide gel and electrically transferred to nitrocellulose membrane (HybondTM-C-extra; Amersham Biosciences). Blots were blocked in 5 % skimmed milk in Tris-buffered saline (TBS) containing 0.05 % Tween-20 (TBS-T), rinsed in TBS-T, then successively incubated with primary antibody, mouse
  • ELISA The accessibility and functionality of recombinant E:DIII proteins was evaluated by their binding activity to the monoclonal anti-His 6 tag antibody, in standard ELISA procedure. In brief, aliquots (100 ⁇ ) of VLP suspension (50 ⁇ ; 10 E 11 VLP/ml) in coating buffer (0.1M NaHC0 3 , pH 9.6) were incubated overnight at 4 °C in 96-well microtiter plates (NUNC, Roskilde, Denmark).
  • the coated wells were blocked with 200 ⁇ of blocking buffer (2 % bovine serum albumin in 0.14 M NaCl, 0.05M Tris-HCl buffer, pH 8.2; TBS-BSA) for 1 h at room temperature (RT), then washed five times with washing buffer (0.05 % Tween-20 in TBS ; TBS-T). Monoclonal anti-His 6 tag antibody was added at dilution 1 :2,500 in TBS-BSA, and incubated for 1 hr at RT. Mouse antisera against the four DENV serotypes, and against the WNV strains were laboratory-generated. Patient sera positive for DENV and WNV infections were used to confirm expressed-DIII protein configuration.
  • IF Immunofluorescence microscopy.
  • Baculovirus-infected Sf9 cell monolayers were harvested at 48 h pi, fixed with 3 % paraformaldehyde in phosphate buffered saline (PBS) and, when required, permeabilized in 0.2 % (v/v) Triton X-100 in PBS.
  • Cells were blocked with 3 % BSA in PBS (PBS-BSA), and His-tagged E :DIII detected by reaction with monoclonal anti-His 6 tag antibody (1 :10,000 in PBS-BSA) and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody (Molecular Probes, Invitrogen).
  • E :DIII and Gag proteins For double labeling of E :DIII and Gag proteins, cell samples were reacted with rabbit anti-Gag antibody (1 : 1,000 in PBS-BSA) and Alexa Fluor® 546-labeled goat anti-rabbit IgG (Molecular Probes, Invitrogen). Cell samples were labeled using the Image-iTTM LIVE Plasma Membrane and Nuclear Labeling Kit (Molecular Probes, Invitrogen), which provides red-fluorescent Alexa Fluor® 594 wheat germ agglutinin (WGA) and blue-fluorescent Hoechst 3342 dye for plasma membrane and nucleus staining, respectively.
  • WGA wheat germ agglutinin
  • Hoechst 3342 dye blue-fluorescent Hoechst 3342 dye for plasma membrane and nucleus staining, respectively.
  • VLP Electron microscopy (EM) and immuno-EM. VLP specimens were processed and observed under the EM as previously described [21]. In brief, VLP were diluted in 20 ⁇ 0.14 M NaCl, 0.05M Tris-HCl buffer, pH 8.2 (Tris-buffered saline; TBS) and adsorbed onto carbon-coated formvar membrane on grids. The grids were incubated with primary antibody (monoclonal anti-His 6 tag antibody) at a dilution of 1 : 50 in TBS for 1 h at room temperature (RT).
  • primary antibody monoclonal anti-His 6 tag antibody
  • the grids were post-incubated with 10-nm colloidal gold-tagged goat anti-mouse IgG antibody (British Biocell International Ltd, Cambridge, UK; diluted to 1 :50 in TBS) for 30 min at RT.
  • the specimens were negatively stained with 1 % sodium phosphotungstate in H 2 0 for 1 min at RT, rinsed again with TBS, and examined under a JEM 1400 JEOL electron microscope equiped with an Orius-Gatan digitalised camera (Gatan France, 78113 Grandchamp).
  • Human CD16 (or FcyRIIIa) is one of the low-affinity receptors for IgG Fc and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). It links IgG-sensitized target cells to CD16-bearing cytotoxic cells, i.e. CD56dim natural killer (NK) cells, a fraction of monocytes and macrophages, and rare T cells, and activates these effector cells.
  • NK cytotoxic cells
  • FcsRIy is a high affinity receptor for IgE receptor, and was abbreviated RIgE in the present study.
  • CD16-RIgE is a chimeric receptor molecule consisting of the ectodomain of CD 16 fused to the transmembrane and cytoplasmic domains of the FcsRIy receptor [41].
  • the cDNA for CD16-RIgE was cloned into the genome of the baculovirus AcMNPV to generate the recombinant AcMNPV-CD16-RIgE (Fig. 1).
  • Sf9 insect cells were infected with AcMNPV-CD16- RlgE, and the level of expression of the recombinant CD16-RIgE glycoprotein was assessed by SDS-PAGE and Western blot analysis of cell lysates.
  • CD16-RIgE was expressed at high levels at 36-72 h post infection (pi) with a maximum at 48 h, as shown by the fuzzy band at 75 kDa, suggesting that this protein was not toxic for Sf9 cells.
  • pi 36-72 h post infection
  • the fuzzy band at 75 kDa
  • the HIV-1 Gag precursor, Pr55Gag is addressed to the plasma membrane or to a late endosomal compartment via its N-myristoyl group and a cluster of basic residues in the matrix domain [13,20,43].
  • Pr55Gag efficiently self-assembles into VLPs budding at the plasma membrane [14,17,18].
  • the CD16-RIgE glycoprotein with is N-terminal signal peptide reaches the cell surface via the Golgi pathway.
  • VSV-G Taking advantage of the capacity of VSV-G to bind to a wide variety of cell surface receptors, combined with the ubiquitous character of VSV-G- pseudotyping and the VSV-G fusiogenic property, VSV-G has been largely used to construct fusiogenic retroviral or lentiviral vectors in mammalian cells [44].
  • AcMNPV- VSV-G was therefore a useful standard to test the capacity of HIV- 1 Gag- VLPs produced in baculovirus-infected insect cells to be pseudotyped by foreign glycoproteins, including VSV-G and CD16-RIgE.
  • Sf9 were therefore co-infected with AcMNPV-Gag and AcMNPV- VSV-G at equal MOI, and extracellular VLPs were collected at 48 h and analyzed by SDS-PAGE and Western blotting.
  • VSV-G was observed to be incorporated in significant amounts in VLPs (Fig. 3 A, ii).
  • VSV-G and CD16-RIgE were detected using two different antibodies, the intensity of the CD16-RIgE signal, visible as a broad and diffuse band at 75 kDa, compared to the dark and sharp band of VSV-G at 62 kDa, suggested a similar efficiency of VLP incorporation for both glycoproteins (compare panels i and ii in Fig. 3A).
  • Sf9 cells were then infected with three recombinant baculoviruses, AcMNPV-Gag, AcMNPV- VSV-G and AcMNPV-CD16-RIgE, and the extracellular VLPs were isolated and analyzed as above.
  • VLPs reacted with both anti- VSV-G and anti-CD 16 antibodies (Fig. 3 A, iii ; rightmost lane), suggesting that both VSV-G and CD16-RIgE glycoproteins were co-incorporated into VLPs.
  • the VLPs pseudotyped with CD16-RIgE represented two separate populations, indicating that HIV-Gag VLPs produced in Sf9 cells could incorporate more than one foreign glycoprotein.
  • Baculovirus vectors were constructed to express these prototype chimeric proteins, AcMNPV-DENi-RIgE, AcMNPV-WNWe-RIgE and AcMNPV- WNKu-RIgE, respectively. All three clones showed a similarly high level of expression of their recombinant proteins in Sf9 cells, migrating with the expected apparent molecular mass of 22-23 kDa (Fig. 5 A).
  • DIII-DEN t -RIgE comprised of 215 residues including the signal peptide (22 residues), the oligo-His tag (6 residues), the Dili domain (123 residues), the transmembrane and cytoplasmic domains of the RIgE (63 residues), and a few leftover residues from the deleted CD 16 ectodomain.
  • Fig. 5 B shows that their trafficking pathway in Sf9 cells was the same as in HEK-293.
  • Fig. 5 C shows a confocal microscopy image shows a high resolution image of the expressed DIII-WNWe protein on the cell plasma membrane.
  • each VLP pseudotype was analyzed by ELISA using patient sera positive for DENV-1, WNKu or WNWe.
  • Fig. 8 there was a significant reactivity of the E:DIII-pseudotyped VLPs with their specific antibodies.
  • the Domain III of the envelope glycoprotein of flaviviruses has a structure resembling that of an immunoglobulin constant domain and contributes the loops which are exposed on the exterior of the mature virion [46,47]. Exposure of West Nile and Dengue virus structural domain Dili at the surface of VLP is logical in view of previous observations that this domain is highly immunogenic and capable of inducing a robust immune response and a high level of neutralising antibodies [1-5]. Antibodies binding to epitopes on E:DIII have been detected in sera of patients for both West Nile and Dengue virus [48-52]. As a prototype, the E:DIII of Dengue virus serotype 1 was chosen out of the four serotypes.
  • WNV prototypes Two extensively studied strains of West Nile virus, the Wengler and Kunjin strains, were selected as WNV prototypes.
  • the Wengler strain is of lineage 2 and was isolated from Nigeria, while the Kunjin strain is of lineage 1 and was isolated in Australia.
  • Strains of WNV from lineage 1 are generally more virulent and neuro-invasive, but there is a recent trend for lineage 2 WNV to become increasingly encephalitic [53].
  • selection of these two strains of WNV took into account potential variations that could arise due to differences in geographical distribution and virulence.
  • flavivirus cycle involves a mosquito vector as an intermediate host.
  • flavivirus proteins in Sf9 should be similar to their natural biosynthetic pathway and posttranslational modifications in the insect vector,
  • flavivirus envelope glycoprotein domain(s) at the surface of VLPs mimicked the real situation in the viruses, and should be optimal for immunization purposes,
  • VLPs, as other particulate structures are known to play the role of adjuvant of the immune response [27], and this would be advantageous for vaccination strategies.
  • virus pseudotypes have been described and even used used as molecular tools and therapeutic agents, the pseudotyping of viral vectors is not a simple and straightforward process, as the molecular mechanisms involved are diverse and not fully understood, and many parameters are not perfectly controlled yet [44,54].
  • the complexity of the process has been well documented in HIV-1 virions, in which the recruitment of the viral envelope glycoproteins (SUgpl20/TMgp41) not only requires (i) that the Gag molecules and the SUgpl20/TMgp41 complex meet at the same sites of virus assembly (plasma membrane and multivesicular bodies;
  • the length of the cytoplamic tail was not sufficient to explain the difference in the behaviour of the foreign glycoproteins tested, 106 amino acid residues for CAR, versus 42 for the gamma chain of the RIgE receptor and 31 for VSV-G.
  • Other parameters such as the nature of the matrix domain of the retroviral Gag polyprotein which constitutes the internal protein backbone of the VLPs [16], might control the efficiency of the pseudotyping process of VLPs.
  • retroviral Gag precursor e.g. SIV, MLV, or chimeric MLV-HIV Gag, as well as various Gag mutants [30]
  • SIV SIV
  • MLV chimeric MLV-HIV Gag
  • various Gag mutants [30] could be used to determine the respective contribution of the different structural and functional domains present in the protein partners involved.
  • the CD16-RIgE chimera of the present invention in coexpression with HIV-1 Gag polyprotein in Sf9 ' cells, represents a versatile and useful pseudotyping platform for VLP-display of ectodomains of proteins of interest, in particular for vaccinal purposes.
  • CD16-RIgE a chimeric human membrane glycoprotein consisting of the CD 16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE
  • the extracellular VLPs incorporated CD16-RIgE in their envelope with a high efficiency.
  • the CD 16 ectodomain of CD16-RIgE was replaced by the envelope glycoprotein domain III (E:DIII) of the Wengler and Kunjin strains of West Nile virus (WNWe and WNKu, respectively) and serotype 1 of Dengue virus (DEN ⁇ , as flavivirus prototypes.
  • Each E:DIII domain was equiped with an oligo-histidine tag at its N- terminus.
  • the resulting fusion proteins DIII-WNWe-RIgE, DIII-WNKu-RIgE and DIII-DENi-RIgE were found to traffic to the plasma membrane of human and insect cells, and to be exposed at the cell surface, as their parental clone CD16-RIgE.
  • all three E:DIII-RIgE proteins were found to efficiently form pseudotype VLPs. Immunological and electron microscopic data indicated that the E:DIII domains were accessible at the surface of VLPs, as expected for bona fide pseudotypes.
  • the CD16-RIgE chimera might therefore be proposed as a general VLP-pseudotyping platform, in coexpression with recombinant retroviral Gag polyprotein, for VLP-display of other viral envelope glycoproteins or any other antigen, and their therapeutic application as potential vaccine vectors.
  • the present invention provides a strategy of epitope presentation at the surface of virus-like particles (VLPs).
  • VLPs are retroviral Gag particles which spontaneously assemble into particles and are structurally similar to the native virions but do not contain the viral genetic material.
  • the immunogenic epitopes are exposed on the VLP surface via a membrane-anchored fusion construct to mimic the native antigenic protein as presented by viruses.
  • the protein platform for epitope presentation consists of a chimeric human surface glycoprotein derived from the CD 16 molecule (abbreviated CD 16-RIgE), which is efficiently incorporated into the envelope of the VLPs. It is here shown that the CD 16 ectodomain can be replaced by the envelope glycoprotein domain III of West Nile and Dengue virus, resulting in the exposure of the Envelope (E):DIII epitopes at the surface of the VLP envelope.
  • CD 16-RIgE chimeric human surface glycoprotein derived from the CD 16 molecule
  • SP-CD16-RIgE The only portion left from the CD 16 is its signal peptide (SP) and a couple of amino acids at the junction with the TM domain of RIgE. This is clearly shown in Fig. 1 A.
  • This platform can be used to insert two or more E-DIII domains or full envelope (E) proteins from different flavivirus envelopes in tandem.
  • the recombinant chimera SP-CD16-RIgE can be used as a general VLP- pseudotyping platform for VLP-display of other viral envelope glycoproteins, or other pathogen proteins and their therapeutic application as potential vaccine vectors.
  • This technology of VLP-antigen display/presentation is a major improvement over the conventional "VLP pseudotyping" strategy as the presentation platform can be used for displaying antigenic domains from viral, bacterial or parasitic pathogens.
  • the VLPs are devoid of viral genomes, and their non-replicative nature makes them useful for the design of subunit vaccines which carry the immunological structures of the virus in the absence of infectious genetic material.
  • VLP can induce both humoral- and cellular-mediated immune responses.
  • Immunization regime with E-DIII-RIgE-pseudotyped VLPs of the present invention was based on a protocol previously described and optimized to obtain neutralization antibodies in mice immunized with inactivated flavivirus particles used as the immunogens [1, 2, 3].
  • series of five BALB/C mice were injected intraperitoneally with 100 ⁇ -aliquot per animal of virus stock with purified titre of 10 7 PFU/ml and mixed with complete Freund's adjuvant. This was repeated at weeks 3 and 5 with incomplete Freund's adjuvant, and animals were bled at week 6 [1, 2, 3].
  • E-DIII-DENV1 The simplest explanation for the difference in envelope incorporation into VLP between E-DIII-DENV1 and E-DIII-WNKun, was the slightly lower level of cellular expression of E-DIII-WNKun protein, compared to E-DIII-DENV1.
  • IP infectious to physical particles
  • Animal Balb/C mouse, female, 11 week-old
  • Week 8 Final bleed.
  • ELISA was performed using the viruses as immobilized antigens. Live viruses were coated onto the bottom of the wells in a 96-well MultiSorp plate (Nunc, Denmark) in a Class II Type A2 Biohazard Safety Cabinet. This was achieved by adding 100 ⁇ of 5,000 PFU/ml of virus into each well and incubating the plate overnight at 4°C.
  • Serum diluent was made up of PBS with 0.1% (v/v) Tween 20 (PBST) and 5% (w/v) skimmed milk.
  • Each mouse antiserum raised against the E-DIII protein of DENV1 and KUN was diluted in 1 : 10, 1 :50, 1 : 100 and 1 :200 ratios using 5% skimmed milk.
  • the antiserum raised against the negative control VLP-0 was also similarly diluted.
  • One hundred ⁇ of diluted anti-sera was added into each well such that each diluted antiserum would be added into three wells (triplicates). The plate was then incubated for one hour at 37°C, then 100 ⁇ of diluted (1 :250) secondary anti-IgG-biotin conjugate (Chemicon International, U.S.A.) was added.
  • the plate was incubated for another hour at 37°C and 100 ⁇ of diluted (1 :5,000) strepavidin-HRP conjugate (Chemicon International, U.S.A.) was added into each well. The plate was further incubated for one hour at 37°C, followed by the addition of 100 ⁇ of TMB One substrate solution (Promega Corporation, U.S.A.) into each well. The plate was further incubated for 30 min, and 50 ⁇ of 0.5 M sulfuric acid stop-solution added into each well. All wells were washed with PBST thrice before the addition of any reagent except before the stop solution was added. The absorbance of each well was finally measured at 450 nm.
  • Virus neutralisation plaque reduction neutralisation test (PRNT)
  • Complement C 1 q has been shown to bind to the Fc region of virus- bound antibodies, and could help reduce infection via steric hindrance. Heat- inactivating the antisera used in PRNT would then inactivate Clq and eliminate this confounding factor. Prior to performing PRNT, all the antisera were inactivated by heating them at 56°C for 30 min using a dry-bath. Heat-inactivated antisera were diluted in 1 :10, 1 :50, 1 :100, 1 :200, 1 :400 and 1 :800 ratios and 500 ⁇ of each dilution was incubated with 500 PFU of virus at 37°C for one hour. A control was included in which virus diluent was used instead of antiserum.
  • BH cells were seeded onto 24- well plates and 100 ⁇ of diluted virus- antibody mixtures were transferred in triplicates onto confluent cell monolayers in the wells.
  • a row of mock-infected wells was included as negative controls and a row of neat virus-infected wells served as positive controls. Plates were incubated at 37°C for one hour under 5% carbon dioxide, with rocking every 15 min to ensure even distribution of diluted virus-antibody mixtures.
  • the diluted virus-antibody mixtures were removed and the cell monolayer was washed once with virus diluent.
  • One ml of CMC overlay medium was pipetted into each well. The trays were incubated at 37°C in a humidified C0 2 incubator.
  • ELISA showed that antibodies against Dengue and West Nile (WN) Kunjin viruses were induced in mice after SC administration of E-DIIl-DENVl-VLP and E-DIII-WNKu-VLP, respectively (Fig. 9 and 10).
  • Sera from 2 out of 5 mice were positive for DENV1 virus at all dilutions tested (1 :10 to 1 :200), and reacted in a dose-dependent manner (Fig. 9).
  • Sera from 2 out of 5 mice were positive for WN Kunjin: one serum reacted with WN Kunjin virus at all dilutions tested (1 :10 to 1 :200); another serum was positive at low dilutions (1 :10 & 1 :50 ; Fig. 10).
  • ELISA showed moderate levels of antibodies and binding affinity towards the corresponding live virus particles.
  • mice administered with E-DIII-DENV 1 - VLP and E-DIII-WNKun-VLP were tested in ELISA against five different immobilized virus particles, DENV1, DENV2, DENV3, DENV4 and WN Kunjin.
  • Data showed that all sera bound to the different flaviviruses tested, and at all dilutions used (Fig. 11 and 12).
  • most of the antibodies induced by E-DIII-VLP and detected by ELISA were directed against common region(s) of the E-DIII domains, shared by the different DENV serotypes and by WN Kunjin virus.
  • PRNT showed that sera from 3 out of 5 mice injected with E-DIII- DENV1-VLP had neutralizing antibodies against DENV1 with NA ranging from 20 to 30 % (Fig. 13). Sera from 3 out of 5 mice SC injected with E-DIII- WNKun- VLP had neutralizing antibodies against WN Kunjin virus with NA ranging from 25 to 50 % (Fig. 14). In conclusion, neutralizing antibodies directed against DENV and WN Kunjin virus were induced in mice using SC administration of E-DIII-DENVl-VLP and E-DIII-WNKun-VLP, respectively.
  • mice #4 and #5 were found to develop the highest levels of total anti-DENVl antibodies (refer to Fig. 9), whereas sera of mice #2, #3 and #4 were the highest in NA titer (refer to Fig. 13). Only one animal, mouse #4, had both high levels of total antibodies
  • mice #1 and #4 developed the highest levels of total anti-WN Kunjin antibodies (as assayed by ELISA), whereas sera of mice #2, #3 and #5 showed the highest NA activity (compare Fig. 10 & 14).
  • anti-DENVl neutralizing antibodies induced by administration of mice with E-DIII-DENV1-VLP were bivalent, as they neutralized both serotypes 1 and 2 of dengue virus.
  • Anti-WN Kunjin neutralizing antibodies induced by administration of mice with DIII-WNKu-VLP cross-neutralized DENV serotypes 1 and 2.
  • Virus Res 112 85-94.
  • Pr55gag virus-like particles potent stimulators of innate and acquired immune responses. Mol Immunol 42: 259-277.
  • ADCC Antibody-dependent cellular cytotoxicity
  • Nile virus envelope domain III protein and the cellular receptor alpha Vbeta3 integral J Biol Chem 281 : 1352-1360.
  • RNA is a structural element in retrovirus particles. Proc Natl Acad Sci USA 98: 5246-5251.
  • CSN-5 a component of the COP9 signalosome complex, regulates the levels of UNC-96 and UNC-98, two components of M- lines in Caenorhabditis elegans muscle. Mol. Biol. Cell. 20, 3608-3616.
  • Chipman P. R., Kuhn, R. J., Diamond, M. S., and Rossmann, M. G. (2010b). Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. Proc. Natl. Acad. Sci. USA 107, 18950-18955.

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Abstract

A strategy of pseudotyping HIV-based VLPs consisting of the coexpression in insect cells of retroviral Gag polyprotein and a foreign membrane glycoprotein using two recombinant baculovirus vectors is provided. The nonviral glycoprotein pseudotyping platform consisted of a chimeric human surface glycoprotein derived from the CD16 molecule (abbreviated CD16-RIgE). The CD16 ectodomain was replaced by the envelope glycoprotein domain III of dengue viruses (DENV) and West Nile viruses (WNV), resulting in the exposure of E:DIII epitopes at the surface of the VLP envelope. Sera from mice immunized with E:DIII-pseudotyped VLP showed neutralizing activities against flaviviruses. It is proposed the use of the recombinant chimera CD16-RIgE as a general VLP-pseudotyping platform, in coexpression with the recombinant retroviral Gag polyprotein, for VLP-display of other viral envelope glycoproteins, or other pathogen proteins and their therapeutic application as potential vaccine vectors.

Description

A NOVEL PLATFORM FOR RETRO VIRUS-LIKE PARTICLE (VLP)-DISPLAY OF VACCINE ANTIGENS
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims benefit of, and priority from, SG provisional patent application No. 201001920-6 filed on March 19, 2010, the contents of which are fully incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a platform for retrovirus-like particle (VLP)-display of vaccine antigens.
BACKGROUND
[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention. However, it should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge in any jurisdiction as at the priority date of the application.
[0004] Flaviviruses like West Nile and dengue viruses have established a stronghold in many parts of the tropical and sub-tropical countries world wide. They are one of the most important re-emerging diseases. These viruses are transmitted by mosquito vectors and with global warming, the potential territorial expansion for the spread of these diseases are real. Dengue viruses affects hundreds of millions people each year. To date there are still no vaccines nor anti-virals for human use for either virus. In previous studies, we have demonstrated that the flavivirus envelope (E) domain III (E :DIII) is highly immunogenic and is able to induce potent neutralising antibodies [1-5]. Efficient vaccines are those inducing a robust neutralizing immune response. Presentation of epitopes inducing virus neutralisation (NA) is obviously optimal in whole virus vaccines. Virus-like particles (VLPs) mimicking the natural virions are an advantageous alternative since they are not infectious [6]. This is best illustrated by the VLP -based strategy against the human papilloma virus (HPV), recently approved for human vaccination against HPV infections and HPV-associated cervical lesions which can be cancerous in nature [7,8].
[0005] VLPs are highly organised particulate structures which differ in nature and composition with the virus type, but shared the common feature of being devoid of viral genome, (i) VLPs solely made up of viral capsid proteins can be produced with e.g. HPV, as evoked above, or with hepatitis E virus (HEV) [9]. (ii) On the other hand, VLPs solely consisting of viral envelope glycoproteins have been isolated, and their use as vaccines is envisaged. This is the case for the subviral particles formed by hepatitis B virus (HBV) envelope glycoprotein S, or by chimeric HBV and hepatitis C virus (HCV) envelope proteins [10,11], and influenza virus [12]. (iii) VLPs based on retroviruses or lentiviruses, e.g. human immunodeficiency virus (HIV), represent another type of subviral structure. They are membrane-enveloped particles made up of a backbone of viral polyprotein Gag (or Gag precursor), wrapped by an envelope derived from the host cell plasma membrane.
[0006] The HIV-1 Gag precursor (Pr55Gag) spontaneously assembles into VLPs which bud at the plasma membrane of Gag-expressing cells, e.g. recombinant baculovirus-infected cells, and which are structurally similar to immature virions except that they do not contain any viral genetic material [13-19]. During their budding process, membrane-enveloped viruses bring with them part of the plasma membrane of the producing cells, and some host cell membrane glycoproteins foreign to the virus [20]. Likewise, deletion of the gene encoding the viral envelope protein and substitution by another type of membrane-targeted protein lead to virus pseudotypes. Such pseudotypes have been described in various enveloped viruses, e.g. retroviruses, lentiviruses, vesicular stomatitis virus (VSV), HCV and baculovirus [21- 26].
[0007] One publication describing the construction of the CD16-RIgE clone is by Clemenceau et al. [41]. In this paper, the group made a lentivirus to express the CD16-RIgE protein in B-cells.
SUMMARY
[0008] In a first aspect, the present invention relates to a fusion protein
comprising (i) a CD 16 signal peptide; (ii) an antigenic peptide region downstream of the CD 16 signal peptide; and (iii) an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
[0009] In a second aspect, the present invention relates to a nucleic acid molecule encoding a promoter operably linked to (i) a sequence encoding the fusion protein as described herein; or (ii) a sequence encoding a CD 16 signal peptide upstream of a cloning site and a sequence encoding an IgE receptor transmembrane domain and cytoplasmic tail downstream of the cloning site.
[0010] In a third aspect, the present invention relates to a method of forming a pseudotype virus-like particle, the method comprising coexpressing in a cell (i) a fusion protein as described herein; and (ii) a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein.
[0011] In a fourth aspect, the present invention relates to a pseudotype virus-like particle comprising (i) protein core comprising a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein; (ii) a membrane envelope surrounding the protein core; and (iii) a fusion protein incorporated into the membrane envelope, the fusion protein comprising an antigenic peptide region and an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
[0012] In a fifth aspect, the present invention relates to a vaccine comprising a pseudotype virus-like particle as described herein.
[0013] In a sixth aspect, the present invention relates to use of a vaccine of the fifth aspect for vaccination of a human or animal.
[0014] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] In the figures, which illustrate, by way of example only, embodiments of the present invention:
[0016] Figure 1 is a depiction of a VLP-display of the flavivirus antigenic E :DIII domain. (A), Amino acid sequence of the CD16-RIgE cloning platform. The CD16-RIgE is a chimeric molecule composed of the extracellular domain of CD 16 and the transmembrane domain (TM) and cytoplasmic tail (CT) of the gamma chain of the human high affinity IgE receptor (FceRIy), abbreviated RIgE in the present study. The symbols for the different domains of the amino acid (aa) sequence are the following : signal peptide (amino acid residues 1-22); boxed, ectodomain of CD 16 (aa 23-209); underlined with dotted line, TM domain (aa 210-231); underlined with solid line, CT domain (aa 232-273); shaded residues, deletion (aa 23-206) of the CD 16 ectodomain, replaced by the flavivirus envelope Dili domain. (B), Schematic diagram of the construction of the E:DIII-RIgE chimera (left), and display on the VLP surface (right) by co-expression with HIV-1 Pr55Gag in Sf9 cells doubly infected with baculovirus recombinants AcMPV-DIII-RIgE and AcMNPV-Gag.
[0017] Figure 2 shows coexpression of HIV-1 Pr55Gag and CD16-RIgE in Sf9 cells. Sf9 cells were infected with AcMNPV-Gag alone (left lane) or with AcMNPV- Gag and AcMPV-CD16-RIgE (right lane), at equal MOI each (10 pfu/cell), and harvested at 48 h pi. Whole cell extracts were analysed by SDS-PAGE and Western blotting, using dual color detection: (i) anti-Gag rabbit polyclonal antibody (Ab) and peroxidase-labeled anti-rabbit IgG Ab, and (ii) mouse monoclonal anti-CD 16 antibody and phosphatase-labeled anti-mouse IgG Ab. All Gag products are indicated. The HIV-1 Gag polyprotein precursor is visible as a band at 55 kDa (Pr55Gag), the chimeric CD16-RIgE protein as a band at 75 kDa. m, molecular weight markers.
[0018] Figure 3 shows the pseudotyping of VLP. (A), Western blot analysis. HIV-1 Gag VLPs were produced by Sf9 insect cells and pseudotyped with CD 16- RlgE alone (i), VSV-G alone (ii) or double VSV-G- and CD16-RIgE-pseudotyped VLPs (iii; rightmost lane). (B), Electron microscopy, (i) double VSV-G- and CD 16- RlgE-pseudotyped VLPs, and (inset, ii) nonpseudotyped VLPs. Note the difference between the 'naked' envelope (ii) and 'furry' envelope (i) of VLPs.
[0019] Figure 4 shows the confocal IF microscopy of human cells expressing the E:DIII-RIgE fusion proteins. HEK-293 cells were transfected with pcDNA3 expressing the fusion DIII-WNWe-RIgE, and analysed by fluorescence imaging at 48 h posttransfection without cell permeabilization. Plasma membrane was stained with red-fluorescent WGA, nuclei with blue-fluorescent Hoechst 3342, and DIII-WNKu- RlgE was immunodetected with monoclonal anti-His6 tag antibody and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody. (A), Control cells transduced with empty pcDNA3 plasmid. (B), Cells transduced with pcDN A3 -DIII-WNWe-RIgE. Panels (i) show views from the apical pole; panels (ii) show the corresponding sagittal cell plane reconstructed from the Z-stack images. Note that the DIII-WNWe-RIgE proteins were accessible at the surface of the nonpermeabilized cells, and that they localized in membrane microdomains different from the WGA-reacting regions.
[0020] Figure 5 shows the expression of E:DIII-RIgE fusion proteins in insect cells. (A), Western blot analysis. Sf9 were mock-infected (control lane 1), infected with parental baculovirus vector (control lane 2), or recombinant AcMNPV expressing CD16-RIgE (control lane 3), DIII-DENj-RIgE (lane 4), DIII-WNKu-RIgE (lane 5), and DIII-WNWe-RIgE (lane 6), respectively, and harvested at 48 h pi. The 22-23 kDa band of the envelope E:DIII-RIgE fusion proteins was detected using monoclonal anti-oligoHis tag antibody and complementary phosphatase-labeled antibody. (B), Conventional IF microscopy. Nonpermeabilized Sf9 cells were infected with AcMNPV expressing (i) CD16-RIgE (control, non-tagged fusion protein), (ii) DIII-DENrRIgE, (iii) DIII-WNWe-RIgE, (iv), DIII-WNKu-RIgE. Cells were harvested at 48 h pi and reacted with monoclonal anti-His6 tag antibody and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody. (C), Confocal IF image of DIII-WNWe-RIgE-expressing Sf9 cells showing that the fluorescent signal of the oligoHis tag mainly localized at the cell surface and outlined the cell contour.
[0021] Figure 6 shows the protein analysis of E:DIII-pseudotyped VLPs. VLPs were isolated from Sf9 cells expressing HIV-1 Pr55Gag alone (control VLPs, left side of the blots) or co-expressing Pr55Gag and flavivirus DIII-RIgE fusion construct (right side of the blots). (A), WNKu; (B), DENi . VLPs were purified by .
ultracentrifugation in sucrose-D20 gradient and analyzed by SDS-PAGE and Western blotting. The Gag polyprotein, which constituted the structural backbone of the VLPs, migrated as a major protein species with an apparent MW of 55 kDa (Pr55Gag), the DIII-DENrRIgE and DIII-WNKu-RIgE as a 23 kDa and 22 kDa proteins, respectively. The 23-22 kDa bands were absent from control VLPs. Note the difference in the intensity of Gag and DIII-DENrRIgE and DIII-WNKu-RIgE signals, which had two explanations: (i) Gag was detected using a polyclonal antibody, whereas DIII-DENrRIgE and DIII-WNKu-RIgE were detected with monoclonal anti-His6 tag antibody; (ii) in retroviral particles, 4,000 to 5,000 copies of Gag constitute the protein core, versus only 60 copies of envelope glycoproteins are inserted in the viral envelope. (C), The fractions from sucrose-D20 gradient corresponding to density 1.10-1.20 and containing the VLPs were pooled, further purified and concentrated by pelleting through a sucrose cushion, and analyzed as above. The dual color blot was first reacted with polyclonal antibody against Pr55Gag and secondary peroxidase-labeled anti-rabbit IgG, which revealed the Gag
polyproteins, then with monoclonal ariti-His6 tag antibody and secondary
phosphatase-labeled anti-mouse IgG, which revealed the E:DIII-RIgE fusion proteins, m, molecular weight markers.
[0022] Figure 7 shows the immuno-EM analysis of E :DIII-pseudotyped VLPs. VLPs released from Sf9 cells co-expressing Pr55Gag and flavivirus DIII-RIgE fusion protein, were isolated by ultracentrifugation. Aliquots were adsorbed on EM grids and reacted with monoclonal anti-His6 tag antibody, and secondary 10-nm colloidal gold- labeled anti-mouse IgG. (a, b), DEN]; (c, d), WNKu. Specimens were negatively stained with sodium phosphotungstate. Note that all visible gold grains are found to be associated with membrane-enveloped VLPs of 120-130 nm in diameter [16].
[0023] Figure 8 shows immunological analysis of specificity and 3D
conformation of VLP-displayed E:DIII. Aliquots of control VLP samples (VLP- void), VLP pseudotyped with DIII-DENi-RIgE (VLP-DEN1), or with DIII-WNKu- RlgE (VLP -WNKu) were coated on plates and probed by ELISA for reactivity with patient sera positive for (A) Dengue virus serotype 1, or (B) WNV Kunjin strain virus infection.
[0024] Figure 9 is a graph that shows quantitative evaluation by ELISA of antibodies in sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) on their surface (VLP-DEN1), or with control, nonpseudotyped VLP (VLP-0). The different dilutions of mouse sera were tested against Dengue virus serotype 1 virions adsorbed on the wells. The different symbols indicate the sera from individual animals.
[0025] Figure 10 is a graph that shows quantitative evaluation by ELISA of antibodies in sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) at their surface (VLP-Kun), or with control, nonpseudotyped VLP (VLP-0). The different dilutions of mouse sera were tested against WNKu virions adsorbed on the wells. The different symbols indicate the sera from individual animals. [0026] Figure 11 is a graph that shows antibody specificity by ELISA. Different dilutions of sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) at their surface (VLP-DEN1), were tested against virions of Dengue virus serotypes 1, 2, 3, 4, and West Nile virus strain Kunjin, adsorbed on the wells. Results presented are the mean of triplicate values obtained with sera from 5 different animals ± standard deviation (SD).
[0027] Figure 12 is a graph that shows antibody specificity by ELISA. Different dilutions of sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) on their surface (VLP-Kun), were tested against virions of West Nile virus strain Kunjin, Dengue virus serotypes 1, 2, 3 and 4 adsorbed on the wells. Results presented are the mean of triplicate values obtained with sera from 5 different animals ± standard deviation (SD).
[0028] Figure 13 is a graph that shows evaluation of antibodies against DENV serotype 1 by homologous neutralization (NA) assays. Sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) at their surface (VLP-DEN1), were diluted to 1 :10 and tested against live virions of Dengue virus serotypes 1 in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three
determinations ± SD).
[0029] Figure 14 is a graph that shows evaluation of antibodies against West Nile virus Kunjin by homologous neutralization (NA) assays. Sera from mice immunised with VLP displaying the West Nile virus strain Kunjin (WNKu) envelope domain III (E-DIII) at their surface (VLP-Kun), were diluted to 1 : 10 and tested against live virions of WNKu in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD).
[0030] Figure 15 is a graph that shows neutralization (NA) assays against heterologous DENV serotypes. Different dilutions of sera from mice immunised with
VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) on their surface (VLP-DEN1), were tested against live virions of Dengue virus serotype 2 in
PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD). [0031] Figure 16 is a graph that shows neutralization (NA) assays against heterologous DENV serotypes. Different dilutions of sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) on their surface (VLP-DEN1), were tested against live virions of Dengue virus serotype 3 in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD).
[0032] Figure 17 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses. Different dilutions of sera from mice immunised with VLP displaying the Dengue virus serotype 1 envelope domain III (E-DIII) at their surface (VLP-DEN1), were tested against live virions of West Nile virus strain Kunjin (WNKu) in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD).
[0033] Figure 18 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses. Different dilutions of sera from mice immunised with VLP displaying the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 1 in PRNT assays. N A activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD).
[0034] Figure 19 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses. Different dilutions of sera from mice immunised with VLP the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 2 in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD).
[0035] Figure 20 is a graph that shows neutralization (NA) assays against heterologous Flaviviruses. Different dilutions of sera from mice immunised with VLP the envelope domain III (E-DIII) of West Nile virus strain Kunjin on their surface (VLP-Kun) were tested against live virions of Dengue virus serotype 3 in PRNT assays. NA activity was expressed as percentage of virus neutralization, based on plaque determinations (average of three determinations ± SD). DETAILED DESCRIPTION
[0036] The non-replicative nature of VLPs makes them highly attractive candidates for the design of subunit vaccines which carry the immunological structures of the virus in the absence of infectious genetic material. In addition, VLPs can induce both humoral and cellular-mediated immune responses [27]. Considering that mosquitos are intermediate hosts for West Nile virus (WNV) and dengue virus (DENV), the use of the baculovirus-insect cell expression system for production of VLP and WNV and DENV envelope glycoprotein domains represented a logical approach for the development of VLP vaccines against these flaviviruses. Provided herein is a strategy of pseudotyping HIV-based VLPs involving the co-expression in insect cells of retroviral Gag polyprotein and foreign membrane glycoprotein using two recombinant baculovirus vectors. The nonviral glycoprotein pseudotyping platform utilizes a chimeric human surface glycoprotein derived from the CD 16 molecule (abbreviated CD16-RIgE). It was surprisingly found by the present inventors that CD16-RIgE was incorporated into the envelope of extracellular VLPs with a high efficiency. It is shown herein that CD 16 ectodomain can be replaced by the envelope glycoprotein domain III of WNV and DENV, resulting in the exposure of the E:DIII epitopes at the surface of the VLP envelope. Recombinant chimera CD16-RIgE can be used as a general VLP -pseudotyping platform, in coexpression with recombinant retroviral Gag polyprotein, for VLP-display of other viral envelope glycoproteins, or other pathogen proteins and their therapeutic application as potential vaccine vectors.
[0037] In a first aspect, the present invention relates to a fusion protein comprising (i) a CD 16 signal peptide; (ii) an antigenic peptide region downstream of the CD 16 signal peptide; and (iii) an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
[0038] As used herein, "fusion protein" refers to a protein comprising amino acid sequences that are in addition to, in place of, less than, and/or different from the amino acid sequences encoding the original or native full-length protein or subsequences thereof.
[0039] As used herein, "CD 16 signal peptide" is intended to encompass an amino-terminal extension derived from immature CD 16 protein which directs the protein to be secreted. CD 16 signal peptide directs the fusion proteins of the instant inventions to translocate across the cell membrane. CD 16 protein signal peptide may be proteolytically removed by a signal peptidase during or immediately following membrane translocation of the fusion protein.
[0040] As used herein, "antigenic peptide" refers to a peptide which elicits an immunological response in a subject. Usually, the "immunological response" includes, but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells and/or gamma-delta T cells, and/or virus neutralizing antibodies directed specifically to an antigen or antigens included in the composition or vaccine of interest. Preferably, the host will display either a therapeutic or protective
immunological response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced. Such protection will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and/or a lowered duration of clinical disease or higher viral antibody titer in the tissues or body fluids or excretions of the infected host, or lessened viremia in the blood, or lessened gross or histopathological lesions due to infection.
[0041] As used herein, the term "IgE receptor transmembrane domain" is intended to encompass that a portion of an immunoglobulin E (IgE) receptor protein that resides primarily in a membrane, and the term "IgE cytoplasmic tail" is intended to encompass that portion of an IgE receptor protein that resides within the bounds of a cell, virus, VLP or other membrane-bound body.
[0042] In one embodiment, the antigenic peptide region is not CD 16 ectodomain. As used herein, the term "ectodomain" is intended to encompass that portion of a protein which is located on the outer surface of a cell. For example, the ectodomain of the transmembrane protein CD 16 is that portion of the CD 16 protein which extends from a cell's outer surface into the extracellular space.
[0043] The fusion protein may be suitable for use in forming a pseudotype viruslike particle (VLP).
[0044] In a further embodiment, the antigenic peptide region comprises an antigen useful for vaccination of a human or animal.
[0045] In a further embodiment, the antigenic peptide region comprises a viral, parasitic or bacterial antigen.
[0046] Viral antigens may be from RNA or DNA viruses. Examples of RNA viruses from which a viral antigen may be obtained include, but are limited to, those in the following RNA virus families:
[0047] Arenaviridae, such as lymphcytic choriomeningitis virus (LCM), Lassa virus, Junin, Tacaribe, Pichinde viruses, Machupo virus, and Guanito virus;
Bornaviridae, such as Borna disease virus; Bunyaviradae, such as Hanta virus, California encephalitis virus, Japanese encephalitis virus, LaCrosse virus, Rift Valley fever virus, Bunyavirus, Arbovirus, Nairobi sheep disease virus, Phlebovirus, and Tospoviruses; Caliciviridae, such as Human and animal calici viruses; Coronaviridae, such as SARS Coronavirus; Filoviridae, such as Ebola virus and Marburg virus; Flaviviridae, such as Yellow Fever virus, Dengue Fever virus, West Nile virus, Hepatitis C virus, Pestiviruses, Bovine Viral Diarrhea virus, and Classical Swine Fever virus (and others as indicated below); Nodaviridae, such as Nodaviruses;
Orthomyxoviridae, such as Influenza virus type A, Influenza virus type B, Influenza virus type C, Thogotovirus, and Fowl Plague disease virus; Paramyxoviridae, such as Parainfluenza viruses, Mumps virus, Measles virus, Subacute sclerosing
panencephalitis (SSPE) virus, Respiratory syncytial virus (RSV), Pneumo viruses, "TPMV-like viruses", Newcastle Disease virus, Rinderpest virus, and Canine
Distemper virus; Picornaviridae, such as Human Enteroviruses, including Poliovirus; Coxsackie virus A, Coxsackie virus B, Hepatitis A virus, and Rhinoviruses, Foot and Mouth Disease virus, Enterovirus 70, Apthoviruses, and Cardioviruses; Reoviridae, such as Colorado Tick fever virus, Rotaviruses, Reoviruses, Coltivirus and
Orbiviruses; Retroviridae, such as Human immunodefficiency virus (HIV), Human T- lymphotrophic virus (HTLV), Feline Leukemia virus (FeLV), Friend Leukemia virus (FLV), and MMTV (Mouse Mammary Tumor virus); Rhabdoviridae, such as Rabies virus, and Vesicular Stomatitis virus; and Togaviridae, such as Eastern Equine Encephalitis virus, Western Equine Encephalitis virus, Rubella virus (measles), Alphaviruses, Ross River virus, Astroviruses, Norwalk-like viruses, Hepatitis D and E viruses, Nipah virus, LR1 virus and Benyviruses. [0048] More particularly, viruses in the Flaviviridae from which a viral antigen may be obtained include, for example, those in the genera Flavivirus and Pestivirus, the "Hepatitis C-like viruses", and those in the Yellow fever virus group, Tick-borne encephalitis virus group, Rio Bravo group, Japanese encephalitis group, Tyuleniy group, Ntaya group, Uganda S group, Dengue group, and Modoc group. More specifically, the viruses of the Flaviviridae which may be used in the present invention include, for example, but are not limited to, Gadgets Gully virus, Kyasanur Forest disease virus, Langat virus, including the British, Irish, Louping ill, Spanish and Turkish subtypes, Omsk hemorrhagic fever virus, Powassan virus, Karshi virus, Royal Farm virus, Tick-borne encephalitis virus, including the European, Far Eastern, and Siberian subtypes, Kadam virus, Meaban virus, Saumarez Reef virus, Tyuleniy virus, Aroa virus, Bussuquara virus, Iguape virus, Naranjal virus, Dengue virus 1, Dengue virus 2, Dengue virus 3, Dengue virus 4, Kedougou virus, Cacipacore virus, Japanese encephalitis virus, Koutango virus, Alfuy virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, Usutu virus, Kunjin virus, West Nile virus, Yaounde virus, Kokobera virus, Stratford virus, Bagaza virus, Ilheus virus, Rocio virus, Israel turkey meningoencephalomyelitis virus, Ntaya virus, Tembusu virus, Spondweni virus, Zika virus, Banzi virus, Bouboui virus, Edge Hill virus, Jugra virus, Potiskum virus, Saboya virus, Sepik virus, Uganda S virus, Wesselsbron virus, Yellow fever virus, Entebbe bat virus, Sokoluk virus, Yokose virus, Apoi virus, Cowbone Ridge virus, Jutiapa virus, Modoc virus, Sal Vieja virus, San Perlita virus, Bukalasa bat virus, Carey Island virus, Dakar bat virus, Montana myotis
leukoencephalitis virus, Batu Cave virus, Phnom Penh bat virus, Rio Bravo virus, Cell fusing agent virus, Tamana bat virus, Border disease virus— BD31, Border disease virus— X818, Bovine viral diarrhea virus 1-CP7, Bovine viral diarrhea virus 1-NADL, Bovine viral diarrhea virus 1-Osloss, Bovine viral diarrhea virus 1-SDl, Bovine viral diarrhea virus 2-C413, Bovine viral diarrhea virus 2-New York '93, Bovine viral diarrhea virus 2-strain 890, Classical swine fever virus— Alfort/187, Classical swine fever virus— Alfort-Tubingen, Classical swine fever virus—Brescia, Classical swine fever virus— C, Pestivirus of giraffe, Hepatitis C virus, including genotype 10, genotype 11, genotype la, genotype lb, genotype 2a, genotype 2b, genotype 3a, genotype 4a, genotype 5 a, genotype 6a, and GB virus B, GB virus A, GB virus C, Hepatitis G virus- 1, Dengue virus, Yellow fever virus, St. Louis encephalitis virus, Japanese encephalitis virus, Murray Valley encephalitis virus, West Nile virus, Rocio virus, Tick-borne encephalitis virus, Omsk hemorrhagic fever virus, Kyasunur Forest disease virus, Powassan virus, Pestiviruses, and Hepatitis C virus.
[0049] Examples of DNA viruses from which a viral antigen may be obtained include, but are limited to, those in the following DNA virus families:
[0050] Herpesviridae, such as Herpes Simplex virus, cytomegalovirus, Epstein- Barr virus, Pseudorabies virus and Human Herpes virus 6; Adenovirus; Parvoviridae, such as Human parvovirus B19; Papillomaviridae, such as Human Papillomavirus; and Polyomaviridae, such as JC polyomavirus and BK polyomavirus; Poxviridae, such as Variola virus, Monkeypox virus and Myoma virus.
[0051 ] Examples of parasites from which a parasite antigen may be obtained include, but are limited to, those in the following parasite families:
[0052] Haemonchus, Trichostrongylus, Ostertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophagostomum, Chabertia, Trichuris, Strongylus, Trichonema, Dictyocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostoma, Uncinaria, Toxascaris, Parascaris, Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, Enterobius, Wuchereria, Brugia and Onchocerca.
[0053] More particularly, examples of parasites from which a parasite antigen may be obtained include, but are limited to, the following:
[0054] Plasmodium falciparum, P. vivax, P. ovale, P. malaria; Toxoplasma gondii; Leishmania mexicana, L. tropica, L. major, L. aethiopica, L. donovani, Trypanosoma cruzi, T. brucei, Schistosoma mansoni, S. haematobium, S. japonium; Trichinella spiralis; Wuchereria bancrofti; Brugia malayli; Entamoeba histolytica; Enterobius vermiculoarus; Taenia solium, T. saginata, Trichomonas vaginatis, T. hominis, T. tenax; Giardia lamblia; Babesia bovis, B. divergens, B. microti, Isospore belli, L hominis; Dientamoeba fragiles; Onchocerca volvulus; Ascaris lumbricoides; Necator americanis; Ancylostoma duodenale; Strongyloides stercoralis; Capillaria philippinensis; Angiostrongylus cantonensis; Hymenolepis nana; Diphyllobothrium latum; Echinococcus granulosus, E. multilocularis; Paragonimus westermani, P. caliensis; Chlonorchis sinensis; Opisthorchis felineas, G. Viverini, Fasciola hepatica Sarcoptes scabiei, Pediculus humanus; Phthirius pubis; and Dermatobia hominis.
[0055] Examples of bacteria from which a bacterial antigen may be obtained include, but are limited to, the following:
[0056] Bacteria in general include but are not limited to: P. aeruginosa; E. coli, Klebsiella sp.; Serratia sp.; Pseudomanas sp.; P. cepacia; Acinetobacter sp.; S.
epidermis; E. faecalis; S. pneumonias; S. aureus; Haemophilus sp.; Neisseria Sp.; N. meningitidis; Bacteroides sp.; Citrobacter sp.; Branhamella sp.; Salmonella sp.;
Shigella sp.; S. pyogenes; Proteus sp.; Clostridium sp.; Erysipelothrix sp.; Listeria sp.; Pasteurella multocida; Streptobacillus sp.; Spirillum sp.; Fusospirocheta sp.;
Treponema pallidum; Borrelia sp.; Actinomycetes; Mycoplasma sp.; Chlamydia sp.; Rickettsia sp.; Spirochaeta; Legionella sp.; Mycobacteria sp.; Ureaplasma sp.;
Streptomyces sp.; Trichomoras sp.; and P. mirabilis.
[0057] In a further embodiment, the antigenic peptide region comprises a Dili domain from a flavivirus envelope glycoprotein.
[0058] In a particular embodiment, the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus or Dengue virus.
[0059] Domain III (Dili) of a flavivirus envelope glycoprotein is an
immunoglobulin (Ig)-like domain. For example, Dili of Dengue virus envelope glycoprotein consists of 100 amino acids (residues 303-395) of the C-terminus. This domain has been suggested to be the receptor recognition and binding domain due to a number of reasons. The Ig-like fold present in the Dili protein is commonly associated with structures that have an adhesion function. This domain extends perpendicularly to the surface of the virus with a tip projecting further from the virion surface than any part of the envelope glycoprotein. In addition, studies have demonstrated that both recombinant Dill proteins and antibodies generated against Dili of E protein of flavivirus can inhibit entry of the flavivirus into target cells. In addition, flavivirus with mutations in Dili of the envelope glycoprotein shows either attenuated virulence or the ability to escape immune neutralization [ 1 ] .
[0060] Exemplary strains of West Nile virus include, but are not limited to, West Nile B956, Madagascar 1978, Cyprus 1968, Kunjin 1960, Kunjin 1991, Wengler, Goose Israel 1998, New York 1999, Kenya, Uganda, Senegal 1990, Uganda 1937, Uganda 1959, Central African Republicl 972a, Central African Republic 1972b, Central African Republic 1983, Madagascar 1986 and Madagascar 1988.
[0061] In a particular embodiment, the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus strain Wengler or Kunjin or of Dengue virus serotype 1, 2, 3 or 4.
[0062] In a second aspect, the present invention relates to a nucleic acid molecule encoding a promoter operably linked to (i) a sequence encoding the fusion protein as described herein; or (ii) a sequence encoding a CD 16 signal peptide upstream of a cloning site and a sequence encoding an IgE receptor transmembrane domain and cytoplasmic tail downstream of the cloning site.
[0063] As used herein, the term "nucleic acid molecule," is intended to encompass DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is
"operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
[0064] As used herein, the term "promoter" is intended to encompass a
recognition site of a DNA strand to which the RNA polymerase binds. The promoter forms an initiation complex with RNA polymerase to initiate and drive transcriptional activity. Non-limiting examples of promoters include, for example, constitutive, inducible, tissue-specific and tissue-preferred promoters. Conditions that may induce transcription from an inducible promoter include, for example, any chemicals or compounds, temperature, light wavelength or level. The initiation complex can be modified by activating sequences termed "enhancers" or inhibitory sequences termed "silencers".
[0065] As used herein, the term "cloning site" is intended to encompass a region which allows for the insertion of desired nucleic acid sequences. Typically, the cloning site comprises one or more restriction endonuclease recognition sites. Cloning sites include, but are not limited to, multiple cloning sites or polylinkers. [0066] In one embodiment, the nucleic acid molecule is an expression vector. As used herein, the term "expression vector" is intended to encompass a nucleic acid molecule, preferably a double-stranded DNA molecule, comprising a gene that is expressed in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue- specific regulatory elements and enhancers. These vectors can be designed, using known methods, to contain the elements necessary for directing transcription, translation, or both, of the nucleic acid in a cell to which it is delivered. Known methodology can be used to generate expression constructs that have a protein-coding sequence operably linked with appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques and synthetic techniques. For example, see Sambrook et al. ((2001) Molecular Cloning: a
Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press).
[0067] A variety of expression vector/host systems may be utilized to contain and express the nucleic acid encoding the fusion protein of the present invention. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS.cells (such as COS-7), W138, BHK, HepG2, 3T3, RTN, MDCK, A549, PC 12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the protein are described herein below.
[0068] The secreted peptide is purified from the growth medium by, e.g., the methods used to purify the fusion protein from bacterial and mammalian cell supernatants.
[0069] In a particular embodiment, the nucleic acid is a baculoviral expression vector, wherein the promoter is a promoter that is expressed in insect cells. For example, the cDNA encoding the peptide may be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, Calif.). This vector is then used according to the manufacturer's directions (PharMingen) to infect Spodoptera frugiperda cells in sF9 protein-free media and to produce recombinant protein. The fusion protein may be purified and concentrated from the media using a heparin- Sepharose column (Pharmacia, Piscataway, N.J.) and sequential molecular sizing columns (Amicon, Beverly, Mass.), and resuspended in PBS. SDS-PAGE analysis shows a single band and confirms the size of the fusion protein, and Edman sequencing on a Porton 2090 Peptide Sequencer confirms its N-terminal sequence.
[0070] In a third aspect, the present invention relates to a method of forming a pseudotype virus-like particle, the method comprising coexpressing in a cell (i) a fusion protein as described herein; and (ii) a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein.
[0071] As used herein, "retroviral Gag polyprotein" and "protein precursor of a retroviral Gag polyprotein" are intended to encompass retroviral group specific antigens that constitute retroviral structural proteins.
[0072] In one embodiment, the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SrV), or a chimeric human-animal retroviral Gag polyprotein. As used herein, "HIV- 1 Pr55Gag" is intended to encompass a polyprotein complex comprising pi 7 (matrix) protein, p24 (capsid) protein, p2 (Spl) protein, p7 (nucleocapsid) protein, pi (Sp2) protein, and p6 protein of human immunodeficiency virus- 1 (HIV-1).
[0073] Proteins, polypeptides, and nucleic acids referred to herein are intended to encompass homologues, derivatives, variants, or fragments thereof. A nucleotide sequence or polypeptide sequence is a "homologue" of, or is "homologous" to, another sequence if the two sequences have substantial identity over a specified region and the functional activity of the sequences is conserved (as used herein, the term 'homologous' does not infer evolutionary relatedness). Two polynucleotide sequences or polypeptide sequences are considered to have substantial identity if, when optimally aligned (with gaps permitted), they share at least about 50% sequence identity, or if the sequences share defined functional motifs. In alternative embodiments, optimally aligned sequences may be considered to be substantially identical (i.e. to have substantial identity) if they share at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%), 97%, 98%, 99 identity over a specified region. An "unrelated" or "non-homologous" sequence shares less than 40% identity, though preferably less than about 25 % identity, with a polypeptide or polynucleotide of the invention over a specified region of homology. The terms "identity" and "identical" refer to sequence similarity between two peptides or two polynucleotide molecules. Identity can be determined by comparing each position in the aligned sequences. A degree of identity between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences, i.e. over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the ClustalW program, available at htt ://clustalw. genome.ad.jp, the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al, 1990, J. Mol. Biol 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at http://www.ncbi.nlm.nih.gov/').
[0074] A variant or derivative polypeptide refers to a polypeptide that has been mutated at one or more amino acids, including point, insertion or deletion mutation, but still retains the desired function(s), as well as non-peptides and peptide mimetics which possess the ability to mimic the desired function of the polypeptide. A variant or derivative therefore includes deletions, including truncations and fragments; insertions and additions, including tagged polypeptides and fusion proteins; substitutions, for example conservative substitutions, site-directed mutants and allelic variants; and modifications, including peptoids having one or more non-amino acyl groups (q.v., sugar, lipid, etc.) covalently linked to the peptide and post-translational modifications. As used herein, the term "conserved amino acid substitutions" or "conservative substitutions" refers to the substitution of one amino acid for another at a given location in the peptide, where the substitution can be made without substantial loss of the relevant function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their , size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing. Conservative changes can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, for example, by reaction of a functional side group of an amino acid.
[0075] Variants and derivatives can be prepared, for example, by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of a polypeptide or fragment thereof, and screening for biological activity. Preferably, substitutions are made with conservative amino acid residues, i.e., residues having similar physical, biological or chemical properties. A skilled person will understand how to make such derivatives or variants, using standard molecular biology techniques and methods, described for example in Sambrook et al. ((2001) Molecular Cloning: a Laboratory Manual, 3rd ed., Cold Spring Harbour Laboratory Press), and how to test such derivatives or variants for their desired function, including using techniques as described in the Examples set out herein.
[0076] As used herein, "consists essentially of or "consisting essentially of' means that the sequence includes one or more amino acids at one or both ends of the described sequence, but that the additional amino acids do not materially affect the function of the polypeptide. For example, the polypeptide consisting essentially of a particular sequence may have one, two, three, five or ten amino acids at one or both ends of the described sequence, provided that such a protein still possesses the desired function.
[0077] In a fourth aspect, the present invention relates to a pseudotype virus-like particle comprising (i) protein core comprising a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein; (ii) a membrane envelope surrounding the protein core; and (iii) a fusion protein incorporated into the membrane envelope, the fusion protein comprising an antigenic peptide region and an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
[0078] As used herein, "virus-like particle" ("VLP") is intended to encompass a highly organised particulate structure which differs in nature and composition from the virus type, for example, by being devoid of viral genome and being non- replicative. In one embodiment, VLPs may be comprised of viral capsid proteins, for example from HPV or hepatitis E virus (HEV). In a further embodiment, VLPs may be comprised of viral envelope glycoproteins, for example from hepatitis B virus (HBV) envelope glycoprotein S, or by chimeric HBV and hepatitis C virus (HCV) envelope proteins, or influenza virus. In yet a further embodiment, VLPs may be based on retroviruses or lentiviruses, for example, human immunodeficiency virus (HIV), and may be membrane-enveloped particles made up of a backbone of viral polyprotein Gag (or Gag precursor), wrapped by an envelope derived from the host cell plasma membrane. In a particular embodiment, the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SIV), or a chimeric human-animal retroviral Gag polyprotein.
[0079] As used herein, the terms "pseudotype" or "pseudotyping", are intending to encompass a VLP in which at least one viral envelope protein has been substituted with the fusion protein as described herein.
[0080] In a further embodiment, the antigenic peptide region comprises an antigen useful for vaccination of a human or animal.
[0081] In a further embodiment, the antigenic peptide region comprises a viral, parasitic or bacterial antigen, non-limiting examples of which are described herein.
[0082] In a further embodiment, the antigenic peptide region comprises a Dili domain from a flavivirus envelope glycoprotein.
[0083] In a further embodiment, the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus or Dengue virus.
[0084] In a further embodiment, the Dili domain is the Dill domain from the envelope glycoprotein of West Nile virus strains or of Dengue virus serotype 1, 2, 3 or 4.
[0085] In a further embodiment, the pseudotype virus-like particle is formed by the method of the third aspect. [0086] In a fifth aspect, the present invention relates to a vaccine comprising a pseudotype virus-like particle of the fourth aspect.
[0087] The vaccine of the present invention may be in the form of a
pharmaceutical composition, which may comprise one or more of the fusion protein or pseudotype virus-like particle, any additional active substance or substances or one or more additional therapeutic agents.
[0088] The pharmaceutical composition can be prepared by known methods for the preparation of pharmaceutically acceptable compositions suitable for vaccination of a human or animal, such that an effective quantity of the fusion protein or pseudotype virus-like particle and any additional active substance or substances is combined in a mixture with a pharmaceutically acceptable vehicle, carrier, excipient or diluent. Suitable vehicles, carriers, excipients and diluents are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, albeit not exclusively, solutions or suspensions of the fusion proteins or pseudotype virus-like particles, in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffer solutions with a suitable pH and iso-osmotic with physiological fluids.
[0089] The pharmaceutical composition may be prepared in a physiologically and pharmacologically suitable buffer. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms, and that will maintain the live state of the cells. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences and in The United States Pharmacopeia: The National Formulary (USP 24 NF 19) published in 1999.
[0090] The pharmaceutical composition may further comprise one or more adjuvants. Suitable adjuvants include, but are not limited to, aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc.,
Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), non- metabolizable oil, mineral and/or plant/vegetable and/or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in- water emulsion. The emulsion can be based in particular on light liquid paraffin oil
(European Pharmacopeia type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate/caprate), glyceryl tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifers to form the emulsion. The emulsifers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products, especially L 121. See Hunter et al, The Theory and Practical Application of Adjuvants (Ed.Stewart-Tull, D. E. S.). John Wiley and Sons, NY, pp51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997).
[0091] In a sixth aspect, the present invention relates to use of a vaccine of the fifth aspect for vaccination of a human or animal.
[0092] Accordingly, the fusion proteins, pseudotype virus-like particles and vaccines of the present invention are useful for vaccination of a human or animal. Non-limiting examples of animals include livestock such as cattle, horses, goats, sheep, and pigs; companion animals such as dogs and cats; and domesticated fowl such as chickens, ducks and geese. As used herein, "useful for vaccination of a human or animal" is intended to encompass eliciting an "immunological response", as defined herein.
[0093] Eliciting an immunological response is intended to encompass treating or preventing a disease or condition to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization of the state of disease, prevention of development of disease, prevention of spread of disease, delay or slowing of disease progression, delay or slowing of disease onset, amelioration or palliation of the disease state, and remission (whether partial or total). "Treating" can also mean prolonging survival of a subject beyond that expected in the absence of treatment. "Treating" can also mean inhibiting the progression of disease, slowing the progression of disease temporarily, although more preferably, it involves halting the progression of the disease permanently.
[0094] The pharmaceutical composition of the present invention can be administered in any conventional manner. Examples of administration methods include any that afford access by cells of the immune system to the pharmaceutical composition including parenteral, oral, transdermal, intradermal, intravenous, subcutaneous, intramuscular, intraocular, intraperitoneal, intrarectal, intravaginal, intranasal, intragastrical, intratracheal and intrapulmonarial, or any combination thereof.
[0095] An effective amount of the pharmaceutical composition is administered to the human or animal. The term "effective amount" as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result, for example, to induce protective immunity against a pathogen in the human or animal.
[0096] The dose and frequency of administration of the pharmaceutical composition that is to be used depends on the particular disease or condition being treated or prevented, the severity of the disease or condition, individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of
administration and other similar factors that are within the knowledge and expertise of the health practitioner. These factors are known to those of skill in the art and can be addressed with minimal routine experimentation.
[0097] The present invention is further exemplified by way of the following non- limiting examples.
EXAMPLES
[0098] EXAMPLE 1
[0099] Materials and Methods [00100] Cells, (i) Insect cells. Spodoptera frugiperda Sf9 cells were maintained as monolayers at 28 °C in Grace's insect medium supplemented with 10 % fetal bovine serum (FBS) and antibiotics (Invitrogen). They were infected with
recombinant baculovirus at a multiplicity of infection (MOI) ranging from 5 to 10 PFU/cell, as previously described [15,17,18,28-32]. In co-expression experiments, Sf9 were infected with two recombinant baculoviruses simultaneously at equal MOI [32- 34]. (ii) Mammalian cells. Human embryonic kidney cells (HEK-293) were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and grown in Iscove's medium supplemented with 10 % FBS and 50 mg/ml gentamicin
(Invitrogen).
[00101] Recombinant baculovirus expressing VSV-G , HIV-1 Gag, and VLP production, (i) AcMNPV-VSV-G and AcMNPV-Gag. All foreign genes were inserted into the genome of Autographa californica MultiCapsid NucleoPolyhedrosis Virus (AcMNPV) under the control of a chimeric AcMNPV-GmNPV polyhedrin promoter, as described in previous studies [16-18,30,35]. AcMNPV- VSV-G expressed the attachment glycoprotein G of vesicular stomatitis virus (VSV). AcMNPV-Gag expressed the N-myristoylated full-length Gag polyprotein (Pr55Gag) of HIV-1. (ii) Isolation of extracellular VLP. Sf9 cell culture supernatants were clarified by low- speed centrifugation, then VLP recovered using a two-step procedure comprising sucrose-step gradient centrifugation, followed by ultracentrifugation in linear D20- sucrose gradient [30,34,35]. In brief, (a) VLP were first recovered by pelleting through a cushion of 20 % sucrose in TNE buffer (TNE : 100 mM NaCl, 10 mM Tris- HC1 pH 7.4, 1 mM Na2EDTA) at 30,000 rpm for 1 h at 15 °C in a Kontron TST55.5 rotor. VLP pellets from step (a) were gently resuspended in PBS (0.20-0.25 ml), and (b) further purified by isopycnic ultracentrifugation in sucrose-D20 gradients. Linear gradients (10-ml total volume, 30-50 %, w:v) were centrifuged for 18 h at 28,000 rpm in a Beckman SW41 rotor. The 50 % sucrose solution was made in D20 buffered to pH 7.2 with NaOH, and the 30 % sucrose solution was made in 10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5.7 mM Na2EDTA. Aliquots of 0.4 ml were collected from the top, and proteins analysed by SDS-PAGE, and immunoblot analysis. Protein concentration in samples was determined by Bradford protein assay (Pierce ; Thermo Fisher Scientific Inc., Rockford, IL, USA). (Hi) Titration of VLP. The HIV-1 mature capsid is a fullerene cone composed of approximately 250 hexamers and 12 pentamers of the viral CAp24 protein [36], which accounted for 1,560 copies of CAp24 per core, generated by the proteolytic cleavage of Pr55Gag molecules constituting the immature particle. These data are consistent with the value of 1,500 copies of CAp24 protein per mature core, determined by alternative methods [37,38]. However, less than half of the CAp24 proteins form mature cores, since cryoelectron microscopy and scanning electron microscopy showed that immature HIV-1 particle contains approximately 5,000 copies of closely packed Pr55Gag protein [37,39,40]. Taking this value into account, the VLP titer was estimated by SDS-PAGE analysis of VLP and the intensity of the Pr55Gag signal in Coomassie blue-stained gels, in co- el ectrophoresis with a range of bovine serum albumin samples of known
concentration. A concentration of 1 mg/ml of Pr55Gag protein corresponded to a titer of~ 2 x 10E12 VLP per ml.
[00102] Construction of AcMNPV expressing the chimeric human glycoprotein CD16-RIgE. The plasmid pcDNA3.1/FcyRIIIa/Fc8RlY, expressing the chimeric construct abbreviated CD16-RIgE in was obtained from Henri Vie (INSERM U-609, Nantes; [41]). CD16-RIgE consisted of the fusion of the ectodomain of FcyRIIIa (or CD 16), the human low-affinity receptor for IgG Fc (involved in antibody-dependent cell-mediated cytotoxicity; ADCC), to the transmembrane and cytoplasmic domains of the gamma polypeptide of the human high affinity receptor for IgE, FcsRIy. In terms of functionality, the CD 16 ectodomain was responsible for the recognition of IgG Fc, whereas FceRIy transduced the intracellular signals [41]. The cDNA encoding the CD16-RIgE was then rescued using conventional overlapping PCR method, and inserted into the Nhel and Hindlll sites of the pBlueBac4.5 vector genome (Invitrogen, San Diego, CA). Recombinant AcMNPV-CD16-RIgE were isolated using beta-galactosidase staining of positive Sf9 cells and the blue plaque selection method.
[00103] Dili domain of Flavivirus envelope glycoproteins and construction of recombinant AcMNPV expressing the E :DIII-inserted CD16-RIgE construct. The E :DIII domains of the Dengue virus serotypes 1, 2, 3 and 4 (abbreviated DENj, DEN2, DEN3 and DEN4, respectively), and West Nile Virus strains Sarafend (WNSa), Wengler (WNWe), New York (WNNY) and Kunjin (WNKu) have been cloned into the pET28a vector (Novagen), and expressed in bacterial cells, as described in detail elsewhere [1,2,4,5,42]. All clones contained an oligo-histidine sequence (His6-tag) at their N-terminus. Most of the CD 16 ectodomain was deleted from the
pcDNA3.1/CD16-RIgE, by double cleavage at the Bgl II and EcoR I sites, and substituted with the cDNA fragment of the His6-tagged E :DIII domain from three flavivirus prototypes, DENl5 WNWe and WNKu. The final construct encoded, from the N- to C-terminus, the CD 16 signal peptide, the His6-tag, the E :DIII domain, and the RIgE C-terminal moiety, as depicted in Fig. 1. Each construct was reinserted into the pBlueBac4.5 vector genome, to generate the recombinant baculoviruses
AcMNPV-DENrRIgE, AcMNPV-WNWe-RIgE, and AcMNPV-WN u-RIgE.
[00104] Gel electrophoresis, membrane transfer and antibodies.
Polyacrylamide gel electrophoresis of SDS-denatured protein samples (SDS-PAGE), and immunoblotting analysis have been described in detail in previous studies
[30,34,35]. Briefly, VLP were denatured in SDS/beta-mercaptoethanol-containing loading buffer at 100 °C for 2 min, and proteins were electrophoresed in SDS- denaturing, 10 %-polyacrylamide gel and electrically transferred to nitrocellulose membrane (Hybond™-C-extra; Amersham Biosciences). Blots were blocked in 5 % skimmed milk in Tris-buffered saline (TBS) containing 0.05 % Tween-20 (TBS-T), rinsed in TBS-T, then successively incubated with primary antibody, mouse
monoclonal anti-His6 tag antibody (Tag- 100 antibody; Qiagen SA, Courtaboeuf, France), anti-human CD 16 mouse monoclonal antibody DJ130C (Santa Cruz
Biotechnology) or rabbit anti-Gag antibody. After rinsing with TBS, the blot was incubated with the relevant anti-IgG secondary antibodies, at working dilutions ranging from 1 : 1 ,000 to 1 : 10,000. Anti-HIV- 1 Gag rabbit polyclonal antibody
(laboratory-made ; [35]) was raised in rabbit by injection of bacterially-expressed, GST-fused and affinity-purified carboxy-terminally- truncated Gag protein consisting . of the full-length MA domain and the first seventy-eight residues of the CA domain (Pst I site; gag a sequence). Apparent molecular weights were estimated by
comparison with prestained protein markers (Precison Plus Protein™ Standards, Dual Color; Bio-Rad Laboratories, Inc., Bio-Rad France).
[00105] ELISA. The accessibility and functionality of recombinant E:DIII proteins was evaluated by their binding activity to the monoclonal anti-His6 tag antibody, in standard ELISA procedure. In brief, aliquots (100 μΐ) of VLP suspension (50 μ^ηιΐ ; 10E11 VLP/ml) in coating buffer (0.1M NaHC03, pH 9.6) were incubated overnight at 4 °C in 96-well microtiter plates (NUNC, Roskilde, Denmark). The coated wells were blocked with 200 μΐ of blocking buffer (2 % bovine serum albumin in 0.14 M NaCl, 0.05M Tris-HCl buffer, pH 8.2; TBS-BSA) for 1 h at room temperature (RT), then washed five times with washing buffer (0.05 % Tween-20 in TBS ; TBS-T). Monoclonal anti-His6 tag antibody was added at dilution 1 :2,500 in TBS-BSA, and incubated for 1 hr at RT. Mouse antisera against the four DENV serotypes, and against the WNV strains were laboratory-generated. Patient sera positive for DENV and WNV infections were used to confirm expressed-DIII protein configuration. After washing with TBS-T, aliquots (100 μΐ) of alkaline phosphatase (ALP)-conjugated goat anti-mouse IgG antibody (Sigma) was added to the wells at dilution 1 :3,000 in TBS-BSA and incubated for 1 hr at RT. After extensive washing with TBS-T, the reaction was developed by addition of 100 μΐ p- nitrophenylphosphate substrate (Sigma), and OD measured at 405 nm using an ELISA plate reader at the optimum time.
[00106] Immunofluorescence (IF) microscopy. Baculovirus-infected Sf9 cell monolayers were harvested at 48 h pi, fixed with 3 % paraformaldehyde in phosphate buffered saline (PBS) and, when required, permeabilized in 0.2 % (v/v) Triton X-100 in PBS. Cells were blocked with 3 % BSA in PBS (PBS-BSA), and His-tagged E :DIII detected by reaction with monoclonal anti-His6 tag antibody (1 :10,000 in PBS-BSA) and Alexa Fluor® 488-labeled goat anti-mouse IgG antibody (Molecular Probes, Invitrogen). For double labeling of E :DIII and Gag proteins, cell samples were reacted with rabbit anti-Gag antibody (1 : 1,000 in PBS-BSA) and Alexa Fluor® 546-labeled goat anti-rabbit IgG (Molecular Probes, Invitrogen). Cell samples were labeled using the Image-iT™ LIVE Plasma Membrane and Nuclear Labeling Kit (Molecular Probes, Invitrogen), which provides red-fluorescent Alexa Fluor® 594 wheat germ agglutinin (WGA) and blue-fluorescent Hoechst 3342 dye for plasma membrane and nucleus staining, respectively. For conventional IF microscopy, images were acquired using an Axiovert 135 inverted microscope (Zeiss) equiped with an AxioCam video camera. For confocal microscopy, samples were analyzed using a Nikon AIR fast laser scanning confocal microscope.
[00107] Electron microscopy (EM) and immuno-EM. VLP specimens were processed and observed under the EM as previously described [21]. In brief, VLP were diluted in 20 μΐ 0.14 M NaCl, 0.05M Tris-HCl buffer, pH 8.2 (Tris-buffered saline; TBS) and adsorbed onto carbon-coated formvar membrane on grids. The grids were incubated with primary antibody (monoclonal anti-His6 tag antibody) at a dilution of 1 : 50 in TBS for 1 h at room temperature (RT). After rinsing with TBS, the grids were post-incubated with 10-nm colloidal gold-tagged goat anti-mouse IgG antibody (British Biocell International Ltd, Cardiff, UK; diluted to 1 :50 in TBS) for 30 min at RT. After rinsing with TBS, the specimens were negatively stained with 1 % sodium phosphotungstate in H20 for 1 min at RT, rinsed again with TBS, and examined under a JEM 1400 JEOL electron microscope equiped with an Orius-Gatan digitalised camera (Gatan France, 78113 Grandchamp).
[00108] EXAMPLE 2
[00109] Expression of the chimeric human CD16-RIgE glycoprotein in insect cells
[00110] Human CD16 (or FcyRIIIa) is one of the low-affinity receptors for IgG Fc and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). It links IgG-sensitized target cells to CD16-bearing cytotoxic cells, i.e. CD56dim natural killer (NK) cells, a fraction of monocytes and macrophages, and rare T cells, and activates these effector cells. FcsRIy is a high affinity receptor for IgE receptor, and was abbreviated RIgE in the present study. CD16-RIgE is a chimeric receptor molecule consisting of the ectodomain of CD 16 fused to the transmembrane and cytoplasmic domains of the FcsRIy receptor [41]. The cDNA for CD16-RIgE was cloned into the genome of the baculovirus AcMNPV to generate the recombinant AcMNPV-CD16-RIgE (Fig. 1). Sf9 insect cells were infected with AcMNPV-CD16- RlgE, and the level of expression of the recombinant CD16-RIgE glycoprotein was assessed by SDS-PAGE and Western blot analysis of cell lysates. It was found that CD16-RIgE was expressed at high levels at 36-72 h post infection (pi) with a maximum at 48 h, as shown by the fuzzy band at 75 kDa, suggesting that this protein was not toxic for Sf9 cells. In co-expression of CD16-RIgE and HIV-1 Pr55Gag polyprotein in doubly-infected cells, there was a high level of expression for both recombinant proteins (Fig. 2), indicating that there was no detrimental effect of one protein on the other protein's expression.
[00111] EXAMPLE 3 [00112] Efficient incorporation ofCD16-RIgE into HIV-1 Gag-VLPs
[00113] The HIV-1 Gag precursor, Pr55Gag, is addressed to the plasma membrane or to a late endosomal compartment via its N-myristoyl group and a cluster of basic residues in the matrix domain [13,20,43]. In Sf9 cells, recombinant Pr55Gag efficiently self-assembles into VLPs budding at the plasma membrane [14,17,18]. The CD16-RIgE glycoprotein with is N-terminal signal peptide reaches the cell surface via the Golgi pathway. Next it was verified if the two proteins, Pr55Gag and CD16-RIgE, could co-localize in the same microdomains of the Sf9 cell plasma membrane, and eventually be co-incorporated in budding VLPs, a phenomenon referred to as pseudotyping. Sf9 insect cells were co-infected with AcMNPV-Gag and AcMNPV- CD16-RigE, and the cell culture medium harvested at 48 h pi. Cell-released VLPs were isolated from the extracellular medium and analyzed by.SDS-PAGE and Western blot with anti-CD 16 antibody. A strong signal of CD16-RIgE glycoprotein was found at 75 kDa associated with extracellular HIV-Gag VLPs (Fig. 3 A, i).
[00114] Taking advantage of the capacity of VSV-G to bind to a wide variety of cell surface receptors, combined with the ubiquitous character of VSV-G- pseudotyping and the VSV-G fusiogenic property, VSV-G has been largely used to construct fusiogenic retroviral or lentiviral vectors in mammalian cells [44].
AcMNPV- VSV-G was therefore a useful standard to test the capacity of HIV- 1 Gag- VLPs produced in baculovirus-infected insect cells to be pseudotyped by foreign glycoproteins, including VSV-G and CD16-RIgE. Sf9 were therefore co-infected with AcMNPV-Gag and AcMNPV- VSV-G at equal MOI, and extracellular VLPs were collected at 48 h and analyzed by SDS-PAGE and Western blotting. VSV-G was observed to be incorporated in significant amounts in VLPs (Fig. 3 A, ii). Although VSV-G and CD16-RIgE were detected using two different antibodies, the intensity of the CD16-RIgE signal, visible as a broad and diffuse band at 75 kDa, compared to the dark and sharp band of VSV-G at 62 kDa, suggested a similar efficiency of VLP incorporation for both glycoproteins (compare panels i and ii in Fig. 3A).
[00115] Sf9 cells were then infected with three recombinant baculoviruses, AcMNPV-Gag, AcMNPV- VSV-G and AcMNPV-CD16-RIgE, and the extracellular VLPs were isolated and analyzed as above. VLPs reacted with both anti- VSV-G and anti-CD 16 antibodies (Fig. 3 A, iii ; rightmost lane), suggesting that both VSV-G and CD16-RIgE glycoproteins were co-incorporated into VLPs. The VLPs pseudotyped with CD16-RIgE represented two separate populations, indicating that HIV-Gag VLPs produced in Sf9 cells could incorporate more than one foreign glycoprotein.
[00116] Ultrathin sections of Sf9 cells harvested at 48 h pi were then examined under the EM. VLPs budding at the plasma membrane of triple infected cells (AcMNPV-Gag + AcMNPV-VSV-G + AcMNPV-CD16-RIgE) showed a 'furry' aspect of their surface (Fig. 3B, i), compared to 'naked' VLPs produced by single AcMNPV-Gag-infected cells (Fig. 3B, ii). This confirmed that VLPs could be efficiently pseudotyped with foreign glycoproteins, e.g. CD16-RIgE or/and VSV-G.
[00117] EXAMPLE 4
[00118] Construction of the DIII-DENj-RIgE, DIII-WNWe-RIgE and DIII- WNKu-RIgE fusion proteins and expression in mammalian and insect cells
[00119] The efficient pseudotyping of HIV-Gag VLPs with CD16-RIgE glycoprotein incited us to use it as a platform of insertion of antigenic domains of Flavivirus envelope glycoproteins, and more specifically the domain Dili (E:DIII), identified as the major neutralization domain. DENV serotype 1 (DEN , and West Nile virus strains Wengler (WNWe) and Kunjin (WNKu) E:DIII were used as our three prototype constructs. The E:DIII domain was chosen for its capacity to induce immune response [2,4], and their coding sequences were inserted in that of CD 16- RlgE, in lieu of the deleted CD 16 domain (refer to Fig. 1). When expressed in human cell line HEK-293 using plasmid vector pcDNA3, all three fusion constructs DIII- DENi-RIgE, DIII-WNWe-RIgE and DIII-WNKu-RIgE were found to react with anti- His6 tag antibody in immunofluorescence (IF) microscopy of nonpermeabilized cells, as exemplified by the IF pattern of DIII-WNWe-RIgE-expressing cells (Fig. 4). This indicated that the three fusion constructs followed the same trafficking pathway as the parental CD16-RIgE glycoprotein, and were similarly addressed to the plasma membrane. More importantly, this also indicated that the three Dili domains were accessible at the cell surface and were displayed as the extracellular domain of the DIII-RIgE fusion proteins.
[00120] Baculovirus vectors were constructed to express these prototype chimeric proteins, AcMNPV-DENi-RIgE, AcMNPV-WNWe-RIgE and AcMNPV- WNKu-RIgE, respectively. All three clones showed a similarly high level of expression of their recombinant proteins in Sf9 cells, migrating with the expected apparent molecular mass of 22-23 kDa (Fig. 5 A). This value was consistent with the amino acid sequence, e.g., DIII-DENt-RIgE comprised of 215 residues including the signal peptide (22 residues), the oligo-His tag (6 residues), the Dili domain (123 residues), the transmembrane and cytoplasmic domains of the RIgE (63 residues), and a few leftover residues from the deleted CD 16 ectodomain. In IF microscopy of nonpermeabilized Sf9 cells, the N-terminal oligo-His tag of DIII-DEN]-RIgE, DIII- WNWe-RIgE and Dili- WNKu-RIgE chimeric proteins was found to react the anti- oligoHis antibody at the cell surface (Fig. 5 B), indicating that their trafficking pathway in Sf9 cells was the same as in HEK-293. Fig. 5 C shows a confocal microscopy image shows a high resolution image of the expressed DIII-WNWe protein on the cell plasma membrane.
[00121] EXAMPLE 5
[00122] VLP incorporation of the DIII-DENi-RIgE, DIII-WNWe-RIgE and DIII-WNKu-RIgE fusion proteins
[00123] In order to test the capacity of each fusion construct to pseudotype HIV-1 Gag-VLPs, Sf9 cells were co-infected with two baculoviruses, AcMNPV-Gag required to provide the VLP backbone, and AcMNPV-DIII-DENrRIgE, AcMNPV- DIII- WNWe-RIgE or AcMNPV-DIII- WNKu-RIgE to generate the VLP pseudotypes. Extracellular VLPs were isolated from the extracellular milieu at 48 h pi, using ultracentrifugation in sucrose-D20 gradient. The Gag protein and the oligo-His-DIII signals overlapped in the same gradient fractions, and these fractions corresponded to the apparent density of VLPs (p = 1.15-1.20 ; Fig. 6 A, B). These gradient fractions were pooled and subjected to an ultracentrifugation through a 20 % sucrose cushion [45]. Again, the Dili signal was found in the pelletable fraction, associated with the Gag signal of VLP pellet (Fig. 6 C). This indicated that the DIII-DENj-RIgE, DIII- WNWe-RIgE or DIII-WNKu-RIgE fusion proteins were bona fide components of the VLPs, and not carried over during the isolation process.
[00124] EXAMPLE 6
[00125] Immunological characterization of the E:DIII-VLP pseudotypes [00126] The accessibility of the Dili antigen at the surface of VLPs was examined by immunoelectron microscopy (immuno-EM), using anti-His6 tag antibody to probe for the N-terminal tag of the fusion proteins incorporated in the VLP envelope. As shown with semi-purified VLPs incompletely separated from particles of baculoviral vector, immunogold labeling was found specifically associated with VLPs, and not with baculovirions (Fig. 7). This clearly demonstrated that the three fusion proteins DIII-DENj-RIgE, DIII-WNWe-RIgE and DIII-WNKu- RIgE were indeed inserted in the envelope of HIV- 1 Gag- VLPs (120 to 130 nm in diameter), and that their Dili domains were exposed at the surface of VLPs and immunologically reactive.
[00127] EXAMPLE 7
[00128] Antigenic specificity and correct folding of VLP-displayed flavivirus E:DIII domains
[00129] In order to determine whether the Dili domains of the fusions DIII- DENrRIgE, DIII-WNWe-RIgE and DIII-WNKu-RIgE displayed at the surface of the VLPs adopted a correct three-dimensional (3D) conformation, each VLP pseudotype was analyzed by ELISA using patient sera positive for DENV-1, WNKu or WNWe. In spite of the relatively high level of background signal of control VLPs devoid of E:DIII with these human sera (in particular the anti-Kunjin serum), there was a significant reactivity of the E:DIII-pseudotyped VLPs with their specific antibodies (Fig. 8). This confirmed the accessibility of the E:DIII domain displayed by VLPs previously shown by IF and immuno-EM using the anti-His6 tag monoclonal antibody. The results also indicated that E:DIII domains of Dengue and West Nile viruses were inserted as the ectodomain of the fusion protein with RIgE folded properly and retained their original antigenicity.
[00130] DISCUSSION OF EXAMPLES 1 TO 7
[00131] The Domain III of the envelope glycoprotein of flaviviruses has a structure resembling that of an immunoglobulin constant domain and contributes the loops which are exposed on the exterior of the mature virion [46,47]. Exposure of West Nile and Dengue virus structural domain Dili at the surface of VLP is logical in view of previous observations that this domain is highly immunogenic and capable of inducing a robust immune response and a high level of neutralising antibodies [1-5]. Antibodies binding to epitopes on E:DIII have been detected in sera of patients for both West Nile and Dengue virus [48-52]. As a prototype, the E:DIII of Dengue virus serotype 1 was chosen out of the four serotypes. Similarly, two extensively studied strains of West Nile virus, the Wengler and Kunjin strains, were selected as WNV prototypes. The Wengler strain is of lineage 2 and was isolated from Nigeria, while the Kunjin strain is of lineage 1 and was isolated in Australia. Strains of WNV from lineage 1 are generally more virulent and neuro-invasive, but there is a recent trend for lineage 2 WNV to become increasingly encephalitic [53]. Thus, selection of these two strains of WNV took into account potential variations that could arise due to differences in geographical distribution and virulence.
[00132] Selection of HIV-1 Gag polyproteiri for pseudotyped VLP production was based on the following factors, (i) The first reason was the high productivity of VLPs when the recombinant HIV-1 gag gene was expressed in the baculovirus/insect cell system, compared to other systems [14]. We have shown in previous studies that in AcMNPV-Gag-infected Sf9 cells, 2 x 10E8 to 5 x 10E8 Gag molecules/cell were produced and released into the extracellular medium at 48 h pi. This corresponds to 5 x 10E4 to 1 x 10E5 VLPs per cell [17,18], if one considers that 5 x 10E3 Gag molecules are required to form one VLP [37,40]. (ii) Another factor for producing VLPs pseudotyped with flavivirus envelope glycoprotein (or neutralization domain thereof) in insect cells was that flavivirus cycle involves a mosquito vector as an intermediate host. Thus, the expression of flavivirus proteins in Sf9 should be similar to their natural biosynthetic pathway and posttranslational modifications in the insect vector, (iii) In terms of epitope presentation, displaying flavivirus envelope glycoprotein domain(s) at the surface of VLPs mimicked the real situation in the viruses, and should be optimal for immunization purposes, (iv) Lastly, VLPs, as other particulate structures, are known to play the role of adjuvant of the immune response [27], and this would be advantageous for vaccination strategies.
[00133] It was found herein that the Dili domains of the envelope glycoprotein of dengue virus serotype 1, and of the West Nile virus strains Wengler and Kunjin were all addressed to the plasma membrane of human and insect cells, provided that they were fused to the signal peptide of CD16 at their N-terminus, and to the transmembrane and cytoplasmic domains of the gamma chain of the human high affinity receptor for IgE at their C-terminus. More importantly, when DIII-DENr RlgE, DIII-WNWe-RIgE and DIII-WNKu-RIgE were coexpressed with HIV-1 Gag precursor in insect cells, they were efficiently incorporated onto the envelope of VLPs, and released into the extracellular medium. Immunological analyses indicated that the Dili domain of all three chimeric constructs was accessible at the surface of the VLPs, and was immunologically reactive against its specific antibodies. None of the three fusion proteins DIII-DENi-RIgE, DIII-WNWe-RIgE and DIII-WNKu-RIgE seemed to be glycosylated: their Dili domain does not carry any glycosylation site, and all fusion proteins migrated as sharp bands in SDS-PAGE, the molecular weight of which were consistent with their amino acid sequence. The signal peptide of CD 16 was removed from the VLP-displayed fusion proteins since all three reacted with anti- oligoHis antibody specific for tag located at the N-terminus.
[00134] Although several virus pseudotypes have been described and even used used as molecular tools and therapeutic agents, the pseudotyping of viral vectors is not a simple and straightforward process, as the molecular mechanisms involved are diverse and not fully understood, and many parameters are not perfectly controlled yet [44,54]. The complexity of the process has been well documented in HIV-1 virions, in which the recruitment of the viral envelope glycoproteins (SUgpl20/TMgp41) not only requires (i) that the Gag molecules and the SUgpl20/TMgp41 complex meet at the same sites of virus assembly (plasma membrane and multivesicular bodies;
MVBs), but (ii) also requires the legitimate interaction of the cytoplasmic tail of the TMgp41 with the N-terminal region of the matrix (MA) domain of Pr55Gag
[13,43,55], either directly or indirectly, through an intermediate linker consisting of TIP47 [56].
[00135] Pseudotyping of retroviral VLPs produced in insect cells also results from a complex mechanism, which implies the correct trafficking of recombinant retroviral Gag precursor and foreign envelope protein to the site of VLP assembly, and the budding and egress of membrane-enveloped VLPs from the cell surface. Little is known of the cellular functions involved in these processes in insect cells, compared to mammalian cells. Three types of scenarios were observed in recombinant baculovirus-infected insect cells expressing HIV-1 Pr55Gag. (i) Pseudotyping with CAR, the human receptor for Coxsackievirus and adenovirus, was found to be restricted to the baculovirus particles and was not detected in VLPs [21]. (ii) On the opposite, CD16-RIgE was preferentially incorporated into VLPs, and never recovered from baculovirus particles, as shown in the present study, (iii) The third case was exemplified by VSV-G, which pseudotyped both baculovirions and VLPs with the same efficiency. The length of the cytoplamic tail was not sufficient to explain the difference in the behaviour of the foreign glycoproteins tested, 106 amino acid residues for CAR, versus 42 for the gamma chain of the RIgE receptor and 31 for VSV-G. Other parameters, such as the nature of the matrix domain of the retroviral Gag polyprotein which constitutes the internal protein backbone of the VLPs [16], might control the efficiency of the pseudotyping process of VLPs.
[00136] Other types of retroviral Gag precursor, e.g. SIV, MLV, or chimeric MLV-HIV Gag, as well as various Gag mutants [30], could be used to determine the respective contribution of the different structural and functional domains present in the protein partners involved. The CD16-RIgE chimera of the present invention, in coexpression with HIV-1 Gag polyprotein in Sf9' cells, represents a versatile and useful pseudotyping platform for VLP-display of ectodomains of proteins of interest, in particular for vaccinal purposes.
[00137] It was observed that when HIV-1 Pr55Gag polyprotein was coexpressed in insect cells with CD16-RIgE, a chimeric human membrane glycoprotein consisting of the CD 16 ectodomain fused to the transmembrane domain and cytoplasmic tail of the gamma chain of the high affinity receptor of IgE, the extracellular VLPs incorporated CD16-RIgE in their envelope with a high efficiency. Accordingly, the CD 16 ectodomain of CD16-RIgE was replaced by the envelope glycoprotein domain III (E:DIII) of the Wengler and Kunjin strains of West Nile virus (WNWe and WNKu, respectively) and serotype 1 of Dengue virus (DEN^, as flavivirus prototypes. Each E:DIII domain was equiped with an oligo-histidine tag at its N- terminus. The resulting fusion proteins DIII-WNWe-RIgE, DIII-WNKu-RIgE and DIII-DENi-RIgE, were found to traffic to the plasma membrane of human and insect cells, and to be exposed at the cell surface, as their parental clone CD16-RIgE. Upon coexpression with HIV-1 Pr55Gag, all three E:DIII-RIgE proteins were found to efficiently form pseudotype VLPs. Immunological and electron microscopic data indicated that the E:DIII domains were accessible at the surface of VLPs, as expected for bona fide pseudotypes. Importantly, the E:DIII ectodomains were recognized by their specific antibodies, implying that they adopted their normal 3D structure and overall conformation. The CD16-RIgE chimera might therefore be proposed as a general VLP-pseudotyping platform, in coexpression with recombinant retroviral Gag polyprotein, for VLP-display of other viral envelope glycoproteins or any other antigen, and their therapeutic application as potential vaccine vectors.
[00138] In conclusion, the present invention provides a strategy of epitope presentation at the surface of virus-like particles (VLPs). The VLPs are retroviral Gag particles which spontaneously assemble into particles and are structurally similar to the native virions but do not contain the viral genetic material. The immunogenic epitopes are exposed on the VLP surface via a membrane-anchored fusion construct to mimic the native antigenic protein as presented by viruses. The protein platform for epitope presentation consists of a chimeric human surface glycoprotein derived from the CD 16 molecule (abbreviated CD 16-RIgE), which is efficiently incorporated into the envelope of the VLPs. It is here shown that the CD 16 ectodomain can be replaced by the envelope glycoprotein domain III of West Nile and Dengue virus, resulting in the exposure of the Envelope (E):DIII epitopes at the surface of the VLP envelope.
[00139] The cloning and VLP-display protein platform of the present invention could be abbreviated by the acronym: SP-CD16-RIgE. The only portion left from the CD 16 is its signal peptide (SP) and a couple of amino acids at the junction with the TM domain of RIgE. This is clearly shown in Fig. 1 A.
[00140] When the whole CD 16 ectodomain is deleted and replaced with the Flavi virus Dili domain, the resulting molecule behaves exactly as the parental CD 16- RlgE molecule, and also generates a DIII-RIgE-VLP pseudotype. It is here shown that this platform can be used generically to insert other pathogen proteins for vaccine purposes as with the Flavivirus Dili.
[00141] This platform can be used to insert two or more E-DIII domains or full envelope (E) proteins from different flavivirus envelopes in tandem.
[00142] The recombinant chimera SP-CD16-RIgE can be used as a general VLP- pseudotyping platform for VLP-display of other viral envelope glycoproteins, or other pathogen proteins and their therapeutic application as potential vaccine vectors. [00143] This technology of VLP-antigen display/presentation is a major improvement over the conventional "VLP pseudotyping" strategy as the presentation platform can be used for displaying antigenic domains from viral, bacterial or parasitic pathogens. The VLPs are devoid of viral genomes, and their non-replicative nature makes them useful for the design of subunit vaccines which carry the immunological structures of the virus in the absence of infectious genetic material. In addition, VLP can induce both humoral- and cellular-mediated immune responses.
[00144] EXAMPLE 8
[00145] METHODS
[00146] Mice immunization regime conducted
[00147] Immunization regime with E-DIII-RIgE-pseudotyped VLPs of the present invention was based on a protocol previously described and optimized to obtain neutralization antibodies in mice immunized with inactivated flavivirus particles used as the immunogens [1, 2, 3]. In the cited protocol, series of five BALB/C mice were injected intraperitoneally with 100 μΐ-aliquot per animal of virus stock with purified titre of 107 PFU/ml and mixed with complete Freund's adjuvant. This was repeated at weeks 3 and 5 with incomplete Freund's adjuvant, and animals were bled at week 6 [1, 2, 3].
[00148] Calculation of the doses necessary for immunization with E-DIII- RlgE-pseudotyped VLPs
[00149] (i) Quantitation of incorporation of E-DIII into pseudotyped VLPs. The amounts of E-DIII-RIgE exposed at the surface of VLPs were quantitatively determined by SDS-PAGE analysis and gel scanning. VLPs were co-electrophosed in SDS-polyacrylamide gels with a range of bovine serum albumin standard samples of known concentrations. Gels were stained with Coomassie blue and bands of Gag and envelope Dili domain (E-DIII) proteins were scanned and quantitated by
densitometric analysis, using the ImageJ program (NIH) and a quantification method cited in [57] and detailed in the following Web site: https :// www. lukemiller. org. It was found that a protein ratio ranging between 15 and 30 ng of E-DIII-RIgE per μg total Gag protein (^g Gag protein corresponds to 2 x 109 purified VLPs), and a calculated mean number of 360 ± 30 copies (m ± SEM ; n= 4) per VLP for E-DIII- DENV1 (dengue virus 1), and 240 ± 25 copies (m ± SEM ; n= 4) per VLP for E-DIII- WNKun West Nile virus, kunjin). The simplest explanation for the difference in envelope incorporation into VLP between E-DIII-DENV1 and E-DIII-WNKun, was the slightly lower level of cellular expression of E-DIII-WNKun protein, compared to E-DIII-DENV1.
[00150] (ii) Considering that the infectivity index (ratio of infectious to physical particles; IP : VP) of flaviviruses varies between 1 : 100 and 1 : 1 ,000, it was estimated that the 100 μΐ-dose containing 106 infectious particles (IP) which has been injected in the above cited protocol [1, 2, 3] corresponded to 108 to 109 physical virus particles (VP).
[00151] (iii) Based on the fact that the outer glycoprotein shell of a mature flavivirus particle is formed by 30 rafts of three homodimers of the viral surface protein E, viz. 180 copies of E monomer per virion [58, 59], a range of values of 1.8 xlO10 to 1.8 x 1011 copies of E monomer per dose injected in the above cited protocol were calculated [1, 2, 3].
[00152] (iv) As mentioned above in (i), 1 μg Gag protein corresponds to 2 x 109 VLPs. Then, 10μg Gag protein, which corresponds to 2 x 1010 VLP, would carry 360 x 2 x 1010 = 7.2 xlO12 copies of E-DIII-DENVl, and 240 x 2 x 1010 = 4.8 x 1012 copies of E-DIII-WNKun. Each dose injected per animal consisted therefore of 10- 12 μg VLP, corresponding to 25- to 40-fold the maximal dose of E monomer protein (1.8 x 1011 copies) used in the immunization protocol with inactivated virions [1, 2, 3].
[00153] Immunization regime with E-DIII-pseudotyped VLPs.
[00154] Animal : Balb/C mouse, female, 11 week-old
[00155] Route of injection : subcutaneous, posterior neck
[00156] Series #1 (5 mice ; SEM036 to SEM040) : VLP-0 (control, nonpseudotyped VLPs)
[00157] Series #2 (5 mice ; SEM041 to SEM045) : VLP-DENV1 [00158] Series #3 (5 mice ; SEM046 to SEM050) : VLP-KU
[00159] Immunization schedule :
[00160] Pre-immune sample
[00161] Week 1 : Complete Freund's adjuvant + 10 μ VLP (40 μΐ total volume)
[00162] Week 3 : Incomplete Freund's adjuvant + 10 μ VLP (40 μΐ total volume)
[00163] Week 5: Incomplete Freund's adjuvant + 10 μ VLP (40 μΐ total volume)
[00164] Week 7: Incomplete Freund's adjuvant + \0 μg VLP (40 μΐ total volume)
[00165] Week 8: Final bleed.
[00166] Antibody induction (ELISA) and Virus neutralisation (NA) tests (PRNT)
[00167] Enzyme-linked immunosorbant assay (ELISA)
[00168] In order to test whether the polyclonal antibodies in the mouse anti-sera could bind to homologous (Dengue virus serotype 1 used as our prototype) and heterologous strains of DENV, and WNV (Kunjin strain used as our prototype), ELISA was performed using the viruses as immobilized antigens. Live viruses were coated onto the bottom of the wells in a 96-well MultiSorp plate (Nunc, Denmark) in a Class II Type A2 Biohazard Safety Cabinet. This was achieved by adding 100 μΐ of 5,000 PFU/ml of virus into each well and incubating the plate overnight at 4°C. Any exposed surface on the bottom of wells were blocked with 100 μΐ of 5% (w/v) skimmed milk and incubated at 37°C for 30 min. Serum diluent was made up of PBS with 0.1% (v/v) Tween 20 (PBST) and 5% (w/v) skimmed milk.
[00169] Each mouse antiserum raised against the E-DIII protein of DENV1 and KUN was diluted in 1 : 10, 1 :50, 1 : 100 and 1 :200 ratios using 5% skimmed milk. The antiserum raised against the negative control VLP-0 was also similarly diluted. One hundred μΐ of diluted anti-sera was added into each well such that each diluted antiserum would be added into three wells (triplicates). The plate was then incubated for one hour at 37°C, then 100 μΐ of diluted (1 :250) secondary anti-IgG-biotin conjugate (Chemicon International, U.S.A.) was added. The plate was incubated for another hour at 37°C and 100 μΐ of diluted (1 :5,000) strepavidin-HRP conjugate (Chemicon International, U.S.A.) was added into each well. The plate was further incubated for one hour at 37°C, followed by the addition of 100 μΐ of TMB One substrate solution (Promega Corporation, U.S.A.) into each well. The plate was further incubated for 30 min, and 50 μΐ of 0.5 M sulfuric acid stop-solution added into each well. All wells were washed with PBST thrice before the addition of any reagent except before the stop solution was added. The absorbance of each well was finally measured at 450 nm.
[00170] Virus neutralisation: plaque reduction neutralisation test (PRNT)
[00171] Complement C 1 q has been shown to bind to the Fc region of virus- bound antibodies, and could help reduce infection via steric hindrance. Heat- inactivating the antisera used in PRNT would then inactivate Clq and eliminate this confounding factor. Prior to performing PRNT, all the antisera were inactivated by heating them at 56°C for 30 min using a dry-bath. Heat-inactivated antisera were diluted in 1 :10, 1 :50, 1 :100, 1 :200, 1 :400 and 1 :800 ratios and 500 μΐ of each dilution was incubated with 500 PFU of virus at 37°C for one hour. A control was included in which virus diluent was used instead of antiserum.
[00172] BH cells were seeded onto 24- well plates and 100 μΐ of diluted virus- antibody mixtures were transferred in triplicates onto confluent cell monolayers in the wells. A row of mock-infected wells was included as negative controls and a row of neat virus-infected wells served as positive controls. Plates were incubated at 37°C for one hour under 5% carbon dioxide, with rocking every 15 min to ensure even distribution of diluted virus-antibody mixtures. The diluted virus-antibody mixtures were removed and the cell monolayer was washed once with virus diluent. One ml of CMC overlay medium was pipetted into each well. The trays were incubated at 37°C in a humidified C02 incubator. Plates were incubated for 3-6 days (depending on the virus type) before the overlay medium was removed for staining. The plaques were visualized by staining the monolayer with 0.5% crystal violet in a 25% formaldehyde solution for at least 2 h at room temperature on orbital shaker (Labnet Intl. Inc., USA). The crystal violet solution was removed for proper hazardous chemical disposal and the plates were washed under a running tap to remove residual dye. The plates were finally left to dry and the number of plaques counted.
[00173] RESULTS
[00174] Induction of Flavivirus antibodies in mice immunised with Flavivirus E-DIII-VLP
[00175] ELISA showed that antibodies against Dengue and West Nile (WN) Kunjin viruses were induced in mice after SC administration of E-DIIl-DENVl-VLP and E-DIII-WNKu-VLP, respectively (Fig. 9 and 10). Sera from 2 out of 5 mice were positive for DENV1 virus at all dilutions tested (1 :10 to 1 :200), and reacted in a dose-dependent manner (Fig. 9). Sera from 2 out of 5 mice were positive for WN Kunjin: one serum reacted with WN Kunjin virus at all dilutions tested (1 :10 to 1 :200); another serum was positive at low dilutions (1 :10 & 1 :50 ; Fig. 10). In conclusion, in mice which responded to the antigen, ELISA showed moderate levels of antibodies and binding affinity towards the corresponding live virus particles.
[00176] Binding specificity
[00177] The different sera from mice administered with E-DIII-DENV 1 - VLP and E-DIII-WNKun-VLP were tested in ELISA against five different immobilized virus particles, DENV1, DENV2, DENV3, DENV4 and WN Kunjin. Data showed that all sera bound to the different flaviviruses tested, and at all dilutions used (Fig. 11 and 12). In conclusion, most of the antibodies induced by E-DIII-VLP and detected by ELISA were directed against common region(s) of the E-DIII domains, shared by the different DENV serotypes and by WN Kunjin virus.
[00178] Neutralisation activity (NA)
[00179] PRNT showed that sera from 3 out of 5 mice injected with E-DIII- DENV1-VLP had neutralizing antibodies against DENV1 with NA ranging from 20 to 30 % (Fig. 13). Sera from 3 out of 5 mice SC injected with E-DIII- WNKun- VLP had neutralizing antibodies against WN Kunjin virus with NA ranging from 25 to 50 % (Fig. 14). In conclusion, neutralizing antibodies directed against DENV and WN Kunjin virus were induced in mice using SC administration of E-DIII-DENVl-VLP and E-DIII-WNKun-VLP, respectively.
[00180] Analysis of correlation between antibody levels and virus
neutralization activity
[00181] Comparison of ELISA and PRNT data showed that for DENV 1 , mice #4 and #5 were found to develop the highest levels of total anti-DENVl antibodies (refer to Fig. 9), whereas sera of mice #2, #3 and #4 were the highest in NA titer (refer to Fig. 13). Only one animal, mouse #4, had both high levels of total antibodies
(detected by ELISA) and significant levels of neutralizing antibodies (detected by PRNT; compare Fig. 9 & 13).
[00182] Comparison of ELISA and PRNT data showed that for WN Kunjin, mice #1 and #4 developed the highest levels of total anti-WN Kunjin antibodies (as assayed by ELISA), whereas sera of mice #2, #3 and #5 showed the highest NA activity (compare Fig. 10 & 14).
[00183] In conclusion, there was no correlation between the total levels of antibodies induced by E-DIII-VLPs in immunized animals (as assayed by ELISA) and their capacity to neutralize the live viruses (PRNT). This indicated that only a certain population of antibodies were neutralizing, which was scarcely surprising. NA tests like PRNT evaluated the capacity of antibodies to block the virus replication, and remained the method of choice for evaluating the potency and efficacy of anti- flavivirus vaccines.
[00184] Cross-neutralisation activity
[00185] Neutralisation assays (PRNT) were performed with sera from mice injected with E-DIII-DENVl-VLP (anti-DENVl antibodies) versus two other DENV serotypes, DENV2 and DENV3, and versus West Nile virus Kunjin. Fig. 15 showed some neutralizing activity against DENV2 with anti-DENVl antibodies, as evidenced by a correlation of the average percentages of neutralization with respect to the serum dilutions. This indicated that all sera containing neutralizing antibodies directed towards DENV serotype 1 also neutralized DENV serotype 2 (compare Fig. 13 and Fig. 15). On the other hand, these sera (anti-DENVl antibodies) were not capable of neutralizing DENV serotype 3 and WN unjin virus (Fig. 16 and Fig. 17).
[00186] Sera from mice injected with E-DIII-WNKun-VLP (anti-Kunjin antibodies) were also analyzed against the three DENV serotypes, DENVl, DENV2 and DENV3. As shown in Fig. 18, there was some degree of cross-neutralization activity of DENVl virus with anti-Kunjin antibodies, with an average titer up to 20 % NA at dilution 1 :50.
[00187] This cross NA activity was more conspicuous beween anti-Kunjin antibodies and DENV serotype 2 (Fig. 19), with an average titer up to 30 % NA at dilution 1 :50. Of note, one individual mouse immunized with E-DIII-WNKun-VLP showed a NA effect of DENV2 at 75-80% (Fig. 19). No heterologous NA was observed with anti-Kunjin antibodies and DENV serotype 3 (Fig. 20).
[00188] In conclusion, anti-DENVl neutralizing antibodies induced by administration of mice with E-DIII-DENV1-VLP were bivalent, as they neutralized both serotypes 1 and 2 of dengue virus. Anti-WN Kunjin neutralizing antibodies induced by administration of mice with DIII-WNKu-VLP cross-neutralized DENV serotypes 1 and 2.
[00189] GENERAL CONCLUSIONS
[00190] (i) Induction of virus neutralizing antibodies. Immunisation with flavivirus E-DIII-VLPs showed that neutralizing antibodies against flaviviruses were induced in a significant number of animals: 3 animals out of 5 (~ 60 %) for each vaccine antigen prototype. In some cases, NA titers reached 50% at 1 : 10 serum dilution.
[00191 ] (ii) Importantly, injecting pseudotyped VLP carrying a relatively small domain (domain Dili) of the flavivirus envelope glycoprotein induced neutralizing antibodies in animals. This confirmed that Dili carried a major neutralizing epitope.
[00192] (Hi) Induction of cross-reactive NA antibodies against DENV serotypes 1 and 2. Injecting pseudotyped VLP carrying the Dili domain of the DENV serotype 1 envelope neutralized DENV serotype 1 and DENV serotype 2 as well. [00193] (iv) Induction of cross-reactive NA antibodies between WN Kunjin virus and DENV serotypes 1 and 2. Injection of VLP carrying the Dili domain of the West Nile Kunjin virus envelope resulted in the induction of antibodies neutralizing both DENV serotype 1 and DENV serotype 2. This indicates some common structural and functional features between all Dili domains of flaviviruses, susceptible of evoking immunity in vaccinated animals/humans.
[00194] (v) Correct three-dimensional conformation of the flavivirus E-DIII domain used as immunogen. The data of virus neutralization antibodies induced in immunized animals demonstrates that the flavivirus E-DIII domain expressed at the surface of VLP of the present invention used as the immunogen was in a correct three-dimensional conformation. Taken together, these results demonstrate the feasibility of displaying flavivirus epitopes on the surface of our VLP platform for antigenic presentation.
[00195] All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.
[00196] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.
[00197] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
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Claims

WHAT IS CLAIMED IS:
1. A fusion protein comprising (i) a CD 16 signal peptide; (ii) an antigenic peptide region downstream of the CD 16 signal peptide; and (iii) an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
2. The fusion protein of claim 1, wherein the antigenic peptide region is not CD 16 ectodomain.
3. The fusion protein of claim 1 or claim 2 for use in forming a pseudotype virus-like particle.
4. The fusion protein of any one of claims 1 to 3, wherein the antigenic peptide region comprises an antigen useful for vaccination of a human or animal.
5. The fusion protein of any one of claims 1 to 4, wherein the antigenic peptide region comprises a viral, parasitic or bacterial antigen.
6. The fusion protein of claim 5, wherein the antigenic peptide region
comprises a Dili domain from a flavivirus envelope glycoprotein.
7. The fusion protein of claim 6, wherein the Dili domain is the Dill domain from the envelope glycoprotein of West Nile virus or Dengue virus.
8. The fusion protein of claim 7, wherein the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus strain Wengler or Kunjin or of Dengue virus serotype 1 , 2, 3 or 4.
9. A nucleic acid molecule encoding a promoter operably linked to (i) a sequence encoding the fusion protein of any one of claims 1 to 8; or (ii) a sequence encoding a CD 16 signal peptide upstream of a cloning site and a
10. The nucleic acid molecule of claim 9 that is an expression vector.
11. The nucleic acid molecule of claim 10 that is a baculoviral expression vector, wherein the promoter is a promoter that is expressed in insect cells.
12. A method of forming a pseudotype virus-like particle, the method
comprising coexpressing in a cell (i) a fusion protein of any one of claims 1 to 8; and (ii) a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein.
13. The method of claim 12, wherein the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SIV), or a chimeric human-animal retroviral Gag polyprotein.
14. A pseudotype virus-like particle comprising (i) protein core comprising a retroviral Gag polyprotein or a protein precursor of a retroviral Gag polyprotein; (ii) a membrane envelope surrounding the protein core; and (iii) a fusion protein incorporated into the membrane envelope, the fusion protein comprising an antigenic peptide region and an IgE receptor transmembrane domain and cytoplasmic tail downstream of the antigenic peptide region.
15. The pseudotype virus-like particle of claim 14, wherein the retroviral Gag polyprotein precursor is HIV-1 Pr55Gag, or the Gag polyprotein from other mammalian retroviruses (e.g. MLV, SIV), or a chimeric human- animal retroviral Gag polyprotein.
16. The pseudotype virus-like particle of claim 14 or claim 15, wherein the antigenic peptide region comprises an antigen useful for vaccination of a human or animal. antigen.
18. The pseudotype virus-like particle claim 17, wherein the antigenic peptide region comprises a Dill domain from a flavivirus envelope glycoprotein.
19. The pseudotype virus-like particle of claim 18, wherein the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus or Dengue virus.
20. The pseudotype virus-like particle of claim 19, wherein the Dili domain is the Dili domain from the envelope glycoprotein of West Nile virus strain Wengler or Kunjin or of Dengue virus serotype 1, 2, 3 or 4.
21. The pseudotype virus-like particle of any one of claims 14 to 20 when formed by the method of claim 12.
22. A vaccine comprising a pseudotype virus-like particle of any one of claims 14 to 21.
23. Use of a vaccine of claim 22 for vaccination of a human or animal.
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ITRM20130458A1 (en) * 2013-08-05 2015-02-05 Internat Ct For Genetic En Gineering And ANTI-DENGUE VIRUS GENETIC VACCINE BASED ON THE ECTODOMINS OF THE PROTEIN ENVELOPE
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WO2015019253A3 (en) * 2013-08-05 2015-05-28 International Centre For Genetic Engineering And Biotechnology - Icgeb Anti-dengue virus genetic vaccine based on the envelope protein ectodomains
CN104450631A (en) * 2014-11-11 2015-03-25 中国人民解放军第四军医大学 Enterovirus EV71 type VP1 gene virus-like particle as well as preparation method and application thereof
EP3268394A4 (en) * 2015-03-13 2018-09-19 University of Maryland, Baltimore Universal antibody-mediated biosensor
RU2746486C2 (en) * 2015-03-13 2021-04-14 Юниверсити Оф Мэрилэнд, Балтимор Universal antibody-mediated biosensor
US11440946B2 (en) 2015-03-13 2022-09-13 University Of Maryland, Baltimore Universal antibody-mediated biosensor
WO2017147458A1 (en) * 2016-02-25 2017-08-31 The Trustees Of The University Of Pennsylvania Novel vaccines against zika virus
US11648303B2 (en) 2016-02-25 2023-05-16 The Trustees Of The University Of Pennsylvania Vaccines against zika virus
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