EP1198243A2 - Verwendung von cpg als adjuvans für malariaimpstoff - Google Patents

Verwendung von cpg als adjuvans für malariaimpstoff

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Publication number
EP1198243A2
EP1198243A2 EP00945810A EP00945810A EP1198243A2 EP 1198243 A2 EP1198243 A2 EP 1198243A2 EP 00945810 A EP00945810 A EP 00945810A EP 00945810 A EP00945810 A EP 00945810A EP 1198243 A2 EP1198243 A2 EP 1198243A2
Authority
EP
European Patent Office
Prior art keywords
vaccine
cpg
rts
oligonucleotide
antigen
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.)
Ceased
Application number
EP00945810A
Other languages
English (en)
French (fr)
Inventor
Joseph Cohen
Nathalie Garcon
Gerald Voss
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
Application filed by SmithKline Beecham Biologicals SA filed Critical SmithKline Beecham Biologicals SA
Publication of EP1198243A2 publication Critical patent/EP1198243A2/de
Ceased 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
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium 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
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a novel vaccine formulations and their use in medicine, particularly in the prevention of malaria infections.
  • the present invention is concerned with a CpG oligonucleotide and a malarial antigen.
  • Malaria is one of the world's major health problems with 2 to 4 million people dying from the disease each year.
  • One of the most acute forms of the disease is caused by the protozoan parasite, Plasmodium falciparum which is responsible for most of the mortality attributable to Malaria.
  • the life cycle of P. falciparum is complex, requiring two hosts, man and mosquito for completion.
  • the infection of man is initiated by the inoculation of sporozoites in the saliva of an infected mosquito.
  • the sporozoites migrate to the liver and there infect hepatocytes where they differentiate, via the exoerythrocytic intracellular stage, into the merozoite stage which infects red blood cells (RBC) to initiate cyclical replication in the asexual blood stage.
  • RBC red blood cells
  • the cycle is completed by the differentiation of a number of merozoites in the RBC into sexual stage gametocytes which are ingested by the mosquito, where they develop through a series of stages in the midgut to produce sporozoites which migrate to the salivary gland.
  • the sporozoite stage of P. falciparum has been identified as a potential target of a malaria vaccine.
  • the major surface protein of the sporozoite is known as circumsporozoite protein (CS Protein).
  • This protein from strain 7G8 has been cloned, expressed and sequenced (Dame et al Science 225 (1984) p593).
  • the protein from strain 7G8 is characterised by having a central immunodominant repeat region comprising a tetrapeptide Asn-Ala-Asn-Pro repeated 37 times but interspersed with four minor repeats Asn-Val- Asp-Pro. In other strains the number of major and minor repeats vary as well as their relative position.
  • This central portion is flanked by an N and C terminal portion composed of non-repetitive amino acid sequences designated as the repeatless portion of the CS protein. It has been shown that irradiated sporozoites can provide significant protection against experimental human malaria (Am. J. Trop. Med. Hyg. 24: 297-402, 1975). However, production difficulties makes the use of irradiated sporozoite impractical from the point of view of producing a vaccine.
  • the present invention provides a new, improved malaria vaccines which not only produces a humoral response, but also a cellular immune response.
  • the antigen induces the production of neutralising antibodies against the immunodominant repeat.
  • the antigen should also elicit effector T cell mediated immune responses of the CD4 + and CD8 + cytotoxic T lymphocyte (CTL) type and of the delayed type hypersensitivity type and also, preferably be able to induce T helper (TH) memory cells.
  • CTL cytotoxic T lymphocyte
  • TH T helper
  • WO 93 / 10152 provides a hybrid protein comprising substantially all the C-terminal portion of the CS protein, four or more tandom repeats of the immunodominant region, and the Surface antigen from Hepatitis B virus (HBsAg).
  • the hybrid protein comprises a sequence which contains at least 160 amino acids which is substantially homologous to the C-terminal portion of the CS protein.
  • the CS protein may be ⁇ evoid of the last 12 amino-acids from the C terminal.
  • a protein which comprises a portion of the CS protein of P. falciparum substantially as corresponding to amino acids 210-398 of P. falciparum 7G8 fused in frame via a linear linker to the N-terminal of HBsAg.
  • the linker may comprise a portion of preS2 from HBsAg.
  • RTS RTS,S
  • This hybrid consists of:
  • a methionine-residue encoded by nucleotides 1059 to 1061, derived from the Saccharomyces cerevisiae TDH3 gene sequence. (Musti A.M. et al Gene 1983 25 133-143).
  • Met Ala Pro Three amino acids, Met Ala Pro, derived from a nucleotide sequence (1062 to 1070) created by the cloning procedure used to construct the hybrid gene.
  • RTS* or RTS*,S
  • RTS* or RTS*,S
  • RTS* comprises:
  • TDH3 gene sequence (see Musti et al, loc cit).
  • Trap (or TRAP) from P. falciparum.
  • An apparent homologue of Trap is described in WO92/11868 and relates to an antigen called SSP2 from P. yeolii.
  • International patent application WO 98/ 05355 describes, inter alia, a malaria vaccine based on a combination of Trap and RTS,S.
  • Immunomodulatory oligonucleotides contain unmethylated CpG dinucleotides
  • CpG CpG
  • cytosine-guanosine dinucleotide motifs present in DNA.
  • 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.
  • the central role of the CG motif in i munostimulation was later elucidated in a publication by Krieg, Nature 374, p546 1995. Detailed analysis has shown that the 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.
  • CG pyrimidine pyrimidine and where the CG motif is not methylated.
  • a palindromic sequence is present.
  • the presence of one or more of these immunostimulatory sequence containing oligonucleotides can activate various immune subsets, including natural killer cells (which produce interferon ⁇ and have cytolytic activity) and macrophages (Wooldrige et al Vol 89 (no. 8), 1977).
  • CpG oligonucleotide and a malaria antigen.
  • Vaccine preparation is generally described in Vaccine Design - The subunit and adjuvant approach (Ed. Powell and Newman) Pharmaceutical Biotechnology Vol. 6 Plenum Press 1995. Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877.
  • the preferred oligonucleotides preferably contain two or more CpG motifs separated by six or more nucleotides.
  • the oligonucleotides of the present invention are typically deoxynucleotides.
  • the intemucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorodithioate bond, although phosphodiester and other intemucleotide bonds are within the scope of the invention including oligonucleotides with mixed intemucleotide linkages.
  • the sequences preferably contain all phosphorodithioate modified intemucleotide linkages.
  • Preferred oligonucleotides have the following sequences:
  • a + in the Thio column indicates the presence of a thioate modification.
  • 'Mix' indicates a mixture of thioate modification and sequence without thioate modification (the asterisks indicate the linkages with a thioate modification).
  • a - in the Thio column indicates absence of a thioate modification.
  • a + in the CpG column indicates a the presence of a CpG motif and a - in the CpG column indicates absence of a CpG motif.
  • WD 1005 contains a GpC rather than a CpG motif, thus it is marked with a - in the CpG column of the table.
  • WD 1007 contains a palindromic motif (GACGTC) as well as other non-palindromic CpG sequences. This is also within the scope of a CpG oligonucleotide as the term is used in the present application.
  • oligonucleotides utilised in the present invention may be synthesized by any method known in the art (eg EP 0 468 520). Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US patent 5,666,153, US patent 5,278,302 and WO95/26204.
  • each vaccine does 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.
  • a method for the prevention or amelioration of plasmodium infection in a patient comprising administering an effective amount of either a malaria antigen and a CpG oligonucleotide (as hereinabove defined) or an effective amount of the CpG oligonucleotide followed after a suitable time by an effective amount of a malaria antigen.
  • kits comprising effective amounts of a CpG oligonucleotide- containing formulation for use as a priming formulation for pre-administration to human patients and a malaria antigen for injection at some suitable time later, as described hereinabove.
  • Preferred CpG oligonucleotides are those indicated in the table hereinabove.
  • the CpG will be present in the range 10 ⁇ g per dose to 1000 ⁇ g, preferably 10-100 ⁇ g, especially 25-75 ⁇ g, for example 50 ⁇ 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.
  • an aluminium salt preferably aluminium hydroxide
  • it is generally present in the range of 50 to 100 ⁇ g, preferably 100 to 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 9517210, optionally in the presence of a sterol, such as cholesterol as described for example in PCT/EP96/01464.
  • the vaccine of the invention may also comprise monophosphoryl lipid A and derivatives thereof known in the art.
  • a preferred derivative is 3 de-O-acylated monophosphoryl lipid A, described in British Patent No. 2220211.
  • vaccine formulations of the present invention may additionally comprise other pharmaceutical excipients or immunostimulants.
  • the vaccine formulation additionally comprises an aluminium salt, preferably aluminium hydroxide.
  • CTL cytotoxic T lymphocyte
  • Formulations were prepared 3 days before each injection. All incubations were carried out at room temperature with agitation.
  • CpG/alum group 1 (SOO ⁇ l/dose) RTS, S (8.7 ⁇ g) and gpl20 (8.7 ⁇ g) were adsorbed on 100 ⁇ g of Al(OH) 3 or AlPO 4 for 1 hour
  • the formulation was buffered with a 10-fold concentrated PO 4 /NaCl pH 6.8 solution before addition of lOO ⁇ g of CpG (WD 1001). After 15 min, 50 ⁇ g/ml of fhiomersal was added as preservative.
  • RTS,S (8.7 ⁇ g) and Gpl20(8.7 ⁇ g) were diluted in PBS pH 6.8 before addition of lOO ⁇ g of CpG (WDIOOI). After 5 min, 50 ⁇ g/ml of thiomersal was added as preservative.
  • mice Nine Balb/C mice per group received into the hind footpads 100 ⁇ l vaccine twice at a two-week-interval. Two weeks later spleen cells were harvested and used to determine the induction of HBsAg-specific CTL.
  • CTL analysis cells were cultured for 7 days in 6-well plates in the presence of 10 ⁇ g per ml of synthetic peptide pCMI003 corresponding to an HBsAg CTL epitope (Schirrnbeck et al. , 1995). At the end of the culture period cells were assessed in duplicate for HBsAg-specific cytolytic activity in standard [ 51 Cr] -release assays using control and S-transfected P815 cells.
  • Formulations were prepared one day before each injection. All incubations were carried out at room temperature with agitation.
  • RTS,S (50 ⁇ g) was adsorbed on 500 ⁇ g of Al(OH) 3 for 1 hour The formulation was buffered with a 10-fold concentrated PO 4 /NaCl pH 6.8 solution before addition of 500 ⁇ g of CpG (WDIOOI). After 15 min, 50 ⁇ g/ml of thiomersal was added as preservative.
  • RTS,S (50 ⁇ g) was diluted in PO 4 /NaCl buffer pH 6.8 before addition of 500 ⁇ g of CpG WDIOOI . After 15 min, 50 ⁇ g/ml of thiomersal was added as preservative.
  • rhesus monkeys (Macaca mulatto.) per group were immunized twice intramuscularly with 500 ⁇ l of vaccine at a four- week-interval.
  • Sera and peripheral blood mononuclear cells were taken at several occasions.
  • HBsAg-specific antibodies in monkey sera were determined in a radio immuno assay (RIA, Abbott) according to the manufacturer's instructions.
  • Figure 1 CTL activity of spleen cells from immunized mice. Effector cell activity was assessed by examining 51 Cr release of P815 cells (open circles) or s-transfected P815 cells (closed circles).
  • Figure 2 HBsAg-specific antibody responses in immunized rhesus monkeys. Specific antibodies were evaluated using a commercially available RIA. Individual values from multiple time points for each animal are shown in the table, and group averages are shown in the table and as a graphic.
  • FIG. 3 RTS,S-specif ⁇ c lymphoproliferation in immunized rhesus monkeys 6 days post second immunization. PBMC were stimulated with RTS,S antigen and lymphoproliferative responses were measured by 3 H-thymidine incorporation. Results are expressed in cpm and as SI.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
EP00945810A 1999-06-29 2000-06-23 Verwendung von cpg als adjuvans für malariaimpstoff Ceased EP1198243A2 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB9915204.3A GB9915204D0 (en) 1999-06-29 1999-06-29 Vaccine
GB9915204 1999-06-29
PCT/EP2000/005841 WO2001000231A2 (en) 1999-06-29 2000-06-23 Use of cpg as an adjuvant for malaria vaccine

Publications (1)

Publication Number Publication Date
EP1198243A2 true EP1198243A2 (de) 2002-04-24

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP00945810A Ceased EP1198243A2 (de) 1999-06-29 2000-06-23 Verwendung von cpg als adjuvans für malariaimpstoff

Country Status (6)

Country Link
EP (1) EP1198243A2 (de)
AU (1) AU5977700A (de)
CA (1) CA2376926A1 (de)
GB (1) GB9915204D0 (de)
HK (1) HK1047034A1 (de)
WO (1) WO2001000231A2 (de)

Families Citing this family (12)

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Publication number Priority date Publication date Assignee Title
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
SI1077722T1 (sl) 1998-05-22 2007-02-28 Ottawa Health Research Inst Metode in produkti za induciranje sluznicne imunosti
GB9908885D0 (en) * 1999-04-19 1999-06-16 Smithkline Beecham Biolog Vccine
US20030129251A1 (en) 2000-03-10 2003-07-10 Gary Van Nest Biodegradable immunomodulatory formulations and methods for use thereof
US7129222B2 (en) 2000-03-10 2006-10-31 Dynavax Technologies Corporation Immunomodulatory formulations and methods for use thereof
US7306806B2 (en) 2001-01-26 2007-12-11 United States Of America As Represented By The Secretary Of The Army Recombinant P. falciparum merozoite protein-142 vaccine
CA2462946C (en) * 2001-01-26 2014-04-29 Jeffrey A. Lyon Recombinant p. falciparum merozoite protein-142 vaccine
WO2003015816A1 (en) * 2001-08-10 2003-02-27 Dynavax Technologies Corporation Immunomodulatory oligonucleotide formulations and methods for use thereof
CA2388049A1 (en) 2002-05-30 2003-11-30 Immunotech S.A. Immunostimulatory oligonucleotides and uses thereof
CN100455600C (zh) * 2002-08-14 2009-01-28 阿维迪斯公司 包含支架,佐剂和抗原的异源多聚体化合物及其用途
JP4199988B2 (ja) * 2002-11-14 2008-12-24 大日本印刷株式会社 化粧シート
JP5814507B2 (ja) 2006-09-07 2015-11-17 グラクソスミスクライン バイオロジカルズ ソシエテ アノニム ワクチン

Citations (1)

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WO1993010152A1 (en) * 1991-11-16 1993-05-27 Smithkline Beecham Biologicals S.A. HYBRID PROTEIN BETWEEN CS FROM PLASMODIUM AND HBsAG

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AU1145699A (en) * 1997-09-05 1999-03-22 Smithkline Beecham Biologicals (Sa) Oil in water emulsions containing saponins
KR100629028B1 (ko) * 1998-10-16 2006-09-26 글락소스미스클라인 바이오로지칼즈 에스.에이. 애쥬번트 시스템 및 백신
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Also Published As

Publication number Publication date
GB9915204D0 (en) 1999-09-01
CA2376926A1 (en) 2001-01-04
WO2001000231A2 (en) 2001-01-04
AU5977700A (en) 2001-01-31
WO2001000231A3 (en) 2001-07-05
HK1047034A1 (zh) 2003-02-07

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