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EP1171158A2 - Vaccin - Google Patents

Vaccin

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Publication number
EP1171158A2
EP1171158A2 EP00926986A EP00926986A EP1171158A2 EP 1171158 A2 EP1171158 A2 EP 1171158A2 EP 00926986 A EP00926986 A EP 00926986A EP 00926986 A EP00926986 A EP 00926986A EP 1171158 A2 EP1171158 A2 EP 1171158A2
Authority
EP
European Patent Office
Prior art keywords
vaccine
cpg
antigen
rsv
alum
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Withdrawn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
EP00926986A
Other languages
German (de)
English (en)
Inventor
M. SmithKline Beecham Biologicals SA DESCHAMPS
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 GBGB9909077.1A external-priority patent/GB9909077D0/en
Priority claimed from GBGB9915106.0A external-priority patent/GB9915106D0/en
Application filed by SmithKline Beecham Biologicals SA filed Critical SmithKline Beecham Biologicals SA
Publication of EP1171158A2 publication Critical patent/EP1171158A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P31/04—Antibacterial agents
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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 relates to new vaccine formulations comprising a
  • Respiratory Syncytial Virus (RSV) antigen and a 'CpG' containing immunostimulating oligonucleotide methods for preparing it and its use in therapy.
  • the present invention relates to new combination vaccines for administration to children, to adults and to the elderly.
  • RSV Human Respiratory Syncytial Virus
  • RSV Paramyxoviridiae family of viruses and causes lower respiratory tract illness, particularly in young children and babies. Recent report suggests that RSV is an important pathogen in adults, particularly the elderly.
  • RSV is an enveloped virus with a non-segmented, negative strand ribonucleic acid (RNA) genome of 15,222 nucleotides that codes for 10 messenger
  • RNA negative strand ribonucleic acid
  • RNAs each coding for a single polypeptide.
  • Three of the ten proteins are transmembrane surface proteins: the G (attachment), F (fusion) and SH proteins.
  • M and M2 are virion matrix proteins
  • N, P and L three proteins are components of the nucleocapsid
  • NS1 and NS2 are nonstructural proteins
  • strain A Two antigenically distinct strains of RSV exist, designated strain A and B.
  • RSV occurs in seasonal outbreaks, peaking during the winter in temperate climates and during the rainy season in warmer climates. Wherever the area, RSV tends to have a regular and predictable pattern and other respiratory viral pathogens that occur in outbreaks are rarely present concurrently. RSV is a major cause of serious lower respiratory tract disease in children.
  • RSV infection usually occurs in children younger than one year of age; 95% of children have serologic evidence of past infection by two years of age and 100% of the population do so by adulthood.
  • RSV Symptomatic reinfection occurs throughout life and it has become increasingly apparent that RSV is an important adult pathogen as well, especially for the elderly. RSV infection is almost certainly under-diagnosed in adults, in part because it is considered to be an infection of children. Consequently, evidence of the virus in adults is not sought in order to explain respiratory illness. In addition, RSV is difficult to identify in nasal secretions from individuals who have some degree of partial immunity to the virus, as do the large majority of adults. Young to middle-age adults typically develop a persistent cold-like syndrome when infected with RSV. Elderly individuals may develop a prolonged respiratory syndrome which is virtually indistinguishable from influenza, with upper respiratory symptoms which may be accompanied by lower respiratory tract involvement, including pneumonia.
  • RSV infection is a predictable cause of serious illness among elderly patients residing in the community. Similar to hospitalisations for influenza A, those related to RSV infections were associated with substantial morbidity, as evidenced by prolonged hospital stays, high intensive care admission rates, and high ventilatory support rates.
  • RSV has two envelope glycoproteins, the F protein having a molecular weight of 68,000 to 70,000 Daltons and a larger G glycoprotein having a molecular weight of 84,000 to 90,000 Daltons (Collins et al J. of Virology Vol 49 pp 572-578 (1984)).
  • FG fusion proteins typically comprising the extracellular domain of both proteins, are known (US 5,194,595 Upjohn).
  • Vaccine preparations comprising FG constructs and 3D-MPL are described in WO 98/18819 (SmithKline Beecham Biologicals s.a.).
  • Immunomodulatory oligonucleotides contain unmethylated CpG dinucleotides ("CpG”) and are known (WO 96/02555, EP 468520).
  • CpG is an abbreviation for cytosine-guanosine dinucleotide motifs present in nucleic acid.
  • synthetic oligonucleotides derived from BCG gene sequences were shown to be capable of inducing immunostimulatory effects (both in vitro and in vivo). The authors of these studies concluded that certain palindromic sequences, including a central CG motif, carried this activity.
  • CG motif has to be in a certain sequence context, and that such sequences are common in bacterial DNA but are rare in vertebrate DNA.
  • the present invention provides a vaccine preparation comprising an immunostimulatory CpG oligonucleotide and a RSV antigen.
  • the RSV antigen is a RSV envelope glycoprotein or derivative thereof derived from, preferably, strain A. More preferably the antigen is selected from F glycoprotein, G glycoprotein or a FG fusion protein or immunogenic derivatives thereof. Alternatively the RSV antigen may be for example inactivated virus.
  • immunogenic derivatives include wherein the protein is devoid of the transmembrane domain ie F ⁇ tm, G ⁇ tm and F ⁇ tm G ⁇ tm.
  • the signal sequence is deleted from the G protein. It is preferred that at least about
  • F or G protein 50% or at least about 80% (contiguous sequence) of the extracellular domain of the F or G protein is present.
  • Particular examples include 1-526 or 1-489 amino acid of the F protein, amino acid 69-298 or alternative positions 97-279 of the G protein, and F,.. 526 G 69 . 29g fusion protein.
  • An alternative fusion comprises 1-489 amino acid from F followed by 97-279 of G protein.
  • the preferred oligonucleotides preferably contain two or more CpG motifs separated by six or more nucleotides.
  • the oligonucleotides of the present invention are typically between 15-45 oligonucleotides in length and are typically deoxynucleotides.
  • the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention including oligonucleotides with mixed internucleotide linkages.
  • Preferred oligonucleotides have the following sequences: The sequences preferably contain all phosphorothioate modified internucleotide linkages.
  • WD0002 TCT CCC AGC GTG CGC CAT WD0003: ACC GAT AAC GTT GCC GGT GAC G
  • the CpG oligonucleotides utilised in the present invention may be synthesized by any method known in the art (eg as described in EP 468520). Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US5,666,153, US5,278,302 and WO95/26204.
  • CpG and aluminium salt such as aluminium hydroxide or aluminium phosphate (Alum) are both present this synergistically enhances anti RSV antigen specific antibody.
  • a combination adjuvant significantly enhances the levels of IgG2a antibody, a marker of a THl response.
  • the adjuvant combination of a CpG oligonucleotide and an aluminium salt allows a cell mediated response as determined by specific lymphoproliferation.
  • a vaccine formulation comprising a RSV antigen and an immunostimulatory CpG oligonucleotide in combination with an aluminium salt.
  • the aluminium salt is aluminium hydroxide.
  • vaccine excipients may be added to the formulation for the invention.
  • Preferred additional immunostimulants include for example saponin adjuvants, such as QS21.
  • an aqueous solution of the protein(s) can be used directly for mixing with the adjuvant.
  • the protein can be lyophilised.
  • the antigens of the present invention may be expressed in any suitable host, such as bacterial, mammalian, insect, yeast and fungal cells. The use of insect cells such as Sf9 cells is described by Du et al, BIO/TECHNOLOGY 12, 1994, 813-818.
  • the proteins of the invention are expressed in insect cells using a recombinant baculovirus (Wathen et al J.Gen Virol 1989, 70 pp2625-2635). Most preferably however the proteins of the invention are produced in eukaryotic cells, particularly in Chinese Hamster Ovary (CHO) cells and Vero cells and purified by the method as disclosed in WO98/18819 (SmithKline Beecham Biologicals s.a.). In a preferred embodiment of the invention the antigen is produced by expression in mammalian cells from a DNA sequence having optimised mammalian codon usage.
  • Optimisation of the codon usage involves the replacement of at least one non-preferred or less preferred codon in a natural gene encoding a protein by a preferred codon encoding the same amino acid.
  • Mammalian genes expressed at high levels typically have C or G at their degenerative position (third base in the codon) whereas the RSV or more generally paramyxoviridae codons have A or T.
  • At least one codon, and more preferably all the codons of the RSV protein can be changed to best fit mammalian cell usage, that is, the one (or ones) that is the most prevalent as shown below.
  • the antigen according to the invention may be expressed as a fusion protein.
  • the antigen may be a heterochimeric fusion of an RSV envelope antigen or an immunogenic derivative thereof with an antigen from a different pathogen.
  • Particular examples include fusions with envelope antigens or immunogenic derivatives from other Paramyxoviruses eg parainfluenza viruses (PIV-1, 2 and 3) or mumps virus or measles virus.
  • the invention also provides DNA encoding such a protein or immunogenic derivative thereof in which the codon usage of one or more nucleic acids has been substantially optimised and a process for expressing said DNA in a CHO or insect cell.
  • the antigens may also be made using a recombinant live microorganism, such as a virus or bacterium as the expression system.
  • a recombinant live microorganism such as a virus or bacterium as the expression system.
  • the gene of interest can be inserted into the genome of a live recombinant virus or bacterium. Inoculation and in vivo infection with this live vector will lead to in vivo expression of the antigen and induction of immune responses.
  • Viruses and bacteria used for this purpose are for instance: poxviruses (e.g; vaccinia, fowlpox, canarypox), alphaviruses (Sindbis virus, Semliki Forest Virus, Dialoguelian Equine Encephalitis Virus), adeno viruses, adeno- associated virus, picornaviruses (poliovirus, rhinovirus), herpesviruses (varicella zoster virus, etc), Listeria, Salmonella , Shigella, BCG. These viruses and bacteria can be virulent, or attenuated in various ways in order to obtain live vaccines. Such live vaccines when formulated with a CpG oligo nucleotides also form part of the invention.
  • poxviruses e.g; vaccinia, fowlpox, canarypox
  • alphaviruses Semliki Forest Virus, Kunststoffuelian Equine Encephalitis Virus
  • adeno viruses a
  • the invention further relates to methods for constructing and expressing the proteins of the invention and methods to optimise the codon usage of the nucleic acid sequences which encode such proteins.
  • the antigens of the present invention may also be presented as a nucleic acid encoding said antigen and formulated with a CpG oligonucleotide.
  • the antigens of the present invention are not necessarily recombinant subunit anitigens.
  • the RSV antigen may for example be in the form of inactivated whole virus.
  • Such inactivated virus may be produced from RSV which has been attenuated eg by passaging or by genetic manipulation.
  • a recombinant RSV may be used which has been engineered to contain an envelope antigen from a different strain of RSV eg an RSV strain A backbone with an RSV B envelope protein, optionally attenuated.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, USA 1978 and in Vaccine Design: The Subunit and Adjuvant Approach, edited by Powell and Newman, Plenum Press, New York, 1995. Encapsulation within liposomes is described, for example, by Fullerton, US Patent 4,235,877.
  • each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccinees. Such amount will vary depending upon which specific immunogen is employed and how it is presented. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of protein, preferably 2-100 ⁇ g, most preferably 5-50 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several booster immunisations adequately spaced.
  • the CpG will be present in the range lOO ⁇ g per dose to 3000 ⁇ g, preferably 250-750 ⁇ g, such as 500 ⁇ g per dose.
  • the vaccine used in the present invention may comprise a carrier such as an aluminium salt, eg aluminium hydroxide (Al(OH 3 ), aluminium phosphate or aluminium phosphate sulfate (alum), or a non-toxic oil in water emulsion or a mixture thereof.
  • a carrier such as an aluminium salt, eg aluminium hydroxide (Al(OH 3 ), aluminium phosphate or aluminium phosphate sulfate (alum), or a non-toxic oil in water emulsion or a mixture thereof.
  • aluminium salt preferably aluminium hydroxide
  • carrier it is generally present in the range of 50 to lOO ⁇ g (human: 500 to lOOO ⁇ g) preferably 500 ⁇ g per dose.
  • Non-toxic oil in water emulsions preferably contain a non-toxic oil, eg squalene and an emulsifier such as polysorbitan monoleate (Tween 80), in an aqueous carrier such as phosphate buffered saline.
  • a non-toxic oil eg squalene and an emulsifier such as polysorbitan monoleate (Tween 80)
  • an aqueous carrier such as phosphate buffered saline.
  • the vaccine used in the present invention may comprise an additional adjuvant, preferably a saponin adjuvant such as QS21 as described for example in WO 95/17210, optionally in the presence of a sterol, such as cholesterol as described for example in PCT/EP96/01464.
  • a saponin adjuvant such as QS21 as described for example in WO 95/17210
  • a sterol such as cholesterol as described for example in PCT/EP96/01464.
  • the vaccine formulation of the invention additionally comprises a Streptococcus pneumoniae antigen.
  • Streptococcus pneumoniae is a gram positive bacteria responsible for considerable morbidity and mortality, particularly in the young and aged.
  • the bacteria may become invasive, infecting the lower lungs and causing pneumonia.
  • the rate of pneumococcal pneumonia in the US for persons over 60 years of age is estimated to be 3 to 8 per 100,000. In 20% of cases this leads to bacteremia, and other manifestations such as meningitis, with a mortality rate close to 30% even with antibiotic treatment.
  • a 17 - valent pneumococcal vaccine (Moniarix) is known, based on the purified polysaccharides of the pneumococcal serotypes most commonly involved in invasive disease. The method of purification of these polysaccharides was disclosed in European Patent 72513 Bl. Vaccine efficacy trials with lower valent vaccines demonstrated a 70 to 90% efficacy with respect to serotypes present in the combination. Case controlled studies in the US in persons > 55 years using a 14 valent vaccine demonstrated 70% efficacy (Mills and Rhoads 1996). Inclusion of additional polysaccharides (to make a 23-valent pneumococcal vaccine) were accepted on the basis of an adequate serological response, even though there was clinical efficacy data lacking (Brown 1995).
  • Pneumococcal polysaccharides can be rendered more immunogenic by chemically coupling them to protein carriers, and clinical efficacy trials are being performed to verify this concept for efficacy in preventing infant Otitis media.
  • conjugation methods generally used for producing immunogenic polysaccharide constructs: (1) direct conjugation of carbohydrate and protein; and (2) indirect conjugation of carbohydrates and protein via a bifunctional linker or spacer reagent.
  • both direct and indirect conjugation require chemical activation of the carbohydrate moiety prior to derivatisation. See for example US 5,651,971 and Dick & Beurret, "Glycoconjugates of Bacterial Carbohydrate Antigens," Conjugate Vaccines, J.M. Cruse & R.E. Lewis (eds), Vol. 10, 48 - 114 (1989).
  • the Streptococcus pneumoniae component in a vaccine of the present invention will comprise polysaccharide antigens (preferably conjugated), wherein the polysaccharides are derived from at least four serotypes of pneumococcus.
  • the four serotypes include 6B, 14, 19F and 23F. More preferably, at least 7 serotypes are included in the composition, for example those derived from serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F.
  • At least 11 serotypes are included in the composition, for example the composition in one embodiment includes capsular polysaccharides derived from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F (preferably conjugated).
  • at least 13 polysaccharide antigens are included, although further polysaccharide antigens, for example 23 valent (such as serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F), are also contemplated by the invention.
  • serotypes 8 and 12F are advantageously included to form a 15 valent vaccine
  • serotypes 6A and 19A are advantageously included to form a 13 valent vaccine.
  • the vaccine composition of the invention additionally comprises a Group B Streptococcus antigen.
  • GBS Group B streptococci
  • la, lb, II and III the four classical serotypes
  • Approximately 90% of these strains express either Rib or alpha, two members of the same family of streptococcal cell surface proteins, and have been shown to confer protective immunity against GBS in animal models.
  • the vaccine composition of the invention additionally comprises one or more of a number of other antigens such as an antigen against influenza virus or PIV-3.
  • other antigens such as an antigen against influenza virus or PIV-3.
  • FG antigen was expressed in CHO cells and purified according to WO98/18819.
  • Al(OH) 3 was diluted in H 2 O before adsorption of the FG (2 ⁇ g) antigen for 30 minutes. Subsequently MPL or CpG was added and incubated for thirty minutes. The formulations were then buffered with 10 fold concentrated PO 4 - NaCl pH 6.8. Thiomersal at 1 mg/ml or phenoxy at 5mg/ml was added as preservative. All incubations were carried out at room temperature with agitation. The formulations were prepared simultaneously for the 2 injections with a 12-day maturation of the finalized formulations before the first injection.
  • mice 9 groups of 10 mice were immunized by different routes (50 ⁇ ) at days 0 and 28 with various formulations (see Table 1). Groups 8 was immunized with live RSV by the intra-nasal route (60 ⁇ l). Sera were obtained at days 28 (28 d Post I) and 42 (14 d Post II). On day 42, spleen cells were taken from 5 mice of all groups for CMI analysis.
  • the assay protocol was as follows: coating overnight at 4°C with 50 ⁇ l of purified
  • OD OD were monitored at 490 nm and the titers determined by Softmaxpro (4 parameters equation) referring to a standard curve and expressed in EU/ml.
  • Controls in test included a pool of randomly chosen human sera (Human pool) and Sandoglobuline (lot 069, generic human IgG produced by Sandoz).
  • Proliferation was evaluated after a 96h incubation of 4xl0 5 cells/well of 96 well plates with 200 ⁇ l of media containing 10 to 0.03 ⁇ g/ml of FG (3-fold dilutions).
  • Cytokine induction was evaluated after 96 h incubation of 2.5x10° cells per well of 24 well with 1 ml of media containing lO ⁇ g to 0.01/ ⁇ g of FG (10-fold dilutions). Supernatants were then harvested to determine the quantity of IFN- ⁇ and IL-5 induced by ELISA following our standard protocol.
  • Group 9 constitutes the negative control for the CMI studies.
  • Groups 6 and 7 constitute controls for the immunogenicity that could be induced by immunization of the mice with the adjuvants alone.
  • the RSV live immunizations was a control for the immune response induced upon natural RSV IN infection (Group 8).
  • CpG- Alum induce a IgGl:Ig2a ⁇ 1 ratio while the three other formulations induce a > 1 ratio.
  • CpG and CpG- Alum induce similar IgGl titers which are two to three times (CpG ⁇ CpG- Alum ⁇ SB ASli) lower than those induced by alum 3D-MPL and Alum.
  • CpG- Alum induce high IgG2a titers which are three to five times higher than those induced by CpG and alum 3D-MPL and thirty to fifty times higher than those induced by FG Alum.
  • the induced FG-specific lymphoproliferation shows the induction of equivalent stimulation indexes for CpG alum, 3D-MPL alum and alum except for FG CpG.
  • FG CpG similarly to the adjuvants alone groups and the na ⁇ ve mice control group, does not induce any detectable FG specific lymphoproliferation.
  • RSV live IN does not induce a sufficiently high immune response for the lymphoproliferation assay to be able to pick it up.
  • the antibody analysis shows a clear linear synergistic effect is observed upon formulating FG with CpG and Alum as compared to FG formulated with either of the adjuvants.
  • FG CpG- Alum Based on the isotype analysis FG CpG- Alum, FG CpG and FG alum MPL all induce a mixed Th profile as measured by the induction of both IgG2a and IgGl antibodies.
  • FG Alum however almost exclusively induces IgGl antibodies, marker of a Th2 response.
  • the results suggest FG CpG- Alum predominantly induce Thl antibody isotypes while FG CpG and to a greater extend FG alum 3D-MPL predominantly induce Th2 antibody isotypes.
  • the formulation of FG with both CpG and Alum leads to a three-fold increase in IgG2a titer as compared to FG CpG alone.
  • Formulations were prepared 4 days before the first injection.
  • CpG (50, 10 or 2 ⁇ g) was preadsorbed on Al(OH)3 as concentrated monobulk by mixing the immunostimulants with the Aluminium salt one day before the final formulation.
  • Non-adsorbed 11 -valent SP, conjugated to protein D was prepared by mixing the 11 conjugated components by numeric order and adjusting the concentration to 2 ⁇ g/val/ml with NaCl 150mM.
  • the final formulations were prepared by mixing H 2 O and Aluminium salt if needed and preadsorbed CpG.
  • the non-adsorbed 11 -valent polysaccharide (PS) was prepared by mixing the 11 valences by numeric order and adjusting the concentration at 2 ⁇ g/val/ml with NaCl 150mM.
  • the final formulations were prepared by mixing H 2 O and PO-JNaCl buffer and Non-adsorbed 11-valent (0.1 ⁇ g/val) and /or FG (2 ⁇ g). After 5 minutes of incubation CpG was added and incubated for 30min. Thiomersal (l ⁇ g/ml) was then added and allowed to incubate for 30min.
  • mice 19 groups of 10 mice were immunized by the intramuscular route (100 ⁇ l) at days 0 and 28 with various formulations (see Table 4). Sera were obtained at days 42 and 43 (14/15 d Post II). On day 43, spleen cells were taken from 5 mice of the groups immunized with FG containing formulations as well as from 5 naive Balb/c mice (group 24, not immunized).
  • Anti-FG antibodies All humoral results were performed for 10 mice/group (individual response for the anti-FG Ig, IgGl and IgG2a and neutralization titers) and cellular results were presented for 5 mice/group on pool.
  • Murine IgG to pneumococcal polysaccharide types 3, 6B, 7F, 14, 19F and 23F was measured by ELISA in a method adapted from the CDC protocol.
  • This protocol includes the addition of soluble cell wall polysaccharide (CPS) to the sera to inhibit the measurement of CPS antibodies.
  • CPS is a phosphoryl-choline containing teichoic acid common to all pneumococci. It is present under the capsule, and antibodies to it are only weakly protective. Since CPS is linked to the capsular polysaccharide, there is usually 0.5 to 1% CPS contaminating the purified capsular polysaccharide used to coat the ELISA plates. Thus, measurement of the CPS antibodies can confound the interpretation ELISA results with respect to the capsular polysaccharide.
  • the ELISA was performed with polysaccharides coated at 20, 5, 5, 20 and 20 ⁇ g/ml in PBS buffer for types 6B, 7F, 14, 19F and 23F respectively.
  • Sera was pre-mixed with the equivalent of 500 ⁇ g/ml CPS in undiluted sera, and incubated for 30 minutes before addition to the ELISA plate.
  • Murine IgG was detected with Jackson ImmunoLab goat anti-murine IgG (H+L) peroxidase at 1 :5000 dilution.
  • the titration curves were referenced to polysaccharide specific murine monoclonal antibodies of known concentration for each serotype using logistic log comparison by SoftMax Pro.
  • the monoclonal antibodies used were HASP4, PS7/19, PS14/4, PS19/5 and PS23/22 for types 6B, 7F, 14, 19F and 23F respectively.
  • serotype 3 a similar ELISA was done except that immunoplates were first coated with methylated human serum albumin (1 ⁇ g/ml in PBS, 2 hours, 37°C) in order to improve the PS3 coating (2.5 ⁇ g/ml in PBS, overnight, 4°C).
  • PS3/6 was used as standard reference.
  • Spleen cells were isolated 14d Post II from groups immunized with FG containing formulations and from na ⁇ ve mice for use as a negative control for the FG-specific cellular response analysis. Samples were analyzed for both FG-specific IFN- ⁇ and IL-5 cytokines secretion.
  • anti-PS IgG titers were at least similar in animals given the 11 -valent PS conjugate together with FG, compared to the animals immunized with the 11 -valent PS conjugate alone ( Figure 4). This result demonstrated that combining pneumococcal PS conjugates and RSV FG antigen within a CpG or an Alum CpG formulation did not reduce or alter the anti-pneumococcal immune response. Antibody titers to serotypes 7F and 14 were even significantly improved in the presence of FG using the CpG adjuvant formulation (with 2 ⁇ g and 50 ⁇ g CpG doses).
  • Pneumoniae 11 -valent conjugated vaccine does not hamper the induction of the humoral and cellular responses.
  • FG CpG-Alum Based on the isotype analysis FG CpG-Alum, FG CpG and FG alum all induce a mixed Th profile as measured by the induction of both IgG2a and IgGl antibodies. FG alum however induces mostly IgGl antibodies, marker of a Th2 response while FG CpG and FG CpG-alum both induce more IgG2a, marker of a Thl response than IgGl. For both CpG formulations, the greater the CpG dose, the greater the predominance of the Thl marker.
  • Results demonstrated that combining pneumococcal PS conjugates and RSV FG antigen within a CpG or an Alum CpG formulation did not alter the anti-pneumococcal immune response.
  • Antibody titers to serotypes 7F and 14 were even significantly improved in the presence of FG using the CpG adjuvant formulation.
  • the Alum CpG formulations were clearly more potent than CpG given alone at the same dose.
  • 2 ⁇ g CpG was as good an adjuvant as 50 ⁇ g CpG, whereas a clear dose-response was observed in animals given the non- adsorbed CpG formulations.

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Abstract

L'invention concerne une formulation vaccinale comprenant un antigène du virus respiratoire syncytial, ainsi qu'un oligonucléotide CpG immunostimulateur. L'invention concerne également des procédés de préparation de cette formulation vaccinale, ainsi que l'utilisation de celle-ci en médecine. On peut inclure d'autres antigènes dans cette formulation, afin de produire des vaccins combinés, destinés à être administrés à des enfants, des adultes et des personnes âgées.
EP00926986A 1999-04-20 2000-04-17 Vaccin Withdrawn EP1171158A2 (fr)

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GB9915106 1999-06-28
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US20020098199A1 (en) 2000-03-10 2002-07-25 Gary Van Nest Methods of suppressing hepatitis virus infection using immunomodulatory polynucleotide sequences
US20010046967A1 (en) * 2000-03-10 2001-11-29 Gary Van Nest Methods of preventing and treating respiratory viral infection using immunomodulatory polynucleotide
US7157437B2 (en) 2000-03-10 2007-01-02 Dynavax Technologies Corporation Methods of ameliorating symptoms of herpes infection using immunomodulatory polynucleotide sequences
AU2001285421A1 (en) * 2000-08-10 2002-02-18 Henry M. Jackson Foundation For The Advancement Of Military Medicine Vaccine against rsv
CA2388049A1 (fr) 2002-05-30 2003-11-30 Immunotech S.A. Oligonucleotides immunostimulateurs et utilisations connexes
US9107906B1 (en) 2014-10-28 2015-08-18 Adma Biologics, Inc. Compositions and methods for the treatment of immunodeficiency
US10259865B2 (en) 2017-03-15 2019-04-16 Adma Biologics, Inc. Anti-pneumococcal hyperimmune globulin for the treatment and prevention of pneumococcal infection
CN110052237A (zh) * 2019-04-19 2019-07-26 贺州学院 一种多孔重质碳酸钙吸附材料的制备方法
IL296071A (en) * 2020-03-01 2022-11-01 Valneva Austria Gmbh Cpg-adjuvanted sars-cov-2 virus vaccine
EP4114463A4 (fr) 2020-03-01 2023-09-06 Dynavax Technologies Corporation Vaccins à coronavirus comprenant un agoniste de tlr9
US20240027434A1 (en) * 2020-12-16 2024-01-25 Dynavax Technologies Corporation Method for quantifying cpg-containing oligonucleotides in compositions comprising alum

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US6764682B1 (en) * 1994-06-16 2004-07-20 Aventis Pasteur Limited Adjuvant compositions containing more than one adjuvant
US6207646B1 (en) * 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
WO1998018819A1 (fr) * 1996-10-29 1998-05-07 Smithkline Beecham Biologicals S.A. Purification d'antigenes du virus respiratoire syncytial
EP0855184A1 (fr) * 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Composition pharmaceutique comprenant un polynucléotide et un antigène notamment pour la vaccination
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AU4552500A (en) 2000-11-02
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CA2370708A1 (fr) 2000-10-26

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