WO1999004010A1 - Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial - Google Patents

Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial Download PDF

Info

Publication number
WO1999004010A1
WO1999004010A1 PCT/CA1998/000697 CA9800697W WO9904010A1 WO 1999004010 A1 WO1999004010 A1 WO 1999004010A1 CA 9800697 W CA9800697 W CA 9800697W WO 9904010 A1 WO9904010 A1 WO 9904010A1
Authority
WO
WIPO (PCT)
Prior art keywords
rsv
protein
nucleotide sequence
host
vector
Prior art date
Application number
PCT/CA1998/000697
Other languages
English (en)
Inventor
Xiaomao Li
Suryaprakesh Sambhara
Michel H. Klein
Original Assignee
Connaught Laboratories Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Connaught Laboratories Limited filed Critical Connaught Laboratories Limited
Priority to EP98934710A priority Critical patent/EP0996730A1/fr
Priority to BR9815496-6A priority patent/BR9815496A/pt
Priority to CA002296089A priority patent/CA2296089A1/fr
Priority to NZ502626A priority patent/NZ502626A/xx
Priority to JP2000503216A priority patent/JP2001512662A/ja
Priority to AU84278/98A priority patent/AU756222B2/en
Publication of WO1999004010A1 publication Critical patent/WO1999004010A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18522—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18511—Pneumovirus, e.g. human respiratory syncytial virus
    • C12N2760/18534—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention is related to the field of respiratory syncytial virus (RSV) vaccines and is particularly concerned with vaccines comprising nucleic acid sequences encoding the attachment (G) protein of RSV.
  • RSV respiratory syncytial virus
  • Respiratory syncytial virus a negative- strand RNA virus belonging to the Paramyxoviridae family of viruses, is the major viral pathogen responsible for bronchiolitis and pneumonia in infants and young children (ref. 1 - Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure) . Acute respiratory tract infections caused by RSV result in approximately 90,000 hospitalizations and 4,500 deaths per year in the United States (ref. 2).
  • a protective immune response against RSV is thought to require the induction of neutralizing antibodies against the surface fusion (F) and attachment (G) glycoproteins (ref. 4) .
  • F surface fusion
  • G attachment glycoproteins
  • CTL cytotoxic T lymphocytes
  • the G protein (33 kDa) of RSV is heavily O- glycosylated giving rise to a glycoprotein of apparent molecular weight of 90 kDa (ref. 5) .
  • Two broad subtypes of RS virus have been defined: A and B (ref. 6) . The major antigenic differences between these subtypes are found in the G glycoprotein (refs. 3, 7).
  • RSV proteins as vaccines may have obstacles.
  • Parenterally administered vaccine candidates have so far proven to be poorly immunogenic with regard to the induction of neutralizing antibodies in seronegative chimpanzees.
  • the serum antibody response induced by these antigens may be further diminished in the presence of passively acquired antibodies, such as the transplacentally acquired maternal antibodies which most young infants possess .
  • a subunit vaccine candidate for RSV consisting of purified fusion (F) glycoprotein from RSV infected cell cultures and purified by immunoaffinity or ion-exchange chromatography has been described (ref. 8).
  • the vaccine was found to be safe in seropositive children and in three seronegative children (all > 2.4 years of age). The effects of immunization on lower respiratory tract disease could not be determined because of the small number of children immunized.
  • One immunizing dose in seropositive children induced a 4 -fold increase in virus neutralizing antibody titres in 40 to 60% of the vaccinees.
  • a further problem facing subunit RSV vaccines is the possibility that inoculation of seronegative subjects with immunogenic preparations might result in disease enhancement.
  • FI-RSV formalin-inactivated RSV preparation
  • the immune response to immunization with a synthetic RSV FG fusion protein resulted in disease enhancement in rodents resembling that induced by a formalin-inactivated RSV vaccine.
  • Immunization of mice with a recombinant vaccinia virus expressing the RSV G protein resulted in G-specific T cell responses in the lungs which are exclusively recruited from the CD4+T cell sublineage and are strongly Th2-biased.
  • G-specific T cells induce lung haemmorrage, pulmonary neutrophil recruitment (shock lung) , intense pulmonary eosinophilia, and sometimes death in the adoptively transferred murine recipients (ref. 14) .
  • the association of immunization with disease enhancement using certain vaccine preparations including non-replicating antigens suggests caution in their use as vaccines in seronegative humans .
  • Live attenuated vaccines against disease caused by RSV may be promising for two main reasons. Firstly, infection by a live vaccine virus induces a balanced immune response comprising mucosal and serum antibodies and cytotoxic T-lymphocytes . Secondly, infection of infants with live attenuated vaccine candidates or naturally acquired wild-type virus is not associated with enhanced disease upon subsequent natural reinfection. It will be challenging to produce live attenuated vaccines that are immunogenic for younger infants who possess maternal virus-neutralizing antibodies and yet are attenuated for seronegative infants greater than or equal to 6 months of age. Attenuated live virus vaccines also have the risks of residual virulence and genetic instability.
  • plasmid DNA inoculation to express viral proteins for the purpose of immunization may offer several advantages over the strategies summarized above. Firstly, DNA encoding a viral antigen can be introduced in the presence of antibody to the virus itself, without loss of potency due to neutralization of virus by the antibodies. Secondly, the antigen expressed in vivo should exhibit a native conformation and the appropriate glycosylation.
  • the antigen should induce an antibody response similar to that induced by the antigen present in the wild-type virus infection.
  • some processes used in purification of proteins can induce conformational changes which may result in the loss of immunogenicity of protective epitopes and possibly immunopotentiation.
  • the expression of proteins from injected plasmid DNAs can be detected in vivo for a considerably longer period of time than that in virus- infected cells, and this has the theoretical advantage of prolonged cytotoxic T-cell induction and enhanced antibody responses.
  • in vivo expression of antigen may provide protection without the need for an extrinsic adjuvant.
  • the ability to immunize against disease caused by RSV by administration of a DNA molecule encoding an RSV G protein was unknown before the present invention.
  • the efficacy of immunization against RSV induced disease using a gene encoding a secreted form of the RSV G protein was unknown. Infection with RSV leads to serious disease.
  • the present invention relates to a method of immunizing a host against disease caused by respiratory syncytial virus, to non-replicating vectors containing nucleic acid molecules used in immunogenic compositions for such purpose, and to diagnostic procedures utilizing the vectors and nucleic acid molecules.
  • the present invention is directed towards the provision of nucleic acid vaccines encoding the G protein of respiratory syncytial virus.
  • an immunogenic composition for in vivo administration to a host for the generation in the host of protective antibodies to respiratory syncytial virus (RSV) G protein comprising a non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host, and a pharmaceutically-acceptable carrier therefor.
  • a non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein
  • the first nucleotide sequence may be that which encodes a full-length RSV G protein.
  • the first nucleotide sequence may comprise the nucleotide sequence shown in Figure 2 (SEQ. ID No: 1) or encode a full length RSV G protein having the amino acid sequence shown in Figure 2 (SEQ. ID no: 2) .
  • the first nucleotide sequence may be that which encodes an RSV G protein from which the transmembrane coding sequence and sequences upstream thereof are absent.
  • the first nucleotide sequence encoding the truncated RSV G protein may comprise the nucleotide sequence shown in Figure 3 (SEQ.
  • RSV G protein may comprise a nucleotide sequence encoding the truncated RSV G protein having the amino acid sequence shown in Figure 3 (SEQ ID no: 4) .
  • SEQ ID no: 4 The lack of expression of the transmembrane region results in a secreted form of the RSV G protein.
  • the non-replicating vector may further comprise a heterologous signal peptide encoding nucleotide sequence immediately upstream of the 5' -terminus of the first nucleotide sequence.
  • the signal peptide encoding sequence may encode the signal peptide of human tissue plasminogen activator.
  • the promoter sequence may be an immediate early cytomegalovirus (CMV) promoter.
  • CMV immediate early cytomegalovirus
  • the second nucleotide sequence may comprise the human cytomegalovirus Intron
  • the non-replicating vector generally is a plasmid vector.
  • Plasmid vectors encoding the G protein and included in the immunogenic composition provided by this aspect of the invention may specifically be pXL5 or pXL6, constructed and having their characterizing elements, as seen in Figures 4 or 5, respectively.
  • a method of immunizing a host against disease caused by infection with respiratory syncytial virus which comprises administering to the host an effective amount of a non- replicating vector comprising: a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host .
  • a non- replicating vector comprising: a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucle
  • the immunization method may be effected to induce a balanced Thl/Th2 immune response.
  • the present invention also includes a novel method of using a gene encoding respiratory syncytial virus (RSV) G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, to protect a host against disease caused by infection with respiratory syncytial virus, which comprises : isolating the gene; operatively linking the gene to at least one control sequence to produce a non-replicating vector, said control sequence directing expression of the RSV G protein when said vector is introduced into a host to produce an immune response to the RSV G protein, and introducing the vector into the host .
  • RSV respiratory syncytial virus
  • the procedure provided in accordance with this aspect of the invention may further include the step of: operatively linking the gene to an immunoprotection enhancing sequence to produce an enhanced immunoprotection by the RSV G protein in the host, preferably by introducing the immunoprotection enhancing sequence between the control sequence and the gene, including introducing immunostimulatory CpG sequences in the vector.
  • the present invention includes a method of producing a vaccine for protection of a host against disease caused by infection with respiratory syncytial virus (RSV), which comprises: isolating a first nucleotide sequence encoding an RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, operatively linking the first nucleotide sequence to at least one control sequence to produce a non- replicating vector, the control sequence directing expression of the RSV G protein when introduced into a host to produce an immune response to the RSV G protein when expressed in vivo from the vector in a host, operatively linking the first nucleotide sequence to a second nucleotide sequence to increase expression of the RSV G protein in vivo from the vector in a host, and formulating the vector as a vaccine for in vivo administration.
  • RSV respiratory syncytial virus
  • the vector may be a plasmid vector selected from pXL5 and pXL6.
  • the invention further includes a vaccine for administration to a host, including a human host, produced by this method.
  • a respiratory syncytial virus (RSV) G protein in a sample comprising the steps of:
  • the non-replicating vector comprising a first nucleotide sequence encoding an RSV G protein or an RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in the host and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein in vivo from the vector in the host;
  • the non-replicating vector employed to elicit the antibodies may be a plasmid vector pXL5 or pXL6.
  • the invention also includes a diagnostic kit for detecting the presence of a respiratory syncytial virus (RSV) G protein in a sample, comprising:
  • a non-replicating vector capable of generating antibodies specific for the RSV G protein when administered to a host
  • said non- replicating vector comprises a first nucleotide sequence encoding an RSV G protein or an RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in a host, and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein in vivo from the vector in the host;
  • isolation means to isolate the RSV G protein specific antibodies
  • the present invention further is directed to a method for producing antibodies specific for a G protein of a respiratory syncytial virus (RSV) comprising:
  • a non-replicating vector comprising: a first nucleotide sequence encoding a RSV G protein or a RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to said first nucleotide sequence for expression of said RSV G protein in the host, and a second nucleotide sequence located between said first nucleotide sequence and said promoter sequence to increase expression of said RSV G protein in vivo from said vector in the host; and
  • the present invention is also directed to a method for producing monoclonal antibodies specific for a G protein of respiratory syncytial virus (RSV) , comprising the steps of:
  • RSV G protein fragment that generates antibodies that specifically react with RSV G protein, a promoter sequence operatively coupled to the first nucleotide sequence for expression of the RSV G protein in the host and a second nucleotide sequence located between the first nucleotide sequence and the promoter sequence to increase expression of the RSV G protein when in vivo from the vector in a host; (b) administering the vector to at least one mouse to produce at least one immunized mouse;
  • (h) isolating anti-RSV G protein monoclonal antibodies .
  • Such monoclonal antibodies may be used to purify RSV G protein from virus.
  • RSV G protein is used to define a full-length RSV G protein, such proteins having variations in their amino acid sequences including those naturally occurring in various strains of RSV, a secreted form of RSV G protein lacking a transmembrane region, as well as functional analogs of the RSV G protein.
  • a first protein is a "functional analog" of a second protein if the first protein is immunologically related to and/or has the same function as the second protein.
  • the functional analog may be, for example, an immunologically-active fragment of the protein or an immunologically-active substitution, addition or deletion mutant thereof.
  • Figure 1 illustrates a restriction map of the gene encoding a G protein of respiratory syncytial virus (RSV) ;
  • Figure 2 illustrates the nucleotide sequence of a gene encoding a membrane bound form of the G protein of respiratory syncytial virus (SEQ ID No: 1) as well as the amino acid sequence of the RSV G protein encoded thereby (SEQ ID No: 2) ;
  • Figure 3 illustrates the nucleotide sequence of a gene encoding the secreted form of the RSV G protein lacking the transmembrane domain (SEQ ID No: 3) as well as the amino acid sequence of a truncated RSV G protein lacking the transmembrane domain encoded thereby (SEQ ID NO : 4 ) ;
  • Figure 4 shows the construction of plasmid pXL5 containing a gene encoding a full-length membrane attached form of the RSV G protein and containing the CMV Intron A sequence;
  • Figure 5 shows the construction of plasmid pXL6 containing a gene encoding a secreted form of the RSV G protein lacking the transmembrane domain and containing the CMV Intron A sequence as well as a nucleotide sequence encoding a signal peptide of the human tissue plasminogen activator (TPA) ;
  • Figure 6 shows the nucleotide sequence for the plasmid VR-1012 (SEQ ID No. 5) ;
  • Figure 7 shows the nucleotide sequence for the 5 ' untranslated region and the signal peptide of the human tissue plasminogen activator (TPA) (SEQ. ID no: 6) and Figure 8 shows the lung cytokine expression profile in DNA immunized mice after RSV challenge.
  • TPA tissue plasminogen activator
  • the present invention relates generally to polynucleotide, including DNA, immunization to obtain protection against infection by respiratory syncytial virus (RSV) and to diagnostic procedures using particular non-replicating vectors.
  • RSV respiratory syncytial virus
  • several recombinant plasmid vectors were constructed to contain a nucleotide sequence encoding an RSV G protein.
  • the nucleotide sequence of the full length RSV G gene is shown in Figure 2 (SEQ ID No: 1) .
  • Certain constructs provided herein include the nucleotide sequence encoding the full-length RSV G (SEQ ID No : 2) protein while others include an RSV G gene modified by deletion of the transmembrane coding sequence and nucleotides upstream thereof (see Figure 3, SEQ ID No: 3) , to produce a secreted or truncated RSV G protein lacking the transmembrane domain (SEQ ID No. 4) .
  • the nucleotide sequence encoding the RSV G protein is operatively coupled to a promoter sequence for expression of the encoded RSV G protein in vivo .
  • the promoter sequence may be the human immediately early cytomegalovirus (CMV) promoter. This promoter is described in ref. 19. Any other convenient promoter may be used, including constitutive promoters, such as, the Rous Sarcoma Virus LTRs, and inducible promoters, such as the metallothionin promoter, and tissue specific promoters .
  • CMV immediately early cytomegalovirus
  • Any other convenient promoter may be used, including constitutive promoters, such as, the Rous Sarcoma Virus LTRs, and inducible promoters, such as the metallothionin promoter, and tissue specific promoters .
  • the non-replicating vectors provided herein when administered to an animal in the form of an immunogenic composition with a pharmaceutically-acceptable carrier, effect in vivo RSV G protein expression, as demonstrated by an antibody response in the animal to which it is administered. Such antibodies may be used herein in the detection of RSV protein in a sample, as described in more detail below.
  • non- replicating vectors specifically plasmids pXL5 and pXL6, produced anti-G antibodies, virus neutralizing antibodies, a balanced Thl/Th2 response in the lungs post viral challenge and conferred protection in mice against live RSV infection, as seen from the Examples below.
  • the recombinant vector also may include a second nucleotide sequence located adjacent the RSV G protein encoding nucleotide sequence to enhance the immunoprotective ability of the RSV G protein when expressed in vivo in a host .
  • Such enhancement may be provided by increased in vivo expression, for example, by increased mRNA stability, enhanced transcription and/or translation.
  • This additional sequence generally is located between the promoter sequence and the RSV G protein-encoding sequence.
  • This enhancement sequence may comprise the immediate early cytomegalovirus Intron A sequence.
  • the non-replicating vector provided herein may also comprise an additional nucleotide sequence encoding a further antigen from RSV, an antigen from at least one other pathogen or at least one immunomodulating agent, such as a cytokine .
  • Such vector may contain the additional nucleotide sequence in a chimeric or a bicistronic structure.
  • vectors containing the additional nucleotide sequence may be separately constructed and coadministered to a host, along with the non-replicating vectors provided herein.
  • the non-replicating vector may further comprise a nucleotide sequence encoding a heterologous viral or eukaryotic signal peptide, such as the human tissue plasminogen activator (TPA) signal peptide, in place of the endogenous signal peptide for the truncated RSV G protein.
  • a heterologous viral or eukaryotic signal peptide such as the human tissue plasminogen activator (TPA) signal peptide
  • TPA tissue plasminogen activator
  • the immunogenicity of the non-replicating DNA vectors may be enhanced by inserting immunostimulatory CpG sequences in the vector.
  • Immunogenic compositions suitable to be used as vaccines, may be prepared from the RSV G genes and vectors as disclosed herein. The vaccine elicits an immune response in an animal which includes the production of anti-RSV G antibodies.
  • Immunogenic compositions, including vaccines, containing the nucleic acid may be prepared as injectables, in physiologically- acceptable liquid solutions or emulsions for polynucleotide administration.
  • the nucleic acid may be associated with liposomes, such as lecithin liposomes or other liposomes known in the art, as a nucleic acid liposome (for example, as described in WO 9324640, ref. 20) or the nucleic acid may be associated with an adjuvant, as described in more detail below.
  • Liposomes comprising cationic lipids interact spontaneously and rapidly with polyanions, such as DNA and RNA, resulting in liposome/nucleic acid complexes that capture up to
  • compositions for genetic immunization comprising cationic lipids and polynucleotides .
  • Agents which assist in the cellular uptake of nucleic acid such as calcium ions, viral proteins and other transfection facilitating agents, may advantageously be used.
  • Polynucleotide immunogenic preparations may also be formulated as microcapsules, including biodegradable time-release particles.
  • U.S. Patent 5,151,264 describes a particulate carrier of a phospholipid/glycolipid/polysaccharide nature that has been termed Bio Vendels Supra Mole vides (BVSM) .
  • BVSM Bio Vendels Supra Mole vides
  • the particulate carriers are intended to transport a variety of molecules having biological activity in one of the layers thereof.
  • U.S. Patent 5,075,109 describes encapsulation of the antigens trinitrophenylated keyhole limpet hemocyanin and staphylococcal enterotoxin B in 50:50 poly (DL-lactideco-glycolide) .
  • Other polymers for encapsulation are suggested, such as poly (glycolide) , poly (DL-lactide-co- glycolide), copolyoxalates, polycaprolactone, poly (lactide-co-caprolactone) , poly (esteramides) , polyorthoesters and poly (8- hydroxybutyric acid), and polyahhydrides .
  • WO 91/06282 describes a delivery vehicle comprising a plurality of bioadhesive microspheres and antigens.
  • the microspheres being of starch, gelatin, dextran, collagen or albumin.
  • This delivery vehicle is particularly intended for the uptake of vaccine across the nasal mucosae .
  • the delivery vehicle may additionally contain an absorption enhancer.
  • the RSV G gene containing non-replicating vectors may be mixed with pharmaceutically acceptable excipients which are compatible therewith.
  • excipients may include, water, saline, dextrose, glycerol, ethanol, and combinations thereof.
  • the immunogenic compositions and vaccines may further contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants to enhance the effectiveness thereof.
  • Immunogenic compositions and vaccines may be administered parenterally, by injection subcutaneously, intravenously, intradermally or intramuscularly, possibly following pretreatment of the injection site with a local anesthetic.
  • the immunogenic compositions formed according to the present invention may be formulated and delivered in a manner to evoke an immune response at mucosal surfaces.
  • the immunogenic composition may be administered to mucosal surfaces by, for example, the nasal or oral (intragastric) routes.
  • other modes of administration including suppositories and oral formulations may be desirable.
  • binders and carriers may include, for example, polyalkylene glycols or triglycerides.
  • Oral formulations may include normally employed incipients, such as, for example, pharmaceutical grades of saccharine, cellulose and magnesium carbonate.
  • the immunogenic preparations and vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective, protective and immunogenic.
  • the quantity to be administered depends on the subject to be treated, including, for example, the capacity of the individual's immune system to synthesize the RSV G protein and antibodies thereto, and if needed, to produce a cell-mediated immune response.
  • Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are readily determinable by one skilled in the art and may be of the order of about 1 ⁇ g to about 2 mg of the RSV G gene-containing vectors. Suitable regimes for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations.
  • the dosage may also depend on the route of administration and will vary according to the size of the host .
  • a vaccine which protects against only one pathogen is a monovalent vaccine.
  • Vaccines which contain antigenic material of several pathogens are combined vaccines and also belong to the present invention. Such combined vaccines contain, for example, material from various pathogens or from various strains of the same pathogen, or from combinations of various pathogens .
  • Immunogenicity can be significantly improved if the vectors are co-administered with adjuvants, commonly used as 0.05 to 0.1 percent solution in phosphate- buffered saline.
  • adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves.
  • Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses .
  • Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Thus, adjuvants have been identified that enhance the immune response to antigens.
  • adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines.
  • extrinsic adjuvants and other immunomodulating material can provoke potent immune responses to antigens.
  • these include saponins complexed to membrane protein antigens to produce immune stimulating complexes (ISCOMS) , pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS) , as well as monophoryl lipid A, QS 21 and polyphosphazene.
  • ISCOMS immune stimulating complexes
  • MDP muramyl dipeptide
  • LPS lipopolysaccharide
  • the non-replicating vector comprising a first nucleotide sequence encoding an G protein of RSV may be delivered in conjunction with a targeting molecule to target the vector to selected cells including cells of the immune system.
  • the immunogenicity of the non-replicating vector may be enhanced by coadministering plasmid DNA vectors expressing cytokines or chemokines or by coexpressing such molecules in a bis-cistronic or fusion construct.
  • the non-replicating vector may be delivered to the host by a variety of procedures, for example, Tang et al . (ref. 21) disclosed that introduction of gold microprojectiles coated with DNA encoding bovine growth hormone (BGH) into the skin of mice resulted in production of an i-BGH antibodies in the mice, while Furth et al . (ref. 22) showed that a jet injector could be used to transfect skin, muscle, fat and mammary tissues of living animals. 2. I ⁇ nunoassays
  • the RSV G genes and vectors of the present invention are useful as immunogens for the generation of anti-G antibodies for use in immunoassays, including enzyme-linked immunosorbent assays (ELISA) , RIAs and other non-enzyme linked antibody binding assays or procedures known in the art.
  • ELISA assays the non- replicating vector first is administered to a host to generate antibodies specific to the RSV G protein.
  • RSV G-specific antibodies are immobilized onto a selected surface, for example, a surface capable of binding the antibodies, such as the wells of a polystyrene microtiter plate.
  • a non-specific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample, may be bound to the selected surface.
  • BSA bovine serum albumin
  • the immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • This procedure may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween.
  • BGG bovine gamma globulin
  • PBS phosphate buffered saline
  • the sample is then allowed to incubate for from about 2 to 4 hours, at temperatures such as of the order of about 20° to 37°C.
  • the sample-contacted surface is washed to remove non-immunocomplexed material.
  • the washing procedure may include washing with a solution, such as PBS/Tween or a borate buffer. Following formation of specific immunocomplexes between the test sample and the bound RSV G specific antibodies, and subsequent washing, the occurrence, and even amount
  • BIOLOGICAL MATERIALS Certain plasmids that contain the gene encoding the RSV G protein and referred to herein have been deposited with the American Type Culture Collection (ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., pursuant to the Budapest Treaty and prior to the filing of this application. Samples of the deposited plasmids will become available to the public upon grant of a patent based upon this United States patent application and all restrictions on access to the deposits will be removed at that time. Samples of the deposited plasmids will be replaced if the depository is unable to dispense viable samples.
  • ATCC American Type Culture Collection
  • This Example describes the construction of vectors containing the RSV G gene.
  • Figure 1 shows a restriction map of the gene encoding the G protein of respiratory syncytial virus and Figure 2 shows the nucleotide sequence of the gene encoding the full-length RSV G protein (SEQ ID No: 1) and the deduced amino acid sequence (SEQ ID No: 2) .
  • Figure 3 shows the gene encoding the secreted RSV G protein (SEQ ID No: 3) and the deduced amino acid sequence (SEQ ID No: 4) .
  • Plasmid pXL5 ( Figure 4) was prepared for the expression of the full-length RSV G protein as follows:
  • RSV G12 Bluescript plasmid containing the cDNA encoding the full-length G protein of a clinical RSV isolate (subgroup A) was used to construct vectors for RSV DNA-G immunization.
  • RSV G12 was digested with Afllll and EcoRI and filled-in with the Klenow subunit of DNA polymerase. The resulting
  • VR-1012 Vical ( Figure 6) previously linearized with EcoRV. This procedure placed the RSV G cDNA downstream of the immediate-early cytomegalovirus (CMV) promoter and Intron A sequences of human cytomegalovirus (CMV) and upstream of the bovine growth hormone (BGH) poly-A site. The junctions of the cDNA fragments in the plasmid construct were confirmed by sequencing analysis. The resulting plasmid was designated pXL5.
  • Plasmid pXL6 ( Figure 5) was prepared for the expression of a secretory RSV G protein as follows:
  • RSV G12 was digested with EcoRI, filled-in with Klenow and digested again with BamHI .
  • the BamHI cleavage resulted in the generation of a cDNA fragment encoding a RSV G protein with N-terminal truncation.
  • This DNA segment was gel-purified and ligated in the presence of a pair of 11 mer oligodeoxynucleotides (5 ' GATCCACTCAG 3') (SEQ ID no : 7)
  • This Example describes the immunization of mice. Mice are susceptible to infection by RSV as described in ref. 24.
  • Plasmid DNA was purified through double CsCl centrifugations .
  • tibialis anterior muscles of BALB/c mice male, 6 to 8 week old (Jackson Lab., Bar Harbor, ME, USA) were bilaterally injected with 2 x 50 ⁇ g (l ⁇ g/ ⁇ L in PBS) of either pXL5, pXL6 or V-1012.
  • the muscles were treated with 2 x 50 ⁇ L (lO ⁇ M in PBS) of cardiotoxin (Latoxan, France) to increase DNA uptake and enhance immune responses, as reported by Davis et al (ref. 23) .
  • mice were boosted with the same dose of plasmid DNA 6 weeks and 13 weeks later, respectively.
  • lOO ⁇ g of the plasmid DNA (2 ⁇ g/ ⁇ L in PBS) of were injected at the base of the tail and boosted 6 weeks and 13 weeks later, respectively.
  • Mice in the positive control group were immunized intranasally (i.n.) with 10 6 plaque forming units (pfu) of a clinical RSV strain of the A2 subtype grown in Hep2 cells kindly provided by Dr. B. Graham (ref. 24).
  • mice Four weeks after the third immunization, mice were challenged intranasally with 10 6 pfu of the RSV A2 strain. Lungs were asceptically removed 4 days later, weighed and homogenized in 2 mL of complete culture medium (ref. 25) . The number of pfu in lung homogenates was determined in duplicate as previously described (ref. 26) using vaccine-quality Vero cells.
  • Example 3 The number of pfu in lung homogenates was determined in duplicate as previously described (ref. 26) using vaccine-quality Vero cells.
  • This Example describes the immunogenicity and protection by polynucleotide immunization.
  • Antisera obtained from immunized mice were analyzed for anti-RSV G IgG antibody titres using specific enzyme-linked immunosorbent assay (ELISA) and for RSV- specific plaque-reduction titres.
  • ELISAs were performed using 96-well plates coated with immunoaffinity-purified RSV G protein (50 ng/mL) and 2-fold serial dilutions of immune sera.
  • a goat anti -mouse IgG antibody conjugated to alkaline phosphatase Jackson ImmunoRes., Mississauga, Ontario, Canada
  • Plaque reduction titres were determined according to Prince et al (ref. 26) using vaccine- quality Vero cells.
  • mice refers to animals with no detectable RSV in the lungs 4 days post viral challenge.
  • plasmids pXL5 and pXL6 were found to be immunogenic following either i.m. or i.d. immunization producing anti-G antibodies and virus neutralizing antibodies.
  • the plasmids pXL5 and pXL6 protected immunized mice against primary RSV infection of the lower respiratory tract .
  • the control vector produced no immune response and did not confer protection.
  • Example 4 This Example describes the determination of the local lung cytokine expression profile in mice immunized with pXL5 and pXL6 after RSV challenge.
  • mice were immunized at 0 and 6 weeks with lOO ⁇ g of pXL5 and 6, prepared as described in Example 1, and challenged with RSV i.n. at 10 weeks.
  • Control animals were immunized with placebo PI -RSV and live RSV and challenged with RSV according to the same protocol.
  • animals were immunized with pXL2 , as described in copending United States Patent Application no. 08/476,397 filed June 7, 1995 (WO 96/40945) and challenged with RSV, also following the same protocol.
  • Four days post viral challenge lungs were removed from immunized mice and immediately frozen in liquid nitrogen.
  • RNA was prepared from lungs homogenized in TRIzol/ ⁇ -mercaptoethanol by chloroform extraction and isopropanol precipitation.
  • Reverse transcriptase- polymerase chain reaction (RT-PCR) was then carried out on the RNA samples using either IL-4, IL-5 or IFN- ⁇ specific primers from CloneTech.
  • the amplified products were then liquid-hybridized to cytokine-specific 32 P- labeled probes from CloneTech, resolved on 5% polyacrylamide gels and quantitated by scanning of the radioactive signals in the gels.
  • Three mouse lungs were removed from each treatment group and analyzed for lung cytokine expression for a minimum of two times. The data is presented in Figure 8 and represents the means and standard deviations of these determinations.
  • the magnitude of the cytokine responses with i.m. pXL6 (RSV G) and pXL2 (RSV F) immunization using the construct expressing a secretory form of the protein (SEC) is significantly higher than that with live RSV immunization.
  • the present invention provides certain novel non-replicating vectors containing genes encoding RSV G proteins, methods of immunization using such vectors and methods of diagnosis using such vectors. Modifications are possible within the scope of this invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Urology & Nephrology (AREA)
  • Pulmonology (AREA)
  • Communicable Diseases (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On décrit des vecteurs à reproduction unique (vecteurs plasmidiques, par exemple) qui contiennent une séquence nucléotidique codant pour une protéine G du virus respiratoire syncytial (VRS), et un promoteur pour ladite séquence, de préférence un promoteur du cyclomégalovirus. Lesdits vecteurs peuvent également contenir une autre séquence nucléotidique adjacente à la séquence codant la protéine G du VRS pour améliorer la capacité d'immunoprotection de cette dernière lorsqu'elle est exprimée in vivo. De tels vecteurs à reproduction unique peuvent être administrés à un hôte, y compris un hôte humain, pour l'immuniser contre l'infection à VRS. Ces vecteurs peuvent également s'utiliser pour produire des anticorps capables de détecter une infection à VRS dans un échantillon.
PCT/CA1998/000697 1997-07-18 1998-07-16 Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial WO1999004010A1 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP98934710A EP0996730A1 (fr) 1997-07-18 1998-07-16 Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial
BR9815496-6A BR9815496A (pt) 1997-07-18 1998-07-16 Vacinas de ácido nucléico que codificam proteìna gde vìrus sincicial respiratório
CA002296089A CA2296089A1 (fr) 1997-07-18 1998-07-16 Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial
NZ502626A NZ502626A (en) 1997-07-18 1998-07-16 Vaccines comprising a nucleic acid vector encoding the G protein of respiratory syncytial virus (RSV)
JP2000503216A JP2001512662A (ja) 1997-07-18 1998-07-16 呼吸器合胞体ウイルスのｇタンパク質をコードする核酸ワクチン
AU84278/98A AU756222B2 (en) 1997-07-18 1998-07-16 Nucleic acid vaccines encoding G protein of respiratory syncytial virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US89644297A 1997-07-18 1997-07-18
US08/896,442 1997-07-18

Publications (1)

Publication Number Publication Date
WO1999004010A1 true WO1999004010A1 (fr) 1999-01-28

Family

ID=25406219

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA1998/000697 WO1999004010A1 (fr) 1997-07-18 1998-07-16 Vaccins d'acide nucleique codant une proteine g du virus respiratoire syncytial

Country Status (8)

Country Link
EP (1) EP0996730A1 (fr)
JP (1) JP2001512662A (fr)
CN (1) CN1174099C (fr)
AU (1) AU756222B2 (fr)
BR (1) BR9815496A (fr)
CA (1) CA2296089A1 (fr)
NZ (1) NZ502626A (fr)
WO (1) WO1999004010A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053767A2 (fr) * 1999-03-05 2000-09-14 Aventis Pasteur Limited Vaccins d'acide nucléique codant pour une protéine du virus respiratoire syncytial
FR2790959A1 (fr) * 1999-03-15 2000-09-22 Pf Medicament Utilisation de fractions membranaires bacteriennes a effet adjuvant, leurs procedes de preparation et composition pharmaceutique les contenant
WO2001021203A1 (fr) * 1999-09-23 2001-03-29 Pierre Fabre Medicament Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale
JP2004500366A (ja) * 1999-12-01 2004-01-08 カイロン　コーポレイション Ｃ型肝炎ウイルス（ｈｃｖ）について特異的な抗体を誘発すること
WO2009038270A1 (fr) * 2007-09-21 2009-03-26 Ewha University - Industry Collaboration Foundation Vaccin contre le virus respiratoire syncytial
US8287870B2 (en) 2008-07-16 2012-10-16 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US8298538B2 (en) 2007-01-04 2012-10-30 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US8309089B2 (en) 2007-01-04 2012-11-13 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
KR101366702B1 (ko) 2010-10-21 2014-02-28 이화여자대학교 산학협력단 호흡기 신시치아 바이러스 백신 조성물 및 그의 제조방법
EP4013782A4 (fr) * 2019-08-12 2023-12-27 Advaccine (Suzhou) Biopharmaceuticals Co. Ltd. Composition immunitaire comprenant un polypeptide g du virus respiratoire syncytial (vrs)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111303278B (zh) * 2013-04-15 2022-09-06 扬森疫苗与预防公司 结合到rsv g蛋白的人类抗体
WO2024139647A1 (fr) * 2022-12-26 2024-07-04 中国医学科学院基础医学研究所 Vaccin à adn de papillomavirus humain de type 16 et son utilisation

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5620896A (en) * 1992-03-23 1997-04-15 University Of Massachusetts Medical Center DNA vaccines against rotavirus infections

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5620896A (en) * 1992-03-23 1997-04-15 University Of Massachusetts Medical Center DNA vaccines against rotavirus infections

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
ELANGO N ET AL: "Resistance to human respiratory syncytial virus ( RSV ) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1986 MAR) 83 (6) 1906-10. JOURNAL CODE: PV3. ISSN: 0027-8424., United States, XP002080966 *
HSU K H ET AL: "Immunogenicity of recombinant adenovirus- respiratory syncytial virus vaccines with adenovirus types 4, 5, and 7 vectors in dogs and a chimpanzee.", JOURNAL OF INFECTIOUS DISEASES, (1992 OCT) 166 (4) 769-75. JOURNAL CODE: IH3. ISSN: 0022-1899., United States, XP002080964 *
JOHNSON T R ET AL: "Priming with secreted glycoprotein G of respiratory syncytial virus ( RSV ) augments interleukin-5 production and tissue eosinophilia after RSV challenge.", JOURNAL OF VIROLOGY, (1998 APR) 72 (4) 2871-80. JOURNAL CODE: KCV. ISSN: 0022-538X., United States, XP002080969 *
NGUYEN T N ET AL: "Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus.", JOURNAL OF BIOTECHNOLOGY, (1995 OCT 16) 42 (3) 207-19. JOURNAL CODE: AL6. ISSN: 0168-1656., Netherlands, XP000561819 *
ROBERTS S R ET AL: "The membrane-associated and secreted forms of the respiratory syncytial virus attachment glycoprotein G are synthesized from alternative initiation codons.", JOURNAL OF VIROLOGY, (1994 JUL) 68 (7) 4538-46. JOURNAL CODE: KCV. ISSN: 0022-538X., United States, XP002080967 *
SCHRIJVER R S ET AL: "Comparison of DNA application methods to reduce BRSV shedding in cattle", VACCINE, vol. 16, no. 2-3, 2 January 1998 (1998-01-02), pages 130-134, XP004098613 *
STOTT E J ET AL: "Human respiratory syncytial virus glycoprotein G expressed from a recombinant vaccinia virus vector protects mice against live-virus challenge.", JOURNAL OF VIROLOGY, (1986 NOV) 60 (2) 607-13. JOURNAL CODE: KCV. ISSN: 0022-538X., United States, XP002080963 *
STOTT E J ET AL: "Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.", JOURNAL OF VIROLOGY, (1987 DEC) 61 (12) 3855-61. JOURNAL CODE: KCV. ISSN: 0022-538X., United States, XP002080965 *
WATHEN M W ET AL: "Characterization of a novel human respiratory syncytial virus chimeric FG glycoprotein expressed using a baculovirus vector.", JOURNAL OF GENERAL VIROLOGY, (1989 OCT) 70 ( PT 10) 2625-35. JOURNAL CODE: I9B. ISSN: 0022-1317., ENGLAND: United Kingdom, XP002080968 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053767A2 (fr) * 1999-03-05 2000-09-14 Aventis Pasteur Limited Vaccins d'acide nucléique codant pour une protéine du virus respiratoire syncytial
WO2000053767A3 (fr) * 1999-03-05 2000-12-28 Connaught Lab Vaccins d'acide nucléique codant pour une protéine du virus respiratoire syncytial
FR2790959A1 (fr) * 1999-03-15 2000-09-22 Pf Medicament Utilisation de fractions membranaires bacteriennes a effet adjuvant, leurs procedes de preparation et composition pharmaceutique les contenant
WO2001021203A1 (fr) * 1999-09-23 2001-03-29 Pierre Fabre Medicament Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale
FR2798857A1 (fr) * 1999-09-23 2001-03-30 Pf Medicament Utilisation d'une proteine de membrane ompa d'enterobacterie associee a un peptide immunogene du vrs pour la preparation de vaccins administrables par voie nasale
JP2004500366A (ja) * 1999-12-01 2004-01-08 カイロン　コーポレイション Ｃ型肝炎ウイルス（ｈｃｖ）について特異的な抗体を誘発すること
US9217028B2 (en) 2007-01-04 2015-12-22 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US9611316B2 (en) 2007-01-04 2017-04-04 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US8298538B2 (en) 2007-01-04 2012-10-30 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US8309089B2 (en) 2007-01-04 2012-11-13 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US9149524B2 (en) 2007-01-04 2015-10-06 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
US8545848B2 (en) 2007-01-04 2013-10-01 Institute For Research In Biomedicine Human cytomegalovirus neutralising antibodies and use thereof
KR100938105B1 (ko) * 2007-09-21 2010-01-21 이화여자대학교 산학협력단 호흡기 신시치아 바이러스 백신
WO2009038270A1 (fr) * 2007-09-21 2009-03-26 Ewha University - Industry Collaboration Foundation Vaccin contre le virus respiratoire syncytial
US8765132B2 (en) 2008-07-16 2014-07-01 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9527902B2 (en) 2008-07-16 2016-12-27 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US8603480B2 (en) 2008-07-16 2013-12-10 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9127049B2 (en) 2008-07-16 2015-09-08 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US8435524B2 (en) 2008-07-16 2013-05-07 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US8298539B2 (en) 2008-07-16 2012-10-30 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9221897B2 (en) 2008-07-16 2015-12-29 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9249213B2 (en) 2008-07-16 2016-02-02 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9365636B1 (en) 2008-07-16 2016-06-14 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9371372B2 (en) 2008-07-16 2016-06-21 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9491906B2 (en) 2008-07-16 2016-11-15 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US10889632B2 (en) 2008-07-16 2021-01-12 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US8287870B2 (en) 2008-07-16 2012-10-16 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9725502B2 (en) 2008-07-16 2017-08-08 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9796772B2 (en) 2008-07-16 2017-10-24 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9796771B2 (en) 2008-07-16 2017-10-24 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US9803000B1 (en) 2008-07-16 2017-10-31 Institute of Research in Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US10040845B2 (en) 2008-07-16 2018-08-07 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
US10414817B2 (en) 2008-07-16 2019-09-17 Institute For Research In Biomedicine Human cytomegalovirus neutralizing antibodies and use thereof
KR101366702B1 (ko) 2010-10-21 2014-02-28 이화여자대학교 산학협력단 호흡기 신시치아 바이러스 백신 조성물 및 그의 제조방법
EP4013782A4 (fr) * 2019-08-12 2023-12-27 Advaccine (Suzhou) Biopharmaceuticals Co. Ltd. Composition immunitaire comprenant un polypeptide g du virus respiratoire syncytial (vrs)

Also Published As

Publication number Publication date
CA2296089A1 (fr) 1999-01-28
EP0996730A1 (fr) 2000-05-03
NZ502626A (en) 2002-09-27
CN1271389A (zh) 2000-10-25
AU756222B2 (en) 2003-01-09
AU8427898A (en) 1999-02-10
JP2001512662A (ja) 2001-08-28
CN1174099C (zh) 2004-11-03
BR9815496A (pt) 2001-12-18

Similar Documents

Publication Publication Date Title
EP0832253B1 (fr) Vaccins a acides nucleiques du virus respiratoire syncytial
US6486135B1 (en) Nucleic acid respiratory syncytial virus vaccines
US6428324B1 (en) RNA respiratory syncytial virus vaccines
US5691449A (en) Respiratory syncytial virus vaccines
JPH07501707A (ja) キメラ免疫原
JPH03502687A (ja) レスピラトリイ・シンシチアル・ウイルス：ワクチンおよび診断法
AU756222B2 (en) Nucleic acid vaccines encoding G protein of respiratory syncytial virus
AU749345B2 (en) Alphavirus vectors for paramyxovirus vaccines
US6017897A (en) Nucleic acid respiratory syncytial virus vaccines
CN110996998A (zh) 表达rsv g的重组嵌合牛/人副流感病毒3及其用途
MXPA00000636A (en) Nucleic acid vaccines encoding g protein of respiratory syncytial virus
KR20230021115A (ko) 생 약독화된 호흡기 세포융합 바이러스 (live attenuated respiratory syncytial virus)
EP1707633A1 (fr) Vecteurs de type alphavirus comme vaccins contre les paramyxovirus
MXPA00004708A (en) Alphavirus vectors for paramyxovirus vaccines
MXPA00002236A (en) Rna respiratory syncytial virus vaccines

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 98809301.4

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2296089

Country of ref document: CA

Ref document number: 2296089

Country of ref document: CA

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: KR

WWE Wipo information: entry into national phase

Ref document number: PA/a/2000/000636

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 502626

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: 84278/98

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1998934710

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09462816

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1998934710

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWG Wipo information: grant in national office

Ref document number: 84278/98

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1998934710

Country of ref document: EP