EP0550485A1 - Ein adjuvans enthaltende gp160 derivate, impfstoffe auf gp160- oder derivatbasin - Google Patents

Ein adjuvans enthaltende gp160 derivate, impfstoffe auf gp160- oder derivatbasin

Info

Publication number
EP0550485A1
EP0550485A1 EP91916067A EP91916067A EP0550485A1 EP 0550485 A1 EP0550485 A1 EP 0550485A1 EP 91916067 A EP91916067 A EP 91916067A EP 91916067 A EP91916067 A EP 91916067A EP 0550485 A1 EP0550485 A1 EP 0550485A1
Authority
EP
European Patent Office
Prior art keywords
protein
gpl60
hiv
vaccine
oligomer
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP91916067A
Other languages
English (en)
French (fr)
Inventor
Frans Van Wijnendale
Moncef Slaoui
Claudine Bruck
Myriam Francotte
Suzy Kummert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
SmithKline Beecham Biologicals SA
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
Priority claimed from GB909021175A external-priority patent/GB9021175D0/en
Priority claimed from GB919106048A external-priority patent/GB9106048D0/en
Application filed by SmithKline Beecham Biologicals SA filed Critical SmithKline Beecham Biologicals SA
Priority to EP94202409A priority Critical patent/EP0644201A1/de
Publication of EP0550485A1 publication Critical patent/EP0550485A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Retroviruses that is, viruses within the family
  • Retroviridae are a large family of enveloped, icosohedral viruses of about 150 nm having a coiled nucleocapsid within the core structure and having RNA as the genetic material.
  • the family comprises the oncoviruses such as the sarcoma and leukemia viruses, certain immunodeficiency viruses and the lentiviruses.
  • HIV-1 Human immunodeficiency virus type 1
  • LTRs long terminal repeats
  • cracr. pol cracr. pol
  • env genes genes that are potential candidates either alone or in concert as vaccinal agents capable of inducing a protective immune response.
  • the HIV-1 envelope protein is synthesized as a polyprotein precursor which is subsequently glycosylated within infected cells to give a glycoprotein with a mol. weight of 160 kDa (gpl60) , which is processed by proteolysis into a gpl20 external glycoprotein and gp41 transmembrane protein.
  • the proviral DNA sequence of HIV-BH10-2 is described by Ratner et al.., ' 'Human Retroviruses and AIDS" 1989, HIV Sequence Database ed. Gerald Meyers, Los Alamos National Laboratory-.
  • the sequence is 8932 bp long, the env gene being located at 5580-8150 and the cleavage sites therein at 7088 and 7112 corresponding to amino acids 503 and 511 (cleavage after these residues) (numbering according to Los Alamos database) .
  • Suitable replacements for lysine are those similar to lysine in hydrophilicity and size, such as histidine, threonine, serine, asparagine, aspartic acid, glutamine and glutamic acid. Of these, the preferred amino acid is glutamic acid.
  • the preferred protein is both uncleavable due to the mutations introduced and also able of eliciting cross neutralising antibody, which is dependent on the correct folding of the protein.
  • the invention provides the modified protein of the invention, in oligomeric form.
  • a relative molecular weight 640 kDa Based on the molecular weight of gpl60 monomer, this form is believed to be tetrameric. This is advantageous since viral surface proteins naturally exist as oligomers which in-vivo form spikes which protude from the viral surface. As many neutralising epitopes are conformational, it is clearly important to mimic as closely as possible the form of the antigen as it appears naturally, since this will provide the most relevant immune response.
  • substantially pure form is meant at least 75% pure, more preferably 90% pure, more preferably 99% pure.
  • the DNA polymer comprising a nucleotide sequence that encodes the modified protein also forms part of the invention.
  • the recombinant DNA molecule of the invention may be prepared in accordance with the invention by the condensation of appropriate mono-, di- or oligomeric nucleotide units.
  • the preparation may be carried out chemically, enzymatically, or by a combination of the two methods,in vitro or ⁇ j ⁇ vivo as appropriate.
  • the DNA molecule may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al in Biochemistry 1985, 24., 5090-5098.
  • the DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.
  • Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20°-70°C, generally in a volume of 50 ⁇ l or less with 0.1-10 ⁇ g DNA.
  • Enzymatic polymerisation of DNA may be carried out in. vitro using a DNA polymerase such as DNA polymerase I (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10°-37°C, generally in a volume of 50 ⁇ l or less. Fragments can be polymerised and amplified by polymerase chain reaction using Taq polymerase (ref. PCR Protocols 1989 - a guide to Methods and Applications, Ed. M.A. Innis et. a.., Acadamic Press) .
  • Taq polymerase ref. PCR Protocols 1989 - a guide to Methods and Applications, Ed. M.A. Innis et. a.., Acadamic Press
  • Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
  • a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4°C to ambient, generally in a volume of 50 ⁇ l or less.
  • the chemical synthesis of the DNA molecule or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in 'Chemical and Enzymatic Synthesis of Gene Fragments - A Laboratory Manual' (ed. H.G. Gassen and A. Lang), Verlag Chemie, einheim (1982),or in other scientific publications, for example M.J. Gait, H. .D. Matthes, M. Singh, B.S. Sproat, and R.C. Titmas, Nucleic Acids Research, 1982, JL0., 6243; B.S. Sproat and W.
  • DNA polymer which encodes the modified protein may be prepared by site directed mutagenesis of the cDNA which codes for unmodified protein, by conventional methods such as those described by G. Winter et al. in Nature 1982, 299, 756-758 or by Zoller and Smith 1982; Nucl. Acids Res., JLO., 10 6487-6500.
  • the vector may be prepared in accordance with the invention by cleaving a vector to provide a linear DNA segment having a intact replicon, and ligating said linear segment and one or more DNA molecules which, together with said linear 0 segment complete the recombinant DNA molecule of the invention.
  • reducing agent should be avoided, and non ionic detergents such as Decyl PEG-300
  • a preferred affinity chromatography medium is Lentil lectin Sepharose and a preferred immunoaffinity chromatography medium is an anti-gpl60 monoclonal antibody such as 178.1 (WO 90/06358) coupled on a suitable carrier such as glutaraldehyde-activated Trisacryl (IBF) .
  • the modified protein may be adsorbed from the monoclonal antibody in the presence of the detergent octyl glucopyranoside which can be removed by dialysis.
  • the detergents are preferably used at concentrations above their theoretical critical micelle concentration.
  • the modified protein produced in accordance with this invention is useful as a diagnostic agent for detection of exposure to HIV.
  • the modified protein is also useful in vaccines for the prevention of infection or for the inhibition or prevention of disease progression.
  • This invention also relates to a vaccine and pharmaceutical compositions containing the modified protein of this invention.
  • Such compositions will contain an immunoprotective quantity of the modified protein of this invention and maybe prepared by conventional techniques.
  • an aqueous solution of the protein can be used directly.
  • the protein with or without prior lyophilization, can be mixed or absorbed with any of the various known adjuvants.
  • adjuvants include, but are not limited to, aluminium hydroxide, muramyl dipeptide and saponins such as Quil A, 3D-MPL (3Deacylated monophosphoryl lipid A) , or TDM.
  • the protein can be encapsulated within microparticles such as liposomes.
  • the protein can be conjugated to an immuostimulating macromolecule, such as killed Bordetella or a tetanus toxoid.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
  • Encapsulation within liposomes is described, for example, by Fullerton, U.S. Patent 4,235,877.
  • Conjugation of proteins to macromolecules is disclosed, for example, by Likhite, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
  • the amount of the modified protein of the present invention in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and whether or not the vaccine is adjuvanted. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of modified protein, preferably 10-200 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists.
  • the invention further provides modified protein of the invention for use in vaccinating a host and use of modified protein of the invention in the preparation of a vaccine.
  • compositions of the present invention may be used to treat, immunotherapeutically, patients suffering from HIV infections.
  • a method of treating a human susceptible to or suffering from an HIV infection by administering an effective amount of the modified gpl60 as herein described.
  • a pharmaceutical formulation comprising gp 160 or an immunological derivative thereof and 3D Monophosphoryl lipid A (3D-MPL) with a suitable carrier.
  • the gp 160 or an immunological derivative thereof and 3D- MPL are presented in an oil in water emulsion. This system provides enhanced neutralising activity.
  • a pharmaceutical or vaccine formulation comprising gp 160 or an immunological derivative thereof,3D- MPL in an oil in water carrier, said carrier comprising an emulsion of a tetrapolyol and a non toxic mineral oil in a buffered saline solution.
  • the carrier comprises a Pluronic polyol such as Pluronic L121, and squalane or squalene or other metabolisable oils
  • An emulsifer such as Tween 80 or Tween 28 is preferably provided to stabilise the emulsion.
  • the concentration of antigen in the final formulation is preferably between lO ⁇ g to 150 ⁇ g/ml, more preferably between 20 ⁇ g to lOO ⁇ g/ml.
  • the concentration range of adjuvant, 3D-MPL, in the vaccine is preferably between lO ⁇ g to lOO ⁇ g/ml more preferably between 25 to 50 ⁇ g/ml.
  • the present invention further provides the vaccine formulations as herein described for use in medicine, in particular for use in the treatment by immonotherapy and prophylatic treatment of HIV-1 infections such as AIDS or AIDS related complex (ARC) .
  • HIV-1 infections such as AIDS or AIDS related complex (ARC) .
  • a method of producing a vaccine comprising gpl60 or an immunological derivative thereof, 3D monophosphoryl lipid A with a suitable carrier, the method comprising mixing gpl60 or immunological derivative with said carrier and with 3D monophosphoryl lipid A.
  • a method of producing a vaccine as herein described in an oil in water carrier wherein an oil in water emulsion is microfluidized to provide sub micron particles in said emulsion and mixed with gpl60 or immunological derivative thereof and 3D MPL.
  • the 3D-MPL is premixed with the emulsion, thereafter the antigen is mixed into the resulting composition.
  • 3D-MPL may be obtained by the methods disclosed in British Patent 2211502.
  • Suitable carriers in this context comprise oil in water emulsions.
  • the formulations of the present invention provide enhanced neutralising titres when compared with conventional vaccine formulations comprising, alum alone as the adjuvant (the only adjuvant licensed for human use) .
  • immunological derivatives is used herein to include immunogenic fragments of gp 160 which when adjuvanted with 3D Monophosphoryl lipid A are capable of raising neutralising antibodies against HIV-1.
  • this will include, for example the HIV-1 outer membrane glycoprotein gp 120, modified gp 160 as herein described as well as the naturally occurring isolated gp 160. Particularly preferred are those derivatives which are also able to raise a DTH response.
  • the invention provides the modified form of the protein in oligomeric form which when purified under gentle, non-reducing conditions is shown to have an apparent molecular weight of between 600-700 KDa 640 Kd and is believed to be a tetramer. This tetramer may be destabilised by running the protein on SDS gel under non- reducing conditions, which then provides a dimer of 330 kd and a monomer. The dimer may further be reduced under standard reducing conditions to yield the monomer.
  • gp 160 or derivatives thereof may be achieved by methods known in the art. Typically this will involve the cloning and expression of the gene encoding for gp 160 in a suitable host.
  • the production of recombinant gp 160 (rgp 160) in such ways may be achieved using the techniques described in Maniatis et al; Molecular Cloning - A laboratory Manual; Cold Spring Harbour 1982.
  • a variety of eukaryotic cells and expression systems are available for expression of the recombinant DNA molecules.
  • the most widely used among these are yeast, insect and mammalian systems, although the invention is not limited to use of these.
  • Such systems employ a recombinant DNA molecule of the invention, optionally a selection marker and, in some cases, maintenance functions such as an origin of replication.
  • Insect cells which can be used in the invention include Drosophila cells and Lepidoptera cells.
  • Useful Drosophila cells include SI, S2 , S3, KC-O and D. hydei cells. See, for example, Schneider et al., J. Embryo1. EXP. Morph. 27;353 (1972); Schulz et al., Proc. Natl. Acad. Sci USA 83;9428 (1986); Sinclair et al., Mol. Cell. Biol. 5:3208 (1985).
  • Drosophila cells are transfected by standard techniques, including calcium phosphate precipitation, cell fusion, electroporation and viral transfection. Cells are cultured in accordance with standard cell culture procedures in a variety of nutrient media, including, e.g., M3 media which consists of balanced salts and essential amino acids. See, Lindquist, PIS 58:163 (1982).
  • Promoters known to be useful in Drosophila include mammalian cell promoters such as SV40 as well as Drosophila promoters, the latter being preferred, examples of useful Drosophila promoters include the Drosophila metallothionein promoter, the 70 kilodalton heatshock protein promoter (HSP70) and the COPIA LTR. See, for example, DiNocera et al., Proc. Natl. Acad. Sci. USA 80;7095 (1983); McGarry et al.. Cell 4.2;903 (1985) .
  • Useful Lepidoptera cells include cells from Trichoplusia ni., Spodoptera fru ⁇ iperda, Heliothis zea, Auto ⁇ raphica californica, Rachiplusis ou, Galleria melonella, Manduca sexta or other cells which can be infected with Baculoviruses, including nuclear polyhedrosis viruses (NPV) , single nucleocapsid viruses (SNPV) and multiple nucleocapsid viruses (MNPV) .
  • NPV nuclear polyhedrosis viruses
  • SNPV single nucleocapsid viruses
  • MNPV multiple nucleocapsid viruses
  • the preferred Baculoviruses are NPV or MNPV Baculoviruses because these contain the polyhedrin gene promoter which is highly expressed in infected cells.
  • the antigen to be free of detectable contaminating proteins after chromatogrphy on the immunoaffinity column.
  • the final product might be contaminated with trace amounts of monoclonal antibody leaking from the immunoaffinity column. Therefore, the amount of 178.1 antibody in the pure gpl60 was measured by ELISA and ranged from 0.004% to 0.02% of the total amount of protein present in the samples.
  • the vehicle was prepared as follows. Pluronic L121 5% (BASF Wyandotte, New Jersey) (v/v) and 10% squalane (Aldrich) were added to phosphate-buffered saline (PBS) containing 0.4% (v/v) Tweea 80. This mixture was then microfluidized. For microfluidization, the emulsion was cycled ten times through a microfluidizer (Model MHO Microfluidics Corp., Newton, Mass.). The resulting emulsion comprised only submicron particles. One volume of this emulsion was mixed to an equal volume of twice concentrated rgpl60 (either 20 ⁇ g or 100 ⁇ g) and vortexed briefly to ensure complete mixing of the components.
  • a capture enzyme immunoassay (EIA) based on a lysate of HIV- 1 (IIIB) infected cells was used to determine the ELISA titer of the antisera after the first and second boost.
  • the test used is very similar to that published by Moore et al. [Moore J.P. et al., 1989, AIDS 3_:155 (63)].
  • the microplate neutralization assay is based on the detection of HIV gag antigen in indicator cells. Briefly, SupTl cells (Hecht et al., 1984, Science 226:1445) are used as indicator cells.
  • the viral inoculum consists of cell free supernatant of a HIV-1 (IIIB) producing lymphoid cell line. The supernatant is centrifuged at high speed to eliminate cells and cell debris, aliquoted in 1 ml vials and stored at -80°C until use.
  • the sera to be tested are inactivated at 56°C for 30 min. prior to testing.
  • Our negative control consists of a pool of sera from preimmune or adjuvant alone inoculated animals (same species as the sera to be tested) .
  • TCID5 For neutralization, 750 TCID5 are incubated with serial two fold dilutions of the sera for 1 hour at 37°C. SupTl cells are then added (4.10 4 cells/well) and incubated 4 days at 37°C. The cytopathic effect is microscopically monitored, Triton X-100 (1 % final concentration) is added to each well and the plate is frozen. A sandwich ELISA is used to monitor the relative amount of viral antigen produced in the cultures. The plates are coated with an anti p55 monoclonal antibody.
  • Triton X-100 treated samples are incubated in the plate and the presence of gag antigen is visualized by biotinylated HIV-1 + human IgGs followed by a streptavidin peroxidase step.
  • the percentages of reduction of HIV-1 antigen production relative to the control are then evaluated for all the serum dilutions tested. Using a curve fit to the data points by non linear least squares analysis, the serum dilution (if any) giving a 50% reduction in antigen production compared to control, is extrapolated.
  • Table 2 shows the neutralizing antibody titer after tertiary immunization.
  • vaccinia gpl60 of the invention is considered 0 of potential use for HIV-1 vaccination.
  • Rhesus monkeys (Macaca mulatta) weighing 3.5 to 5 kg were randomly assigned into seven groups containing 3 or 4 animals per group.
  • 750 TCID ⁇ are incubated with serial two fold dilutions of the sera for 1 hour at 37°C.
  • SupTl cells are then added (2.10 4 cells/well) and incubated 4 days at 37°C.
  • the cytopathic effect is microscopically monitored, Triton X-100 (1% final concentration) is added to each well and the plate is frozen.
  • a sandwich ELISA is used to monitor the relative amount of viral antigen produced in the cultures.
  • the plates are coated with an anti p55 monoclonal antibody.
  • the above Triton X-100 treated samples are incubated in the plate and the presence of gag antigen is visualized by biotinylated HIV-1 + human IgGs followed by a streptavidin peroxydase step.
  • the percentages of reduction of HIV-1 antigen production relative to the controls are then evaluated for all the serum dilutions tested. Using a curve fit to the da ⁇ a points by linear regression analysis, the serum dilution (if any) giving a 50% reduction in antigen production compared to controls, is extrapolated.
  • Table 4 shows the ELISA and neutralizing antibody titer after the second immunization.
  • the general immunogenicity of HIV rgpl60 in 3D-MPL o/w is very good.
  • the neutralising titre (NT) and ELISA titers observed were extremely good in the group of animals receiving rgpl60 in 3D-MPL o/w (group 4) .
  • NT neutralising titre
  • ELISA titers observed were extremely good in the group of animals receiving rgpl60 in 3D-MPL o/w (group 4) .
  • These data suggest a superior adjuvant effect of the 3D-MPL o/w emulsion.
  • the immune 5 response of animals immunized with gpl60 adsorbed on Alum in the presence of 3D-MPL is poor although some, neutralising antibody is produced.
  • the HIV gpl60 contains an hydrophobic moiety and this probably confers the molecule a high affinity for lipids.
  • the recombinant proteins were formulated with 3D MPL (100 ⁇ g/dose) in an o/w emulsion.
  • the formulation consisted of 0.2% Tween 80, 2.5% Pluronic L121, 5% squalane, 100 ⁇ g 3D MPL and recombinant antigen dose (100 ⁇ g for rgpl60 or rgpl20; in a 1 ml injection volume.
  • chimpanzees vaccinated either with rgpl60 or rgp 120 delivered in a 3D MPL o/w emulsion produced high ELISA and neutralizing titers after 3 immunizations.
  • Neutralizing activity could be detected in 3 sera out of 4 after 2 injections and in 4 out of 4 after a further boost.
  • An increase of the neutralizing titers was observed following a third immunization.
  • the ELISA and neutralizing titers measured after this boost are very similar to those obtained in rhesus monkeys immunized three times with the same rg ⁇ l60 formulation.
  • the microplate neutralization assay is based on the visual evaluation of CPE induced by HIV1 infection in indicator cells. Briefly, SupTl cells (Hecht et al, 1984, Science 226;1445) are used as indicator cells.
  • the viral inoculum consists of cell free supernatant of HIV-1 (IIIB) producing lymphoid cell line. The supernatant is centrifuged at high speed to eliminate cells and cell debris, aliquoted in 1 ml vials and stored at -80°C until use.
  • the sera to be tested are inactivated at 56°C for 30 min. prior to testing.
  • Our negative control consists of a pool of sera from preimmune animals.
  • 750 TCID ⁇ Q are incubated with serial two fold dilutions of the sera for 1 hour at 37°C SupTl cells are then added (2.10 cells/well) and incubated 4 days at 37°C
  • the cytopathic effect is microscopically monitored on day 4 and the neutralizing titers are visually determined.
  • the given neutralization titers correspond to the reciprocal of the dilution of serum giving 80% reduction of syncytia formation as compared to preimmune controls.
  • the visually determined titers are further objectivated on day 7 by measuring cell viability in each well using the MTT assay described by Pauwels et al (J. Virol. Methods 20:309- 5321, 1988) .
  • ELISA TITER corresponds to the reciprocal of the serum dilution giving an absorbance equal to 50% of the mazimal absorbance value (midpointer titer) .
  • TITER corresponds to the reciprocal of the serum dilution giving 80% protection from cytopathic effect of IIIB HIV1 isolate. slash indicates that the neutralizing titer lies between 2 successive dilutions of the serum; for example, 200/ indicates that the titer lies between 200 and
  • ELISA TITER corresponds to the reciprocal of the serum dilution giving an absorbance equal 25 to 50% of the maximal absorbance value (midpointer titer)
  • TITER corresponds to the reciprocal of serum dilution giving 100% protection against the cytopathic effect of HSV2

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Physics & Mathematics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP91916067A 1990-09-28 1991-09-21 Ein adjuvans enthaltende gp160 derivate, impfstoffe auf gp160- oder derivatbasin Withdrawn EP0550485A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP94202409A EP0644201A1 (de) 1990-09-28 1991-09-21 GP160 Derivate und Adjuvans enthaltende Impfstoffe auf Basis von GP160 oder dessen Derivate

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB909021175A GB9021175D0 (en) 1990-09-28 1990-09-28 Novel compounds
GB9021175 1990-09-28
GB9106048 1991-03-21
GB919106048A GB9106048D0 (en) 1991-03-21 1991-03-21 Vaccines

Related Child Applications (1)

Application Number Title Priority Date Filing Date
EP94202409A Division EP0644201A1 (de) 1990-09-28 1991-09-21 GP160 Derivate und Adjuvans enthaltende Impfstoffe auf Basis von GP160 oder dessen Derivate

Publications (1)

Publication Number Publication Date
EP0550485A1 true EP0550485A1 (de) 1993-07-14

Family

ID=26297726

Family Applications (2)

Application Number Title Priority Date Filing Date
EP94202409A Withdrawn EP0644201A1 (de) 1990-09-28 1991-09-21 GP160 Derivate und Adjuvans enthaltende Impfstoffe auf Basis von GP160 oder dessen Derivate
EP91916067A Withdrawn EP0550485A1 (de) 1990-09-28 1991-09-21 Ein adjuvans enthaltende gp160 derivate, impfstoffe auf gp160- oder derivatbasin

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP94202409A Withdrawn EP0644201A1 (de) 1990-09-28 1991-09-21 GP160 Derivate und Adjuvans enthaltende Impfstoffe auf Basis von GP160 oder dessen Derivate

Country Status (10)

Country Link
EP (2) EP0644201A1 (de)
JP (1) JPH06501151A (de)
KR (1) KR100194079B1 (de)
AU (1) AU654970B2 (de)
CA (1) CA2092827A1 (de)
IE (1) IE913385A1 (de)
MX (1) MX9101275A (de)
NZ (1) NZ239948A (de)
PT (1) PT99063B (de)
WO (1) WO1992006113A2 (de)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6197311B1 (en) 1991-07-25 2001-03-06 Idec Pharmaceuticals Corporation Induction of cytotoxic T-lymphocyte responses
US6620414B2 (en) 1992-03-27 2003-09-16 Smithkline Beecham Biologicals (S.A.) Hepatitis vaccines containing 3-0-deacylated monophoshoryl lipid A
CA2156525A1 (en) * 1993-02-19 1994-09-01 Susan Dillon Influenza vaccine compositions containing 3-o-deacylated monophosphoryl lipid a
EP0812593B8 (de) * 1993-03-23 2010-11-10 SmithKline Beecham Biologicals S.A. 3-0-Deazylierte Monophosphoryl Lipid A enthaltende Impfstoff-Zusammensetzungen
US6171596B1 (en) 1993-12-10 2001-01-09 The United States Of America As Represented By The Department Of Health And Human Services Oligomeric HIV-1 envelope glycoproteins
US6039957A (en) * 1993-12-10 2000-03-21 United States Of America, As Represented By The Department Of Health And Human Services Oligomeric HIV-1 envelope glycoproteins
GB9326253D0 (en) * 1993-12-23 1994-02-23 Smithkline Beecham Biolog Vaccines
US6368604B1 (en) 1997-09-26 2002-04-09 University Of Maryland Biotechnology Institute Non-pyrogenic derivatives of lipid A
US6306404B1 (en) 1998-07-14 2001-10-23 American Cyanamid Company Adjuvant and vaccine compositions containing monophosphoryl lipid A
CA2376992A1 (en) * 1999-06-29 2001-01-04 Smithkline Beecham Biologicals S.A. Use of cpg as an adjuvant for hiv vaccine
US6635261B2 (en) 1999-07-13 2003-10-21 Wyeth Holdings Corporation Adjuvant and vaccine compositions containing monophosphoryl lipid A

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL89567A0 (en) * 1988-03-28 1989-09-10 Univ Leland Stanford Junior Mutated hiv envelope protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9206113A2 *

Also Published As

Publication number Publication date
JPH06501151A (ja) 1994-02-10
WO1992006113A2 (en) 1992-04-16
KR930702527A (ko) 1993-09-09
CA2092827A1 (en) 1992-03-29
PT99063B (pt) 1999-07-30
PT99063A (pt) 1992-08-31
MX9101275A (es) 1992-05-04
AU654970B2 (en) 1994-12-01
IE913385A1 (en) 1992-04-08
NZ239948A (en) 1993-07-27
EP0644201A1 (de) 1995-03-22
KR100194079B1 (ko) 1999-06-15
AU8510991A (en) 1992-04-28
WO1992006113A3 (en) 1992-08-20

Similar Documents

Publication Publication Date Title
AP368A (en) Derivatives of gp160 and vaccine based on gp160 or a derivative thereof, containing an adjuvant.
Girard et al. Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1
US7709004B2 (en) Method of producing an HIV-1 immune response
WAGNER et al. Construction, expression, and immunogenicity of chimeric HIV-1 virus-like particles
US5653985A (en) Purified gp120 composition retaining natural conformation
JPH05262667A (ja) ウイルス抗原を用いるワクチン
EP1878742A2 (de) HIV-Peptide, -Antigene, -Impfstoffzusammensetzungen und -Immunoassay sowie Verfahren zur Erkennung von durch HIV induzierten Antikörpern
AU654970B2 (en) Derivatives of gp160 and vaccines based on gp160 or a derivative thereof, containing an adjuvant
IE921875A1 (en) Vaccine and treatment method for human immunodeficiency¹virus
KR0172970B1 (ko) Aids백신에 유용한 키메릭 단백 및 그의 제조방법
EP0519001B1 (de) Gereinigtes gp120, in dem seine natürliche konformation erhalten bleibt
US5696238A (en) Purified GP120 composition retaining natural conformation
EP1791556A2 (de) Modifizierte hiv-1-hüllproteine
US20120039923A1 (en) Modified HIV-1 Envelope Proteins
EP0328390B1 (de) Peptidbehandlung von hartnäckigen Infektionskrankheiten
WO1995032000A1 (en) Hiv polyprotein immunogens
WO1992000098A1 (en) Methods of inducing immune response to aids virus

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930324

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LI LU NL SE

17Q First examination report despatched

Effective date: 19940422

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19941023