CA2267191A1 - Vaccines - Google Patents
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- CA2267191A1 CA2267191A1 CA002267191A CA2267191A CA2267191A1 CA 2267191 A1 CA2267191 A1 CA 2267191A1 CA 002267191 A CA002267191 A CA 002267191A CA 2267191 A CA2267191 A CA 2267191A CA 2267191 A1 CA2267191 A1 CA 2267191A1
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- 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
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- A61K39/12—Viral antigens
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/245—Herpetoviridae, e.g. herpes simplex virus
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- A61K39/12—Viral antigens
- A61K39/29—Hepatitis virus
- A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A61P31/22—Antivirals for DNA viruses for herpes viruses
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- 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
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K2039/55511—Organic adjuvants
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- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- A61K2039/55511—Organic adjuvants
- A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
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- A61K2039/55511—Organic adjuvants
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
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- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- C12N2710/00011—Details
- C12N2710/16011—Herpesviridae
- C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
- C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention relates to a vaccine composition comprising alum, an antigen, an immunologically active saponin fraction and a sterol.
Description
Vaccines The present invention relates to novel vaccine formulations, to methods of their production and to their use in medicine. In particular, the present invention relates to vaccines containing alum. an antigen, an immunologically active fraction derived from the bark of Quillaj a S aponaria Molina such as QS21, and a sterol.
Immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina are known in the art. For example QS21, also known as QA21, an Hplc purified fraction from the Quillaja Saponaria Molina tree and it's method of its production is disclosed (as QA21) in US patent No. 5,057,540. Quillaja saponin has also been disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl. Immun., l985, 77. 409.
However, the use of QS21 as an adjuvant is associated with certain disadvantages. For example when QS21 is injected into a mammal as a free molecule it has been observed that necrosis, that is to say, localised tissue death, occurs at the injection site.
WO 96/33739 discloses vaccine formulations comprising an antigen, an immunologically active fraction derived from the bark of Quillaja Saponaria Molina such as QS21, and a sterol.
It has now surprisingly been found that incorporation of alum in vaccine formulations containing MPL, QS21 and SUV enhances both humoral and cellular responses and that vaccine formulations containing MPL, QS21, SUV and alum are non-toxic with a good reactogenicity profile and have enhanced adjuvant activity. In addition, the combined adjuvant appears to favour TH1 responses.
In a first aspect the invention provides a vaccine adjuvant comprising alum, an immunologically active saponin fraction, and a sterol. By the term 'alum' is meant aluminium hydroxide or aluminium phosphate.
Preferably the adjuvant compositions of the invention contain the immunologically active saponin fraction in substantially pure form. Preferably the compositions of the invention contain QS21 in substantially pure form, that is to say, the QS21 is at least 90% pure, preferably at least 95% pure and most preferably at least 98% pure.
WO 98/15287 PCTlEP97/05578 Other immunologically active saponin fractions useful in compositions of the invention include QA 17/QS 17. Compositions of the invention comprising QS21 and cholesterol show decreased reactogenicity when compared to compositions in which the cholesterol is absent, while the adjuvant effect is maintained. In addition it is known that QS21 degrades under basic conditions where the pH is about 7 or greater.
A further advantage of the present compositions is that the stability of QS21 to base-mediated hydrolysis is enhanced in formulations containing cholesterol.
Preferred sterols include ~i-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 1 lth Edn., page 341, as a naturally occurring sterol found in animal fat. Most preferably the sterol is cholesterol.
Preferred compositions of the invention are those forming a liposome structure, that is to say small unilammelar vesicles (SUV). Compositions where the sterol/immunologically active saponin fraction forms an ISCOM structure also form an aspect of the invention.
The ratio of QS21 : sterol will typically be in the order of 1 : 100 to 1 : 1 weight to weight. Preferably excess sterol is present, the ratio of QS21 :
sterol being at least 1 : 1 w/w. Typically for human administration QS21 and sterol will be present in a vaccine in the range of about 1 p.g to about l00 p.g, preferably about 10 p.g to about 50 p.g per dose of QS21.
The liposomes preferably contain a neutral lipid, for example phosphatidylcholine, which is preferably non-crystalline at room temperature, for example eggyolk phosphatidylcholine, dioleoyl phosphatidylchoiine or dilauryl phosphatidylcholine. The liposomes may also contain a charged lipid which increases the stability of the Iipsome-QS21 structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is preferably 1-20% w/w, most preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), most preferably 20-25%.
Particularly preferred and advantageous compositions of the invention contain MPL (~-deacylated mono-phosphoryl lipid A, also known as 3D-MPL). 3D-MPL is known from GB 2 220 211 (Ribi) as a mixture of 3 types of De-O-acylated monophosphoryl lipid A with 4, ~ or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form is disclosed in International Patent Application 94!2129Z.
Suitable compositions of the invention are those wherein liposomes are S initially prepared without MPL, and MPL is then added, preferably as 100 nm particles. The MPL is therefore not contained within the vesicle membrane (known as MPL out). Compositions where the MPL is contained within the vesicle membrane (known as MPL in) also form an aspect of the invention. The antigen can be contained within the vesicle membrane or contained outside the vesicle membrane.
Preferably soluble antigens are outside and hydrophobic or lipidated antigens are either contained within or outside the membrane.
In a preferred aspect of the invention, liposomes/SUV are first added to the QS21 and then mixed with alum which results in a significant proportion of the binding to the alum (via interaction through the liposomes). Such a formulation, when injected, is expected to result in a slower release of QS21 to the body, due to a depot effect of the alum, than if the QS21 was free or in un-fixed liposomes. The formulation containing MPL, QS21, SUV and alum are particularly advantageous as they are non-toxic and highly immunogenic.
Preferably the vaccine formulations will contain an antigen or antigenic composition capable of eliciting an immune response against a human or animal pathogen. In a first aspect the present invention therefore provides a vaccine composition comprising alum, an antigen, an immunologically active saponin fraction and a sterol.
Antigen or antigenic compositions known in the art can be used in the compositions of the invention, including polysaccharide antigens. antigen or antigenic compositions derived from HIV-1, (such as gp120 or gp160), any of Feline Immunodeficiency virus, human or animal herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV 1 or HSV2, cytomegalovirus (especially human) (such as gB or derivatives thereot~, Varicella Zoster Virus (such as gpI, II or III), or from a hepatitis virus such as hepatitis B virus for example Hepatitis B Surface antigen or a derivative thereof, hepatitis A
virus, WO 98t15287 PCT/EP97/05578 hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as Respiratory Syncytial virus (for example HSRV F and G proteins or immunogenic fragments thereof disclosed in US Patent 5,149,650 or chimeric polypeptides containing immunogenic fragments from HSRV proteins F and G. eg FG
glycoprotein disclosed in US Patent 5,194,595), antigens derived from meningitis strains such as meningitis A. B and C, Streptococcus Pneumonia, human papilloma virus, Influenza virus, Haemophilus Influenza B (Hib), Epstein Barr Virus (EBV), or derived from bacterial pathogens such as Salmonella, Neisseria, Borrelia (for example OspA
or OspB or derivatives thereof), or Chlamydia, or Bordetella for example P.69, PT
and FHA, or derived from parasites such as plasmodium or toxoplasma.
HSV GIycoprotein D (gD) or derivatives thereof is a preferred vaccine antigen. It is located on the viral membrane, and is also found in the cytoplasm of infected cells (Eisenberg R.J. et al; J of Virol 1980 35 428-435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins this is probably the best characterised (Cohen et al J. Virology 60 157-166). In vivo it is known to play a central role in viral attachment to cell membranes. Moreover, glycoprotein D has been shown to be able to elicit neutralising antibodies in vivo and protect animals from lethal challenge. A truncated form of the gD molecule is devoid of the C terminal anchor region and can be produced in mammalian cells as a soluble protein which is exported into the cell culture supernatant. Such soluble forms of gD are preferred. The production of truncated forms of gD is described in EP 0 139 417. Preferably the gD
is derived from HSV-2. An embodiment of the invention is a truncated HSV-2 glycoprotein D of 308 amino acids which comprises amino acids 1 through 306 naturally occurring glycoprotein with the addition Asparagine and Glutamine at the C
terminal end of the truncated protein devoid of its membrane anchor region.
This form of the protein includes the signal peptide which is cleaved to allow for the mature soluble 283 amino acid protein to be secreted from a host cell.
In another aspect of the invention, Hepatitis B surface antigen is a preferred vaccine antigen.
As used herein the expression 'Hepatitis B surface antigen' or 'HBsAg' includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV
surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg antigen (see Tiollais et al, Nature, 317, 489 ( 1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S
sequence as described in the above references and in EP-A- 0 278 940. In particular the HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 12-52 followed by residues 133-145 followed by residues 400 of the L-protein of HBsAg relative to the open reading frame on a Hepatitis B
virus of ad serotype (this polypeptide is referred to as L*; see EP 0 414 374). HBsAg within the scope of the invention may also include the pre-S1-preS2-S
polypeptide described in EP 0 198 474 (Endotronics) or close analogues thereof such as those described in EP 0 304 578 (Nlc Cormick and Jones). HBsAg as herein described can also refer to mutants, for example the 'escape mutant' described in WO
9l/14703 or European Patent Application Number 0 511 855A1, especially HBsAg wherein the amino acid substitution at position 145 is to arginine from glycine.
Normally the HBsAg will be in particle form. The particles may comprise for example S protein alone or may be composite particles, for example (L*,S) where L*
is as defined above and S denotes the S-protein of HBsAg. The said particle is advantageously in the form in which it is expressed in yeast.
The preparation of hepatitis B surface antigen S-protein is well documented.
See for example, Harford et al (I983) in Develop. Biol. Standard 54, page 125, Gregg et a1 (1987) in Biotechnology, 5, page 479, EP 0 226 846, EP 0 299 108 and references therein.
In another embodiment, the vaccine antigen is an RSV antigen. In particular an F/G antigen. US patent 5l94595 (Upjohn) describes chimeric glycoproteins containing immunogenic segments of the F and G glycoproteins of RSV and suggests that such proteins can be expressed from a variety of systems including bacterial, yeast, mammalian (eg CHO cells) and insect cells (using for example a baculovirus).
Wathen et al (J. Gen. Virol. l989, 70, 2625-2635) describes a particular RSV
FG chimeric glycoprotein expressed using a baculovirus vector consisting of amino acids i -489 of the F protein linked to amino acids 97-279 of the G protein.
The fornnulations within the scope of the invention rnay also contain an anti-s tumour antigen and be useful for immunotherapeutically treating cancers.
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 protein 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 how it is presented. Generally, it is expected that each dose will comprise 1-1000 mcg of protein, preferably 2-100 mcg, most preferably 4-40 mcg.
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 immunisation adequately spaced.
The formulations of the present invention may be used for both prophylactic and therapeutic purposes.
Accordingly in a further aspect, the invention therefore provides use of a vaccine composition of the invention for the treatment of human patients. The invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient. In particular, the invention provides a method of treating viral, bacterial, parasitic infections or cancer which comprises administering an effective amount of a vaccine of the present invention to a patient.
The following examples and data illustrates the invention.
WO 98I15287 PCTlEP97/05578 Example 1 Preparation of vaccine containing alum, SUV MPL and QS21 1.1 Method of preparation of SUV
A mixture of lipid (such as phosphatidylcholine either from egg-yolk or synthetic) and cholesterol in organic solvent, is dried down under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as phosphate buffered saline) is then added, and the vessel agitated until all the lipid is in suspension. This suspension is then microfluidised until the liposome size is reduced to 100 nm, and then sterile filtered through a 0.2 p,m filter. Extrusion or sonication could replace this step. Typically the cholesterol: phosphatidycholine ratio is 1:4 (w/w), and the aqueous solution is added to give a final cholesterol concentration of 5 to 50 mg/mi.
1.2 Antigen ( 1-500 p.g, preferably 10-100 pg) is added to alum eg (aluminium hydroxide or aluminium phosphate) ( 100-500 fig) in water. The volume of water is chosen so that the volume of the final formulation is S00 p.l. After incubating for 15-30 minutes, 50 wg of MPL is added in the form of small-particle MPL
(W094/21292).
The MPL is left to adsorb onto the alum for 15-30 minutes at room temperature.
times concentrated phosphate buffered saline ( 1.5 M sodium chloride, 0.5M
sodium phosphate pH 7.5) is then added in such a volume so as to render the final formulation isotonic. This formulation is incubated at room temperature for 15-30 minutes.
QS21 (50 pg) is then added to SUV (containing between 50 and 250 pg cholesterol). This mixture is added to the above alum/antigen/MPL/buffer mixture. If required a bacteriostatic such as thiomersal is added (50 p.g).
Example 2 Table 1 shows the binding of QS21 to alum in the presence and absence of liposomes containing 25% (w/w) in dioleoyl phosphatidylcholine, and using a five-fold excess of cholesterol over QS21.
Formulation SUV ~ p.g QS21 bound 500 ug Alum+50 pg QS21 0 < i 0 500 ug Alum+50 ~g QS21 250 pg chol + 1 mg DOPC > 40 In order to increase the binding of QS21 to alum, the quantity of Iiposomes can be decreased. This decreases the cholesterol:QS2I ratio, however it has been shown that the QS21 remains non-toxic for cholesterol:QS21 ratios of 1:1 and greater.
Table 2 shows that if the quantity of alum is decreased (from 500 p.g to 100 pg) the quantity of QS2I that is bound decreases significantly, and the quantity of MPL that is bound also decreases. By adding less liposomes, yet maintaining a cholesterol:QS21 ratio of 1:1 or greater, increased quantities of QS21 and MPL can be bound to the alum.
Formulation Chol/QS21 ~g QS21 boundpg MPL bound 500 p.g alum + ~0 pg QS21 5l1 42 >48 + 50 pg MPL
l00 pg alum + 50 pg QS21 ~/1 17 >40 + 50 pg MPL
100 p.g alum + 50 pg QS21 2/1 30 >45 + 50 pg MPL
100 pg alum + 50 p.g QS21 1/1 40 >45 + 50 pg MPL
Example 3 The adjuvant effect of a combination of antigen (gD2t from Herpes Simple:c Virus-2 - expressed in CHO cells and comprises 283 amino acid from the mature N-terminal of the mature glycoprotein) with MPL and QS21 in combination with liposomes was tested with and without alum. The formulations were tested in African Green Monkeys.
African Green Monkeys were immunised twice (0, 28 days) with 20 ~g gD2t plus 50 ~g MPL plus 50 qg QS21 with or without liposomes (250 p.g cholesterol plus 1 mg DOPC) and with or without 500 pg alum. On day 42 the immune response was analysed.
The results are outlined below in figures 1 to 4.
The humoral response was measured as IgG against the gD protein. Figure 1 shows that the combination of MPL+QS21+SUV+alum induced higher titres than in the absence of alum, Figure 2 shows that the formulation of the invention provided the superior antigen specific proliferation.
The data shows that incorporation of aluminium hydroxide in vaccine formulations containing MPL and QS21 and SUV enhances both the humoral and cellular responses. This is an unexpected finding since it is generally accepted that aluminium as an adjuvant tends to favour Th2 type responses, yet the results presented here demonstrate that the response contains a significant Th 1 component which is not depressed by the addition of alum.
The formulation containing MPL and QS21 and SUV and alum is non-toxic and highly immunogenic.
Example 4 Production of RSV FG CHO cell derived proteins The plasmid pEE 14-FG contains a chimeric construct comprising of a fusion between amino acid sequences of F (1-525) and G (69-298) and was received from a collaboration with A. BOLLEN (ULB/CRI, Belgium). This FG fusion protein contains a total of 755 amino acids. It starts at the N-terminal signal sequence of F and lacks the C-terminal transmembrane domain (525-574) -anchor domain- of F
glycoprotein. Then, followed the extracellular region of G glycoprotein, without the amino-terminal region that contains the Signal/Anchor domain of G, a typical class II
glycoprotein.
The pEE 14-FG expression plasmid was generated by the insertion of the FG
coding sequence from pNIV2857 (A. Bollen, ULB/CRI, Belgium) as an Asp7181 (blunt) 5' - HindIII (blunt) 3' restriction fragment (2188 bp) into the SmaI
site of pEE 14 (Celltech). A Kozak sequence in lieu of the FG start ATG was generated into the pNIV 2857 construction as follows:
pEE 14---ccc gtacc ATG GAG -----x-----CAG TAG aagct ggg ---pEE 14 (SmaI) Metl Gln(298)Stop Asp718I(klenow) HindIII(klenow) 3 0 5' F( 1-525)-------x--------G(69-298)3' WO 98l15287 PCT/EP97l05578 The F sequence in pEEl4-FG is from SS2 RSV strain, and was kindly made available by Dr. PRINGLE as a cDNA construct in a Vaccinia vector (Baybutt and Pringle, J.Gen.ViroL, 1987, 2789-2796). The G sequence is from A2 RSV strain and was generated from a recombinant G Vaccinia virus obtained from Dr. G. WERTZ
Immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina are known in the art. For example QS21, also known as QA21, an Hplc purified fraction from the Quillaja Saponaria Molina tree and it's method of its production is disclosed (as QA21) in US patent No. 5,057,540. Quillaja saponin has also been disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl. Immun., l985, 77. 409.
However, the use of QS21 as an adjuvant is associated with certain disadvantages. For example when QS21 is injected into a mammal as a free molecule it has been observed that necrosis, that is to say, localised tissue death, occurs at the injection site.
WO 96/33739 discloses vaccine formulations comprising an antigen, an immunologically active fraction derived from the bark of Quillaja Saponaria Molina such as QS21, and a sterol.
It has now surprisingly been found that incorporation of alum in vaccine formulations containing MPL, QS21 and SUV enhances both humoral and cellular responses and that vaccine formulations containing MPL, QS21, SUV and alum are non-toxic with a good reactogenicity profile and have enhanced adjuvant activity. In addition, the combined adjuvant appears to favour TH1 responses.
In a first aspect the invention provides a vaccine adjuvant comprising alum, an immunologically active saponin fraction, and a sterol. By the term 'alum' is meant aluminium hydroxide or aluminium phosphate.
Preferably the adjuvant compositions of the invention contain the immunologically active saponin fraction in substantially pure form. Preferably the compositions of the invention contain QS21 in substantially pure form, that is to say, the QS21 is at least 90% pure, preferably at least 95% pure and most preferably at least 98% pure.
WO 98/15287 PCTlEP97/05578 Other immunologically active saponin fractions useful in compositions of the invention include QA 17/QS 17. Compositions of the invention comprising QS21 and cholesterol show decreased reactogenicity when compared to compositions in which the cholesterol is absent, while the adjuvant effect is maintained. In addition it is known that QS21 degrades under basic conditions where the pH is about 7 or greater.
A further advantage of the present compositions is that the stability of QS21 to base-mediated hydrolysis is enhanced in formulations containing cholesterol.
Preferred sterols include ~i-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 1 lth Edn., page 341, as a naturally occurring sterol found in animal fat. Most preferably the sterol is cholesterol.
Preferred compositions of the invention are those forming a liposome structure, that is to say small unilammelar vesicles (SUV). Compositions where the sterol/immunologically active saponin fraction forms an ISCOM structure also form an aspect of the invention.
The ratio of QS21 : sterol will typically be in the order of 1 : 100 to 1 : 1 weight to weight. Preferably excess sterol is present, the ratio of QS21 :
sterol being at least 1 : 1 w/w. Typically for human administration QS21 and sterol will be present in a vaccine in the range of about 1 p.g to about l00 p.g, preferably about 10 p.g to about 50 p.g per dose of QS21.
The liposomes preferably contain a neutral lipid, for example phosphatidylcholine, which is preferably non-crystalline at room temperature, for example eggyolk phosphatidylcholine, dioleoyl phosphatidylchoiine or dilauryl phosphatidylcholine. The liposomes may also contain a charged lipid which increases the stability of the Iipsome-QS21 structure for liposomes composed of saturated lipids. In these cases the amount of charged lipid is preferably 1-20% w/w, most preferably 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), most preferably 20-25%.
Particularly preferred and advantageous compositions of the invention contain MPL (~-deacylated mono-phosphoryl lipid A, also known as 3D-MPL). 3D-MPL is known from GB 2 220 211 (Ribi) as a mixture of 3 types of De-O-acylated monophosphoryl lipid A with 4, ~ or 6 acylated chains and is manufactured by Ribi Immunochem, Montana. A preferred form is disclosed in International Patent Application 94!2129Z.
Suitable compositions of the invention are those wherein liposomes are S initially prepared without MPL, and MPL is then added, preferably as 100 nm particles. The MPL is therefore not contained within the vesicle membrane (known as MPL out). Compositions where the MPL is contained within the vesicle membrane (known as MPL in) also form an aspect of the invention. The antigen can be contained within the vesicle membrane or contained outside the vesicle membrane.
Preferably soluble antigens are outside and hydrophobic or lipidated antigens are either contained within or outside the membrane.
In a preferred aspect of the invention, liposomes/SUV are first added to the QS21 and then mixed with alum which results in a significant proportion of the binding to the alum (via interaction through the liposomes). Such a formulation, when injected, is expected to result in a slower release of QS21 to the body, due to a depot effect of the alum, than if the QS21 was free or in un-fixed liposomes. The formulation containing MPL, QS21, SUV and alum are particularly advantageous as they are non-toxic and highly immunogenic.
Preferably the vaccine formulations will contain an antigen or antigenic composition capable of eliciting an immune response against a human or animal pathogen. In a first aspect the present invention therefore provides a vaccine composition comprising alum, an antigen, an immunologically active saponin fraction and a sterol.
Antigen or antigenic compositions known in the art can be used in the compositions of the invention, including polysaccharide antigens. antigen or antigenic compositions derived from HIV-1, (such as gp120 or gp160), any of Feline Immunodeficiency virus, human or animal herpes viruses, such as gD or derivatives thereof or Immediate Early protein such as ICP27 from HSV 1 or HSV2, cytomegalovirus (especially human) (such as gB or derivatives thereot~, Varicella Zoster Virus (such as gpI, II or III), or from a hepatitis virus such as hepatitis B virus for example Hepatitis B Surface antigen or a derivative thereof, hepatitis A
virus, WO 98t15287 PCT/EP97/05578 hepatitis C virus and hepatitis E virus, or from other viral pathogens, such as Respiratory Syncytial virus (for example HSRV F and G proteins or immunogenic fragments thereof disclosed in US Patent 5,149,650 or chimeric polypeptides containing immunogenic fragments from HSRV proteins F and G. eg FG
glycoprotein disclosed in US Patent 5,194,595), antigens derived from meningitis strains such as meningitis A. B and C, Streptococcus Pneumonia, human papilloma virus, Influenza virus, Haemophilus Influenza B (Hib), Epstein Barr Virus (EBV), or derived from bacterial pathogens such as Salmonella, Neisseria, Borrelia (for example OspA
or OspB or derivatives thereof), or Chlamydia, or Bordetella for example P.69, PT
and FHA, or derived from parasites such as plasmodium or toxoplasma.
HSV GIycoprotein D (gD) or derivatives thereof is a preferred vaccine antigen. It is located on the viral membrane, and is also found in the cytoplasm of infected cells (Eisenberg R.J. et al; J of Virol 1980 35 428-435). It comprises 393 amino acids including a signal peptide and has a molecular weight of approximately 60 kD. Of all the HSV envelope glycoproteins this is probably the best characterised (Cohen et al J. Virology 60 157-166). In vivo it is known to play a central role in viral attachment to cell membranes. Moreover, glycoprotein D has been shown to be able to elicit neutralising antibodies in vivo and protect animals from lethal challenge. A truncated form of the gD molecule is devoid of the C terminal anchor region and can be produced in mammalian cells as a soluble protein which is exported into the cell culture supernatant. Such soluble forms of gD are preferred. The production of truncated forms of gD is described in EP 0 139 417. Preferably the gD
is derived from HSV-2. An embodiment of the invention is a truncated HSV-2 glycoprotein D of 308 amino acids which comprises amino acids 1 through 306 naturally occurring glycoprotein with the addition Asparagine and Glutamine at the C
terminal end of the truncated protein devoid of its membrane anchor region.
This form of the protein includes the signal peptide which is cleaved to allow for the mature soluble 283 amino acid protein to be secreted from a host cell.
In another aspect of the invention, Hepatitis B surface antigen is a preferred vaccine antigen.
As used herein the expression 'Hepatitis B surface antigen' or 'HBsAg' includes any HBsAg antigen or fragment thereof displaying the antigenicity of HBV
surface antigen. It will be understood that in addition to the 226 amino acid sequence of the HBsAg antigen (see Tiollais et al, Nature, 317, 489 ( 1985) and references therein) HBsAg as herein described may, if desired, contain all or part of a pre-S
sequence as described in the above references and in EP-A- 0 278 940. In particular the HBsAg may comprise a polypeptide comprising an amino acid sequence comprising residues 12-52 followed by residues 133-145 followed by residues 400 of the L-protein of HBsAg relative to the open reading frame on a Hepatitis B
virus of ad serotype (this polypeptide is referred to as L*; see EP 0 414 374). HBsAg within the scope of the invention may also include the pre-S1-preS2-S
polypeptide described in EP 0 198 474 (Endotronics) or close analogues thereof such as those described in EP 0 304 578 (Nlc Cormick and Jones). HBsAg as herein described can also refer to mutants, for example the 'escape mutant' described in WO
9l/14703 or European Patent Application Number 0 511 855A1, especially HBsAg wherein the amino acid substitution at position 145 is to arginine from glycine.
Normally the HBsAg will be in particle form. The particles may comprise for example S protein alone or may be composite particles, for example (L*,S) where L*
is as defined above and S denotes the S-protein of HBsAg. The said particle is advantageously in the form in which it is expressed in yeast.
The preparation of hepatitis B surface antigen S-protein is well documented.
See for example, Harford et al (I983) in Develop. Biol. Standard 54, page 125, Gregg et a1 (1987) in Biotechnology, 5, page 479, EP 0 226 846, EP 0 299 108 and references therein.
In another embodiment, the vaccine antigen is an RSV antigen. In particular an F/G antigen. US patent 5l94595 (Upjohn) describes chimeric glycoproteins containing immunogenic segments of the F and G glycoproteins of RSV and suggests that such proteins can be expressed from a variety of systems including bacterial, yeast, mammalian (eg CHO cells) and insect cells (using for example a baculovirus).
Wathen et al (J. Gen. Virol. l989, 70, 2625-2635) describes a particular RSV
FG chimeric glycoprotein expressed using a baculovirus vector consisting of amino acids i -489 of the F protein linked to amino acids 97-279 of the G protein.
The fornnulations within the scope of the invention rnay also contain an anti-s tumour antigen and be useful for immunotherapeutically treating cancers.
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 protein 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 how it is presented. Generally, it is expected that each dose will comprise 1-1000 mcg of protein, preferably 2-100 mcg, most preferably 4-40 mcg.
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 immunisation adequately spaced.
The formulations of the present invention may be used for both prophylactic and therapeutic purposes.
Accordingly in a further aspect, the invention therefore provides use of a vaccine composition of the invention for the treatment of human patients. The invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient. In particular, the invention provides a method of treating viral, bacterial, parasitic infections or cancer which comprises administering an effective amount of a vaccine of the present invention to a patient.
The following examples and data illustrates the invention.
WO 98I15287 PCTlEP97/05578 Example 1 Preparation of vaccine containing alum, SUV MPL and QS21 1.1 Method of preparation of SUV
A mixture of lipid (such as phosphatidylcholine either from egg-yolk or synthetic) and cholesterol in organic solvent, is dried down under vacuum (or alternatively under a stream of inert gas). An aqueous solution (such as phosphate buffered saline) is then added, and the vessel agitated until all the lipid is in suspension. This suspension is then microfluidised until the liposome size is reduced to 100 nm, and then sterile filtered through a 0.2 p,m filter. Extrusion or sonication could replace this step. Typically the cholesterol: phosphatidycholine ratio is 1:4 (w/w), and the aqueous solution is added to give a final cholesterol concentration of 5 to 50 mg/mi.
1.2 Antigen ( 1-500 p.g, preferably 10-100 pg) is added to alum eg (aluminium hydroxide or aluminium phosphate) ( 100-500 fig) in water. The volume of water is chosen so that the volume of the final formulation is S00 p.l. After incubating for 15-30 minutes, 50 wg of MPL is added in the form of small-particle MPL
(W094/21292).
The MPL is left to adsorb onto the alum for 15-30 minutes at room temperature.
times concentrated phosphate buffered saline ( 1.5 M sodium chloride, 0.5M
sodium phosphate pH 7.5) is then added in such a volume so as to render the final formulation isotonic. This formulation is incubated at room temperature for 15-30 minutes.
QS21 (50 pg) is then added to SUV (containing between 50 and 250 pg cholesterol). This mixture is added to the above alum/antigen/MPL/buffer mixture. If required a bacteriostatic such as thiomersal is added (50 p.g).
Example 2 Table 1 shows the binding of QS21 to alum in the presence and absence of liposomes containing 25% (w/w) in dioleoyl phosphatidylcholine, and using a five-fold excess of cholesterol over QS21.
Formulation SUV ~ p.g QS21 bound 500 ug Alum+50 pg QS21 0 < i 0 500 ug Alum+50 ~g QS21 250 pg chol + 1 mg DOPC > 40 In order to increase the binding of QS21 to alum, the quantity of Iiposomes can be decreased. This decreases the cholesterol:QS2I ratio, however it has been shown that the QS21 remains non-toxic for cholesterol:QS21 ratios of 1:1 and greater.
Table 2 shows that if the quantity of alum is decreased (from 500 p.g to 100 pg) the quantity of QS2I that is bound decreases significantly, and the quantity of MPL that is bound also decreases. By adding less liposomes, yet maintaining a cholesterol:QS21 ratio of 1:1 or greater, increased quantities of QS21 and MPL can be bound to the alum.
Formulation Chol/QS21 ~g QS21 boundpg MPL bound 500 p.g alum + ~0 pg QS21 5l1 42 >48 + 50 pg MPL
l00 pg alum + 50 pg QS21 ~/1 17 >40 + 50 pg MPL
100 p.g alum + 50 pg QS21 2/1 30 >45 + 50 pg MPL
100 pg alum + 50 p.g QS21 1/1 40 >45 + 50 pg MPL
Example 3 The adjuvant effect of a combination of antigen (gD2t from Herpes Simple:c Virus-2 - expressed in CHO cells and comprises 283 amino acid from the mature N-terminal of the mature glycoprotein) with MPL and QS21 in combination with liposomes was tested with and without alum. The formulations were tested in African Green Monkeys.
African Green Monkeys were immunised twice (0, 28 days) with 20 ~g gD2t plus 50 ~g MPL plus 50 qg QS21 with or without liposomes (250 p.g cholesterol plus 1 mg DOPC) and with or without 500 pg alum. On day 42 the immune response was analysed.
The results are outlined below in figures 1 to 4.
The humoral response was measured as IgG against the gD protein. Figure 1 shows that the combination of MPL+QS21+SUV+alum induced higher titres than in the absence of alum, Figure 2 shows that the formulation of the invention provided the superior antigen specific proliferation.
The data shows that incorporation of aluminium hydroxide in vaccine formulations containing MPL and QS21 and SUV enhances both the humoral and cellular responses. This is an unexpected finding since it is generally accepted that aluminium as an adjuvant tends to favour Th2 type responses, yet the results presented here demonstrate that the response contains a significant Th 1 component which is not depressed by the addition of alum.
The formulation containing MPL and QS21 and SUV and alum is non-toxic and highly immunogenic.
Example 4 Production of RSV FG CHO cell derived proteins The plasmid pEE 14-FG contains a chimeric construct comprising of a fusion between amino acid sequences of F (1-525) and G (69-298) and was received from a collaboration with A. BOLLEN (ULB/CRI, Belgium). This FG fusion protein contains a total of 755 amino acids. It starts at the N-terminal signal sequence of F and lacks the C-terminal transmembrane domain (525-574) -anchor domain- of F
glycoprotein. Then, followed the extracellular region of G glycoprotein, without the amino-terminal region that contains the Signal/Anchor domain of G, a typical class II
glycoprotein.
The pEE 14-FG expression plasmid was generated by the insertion of the FG
coding sequence from pNIV2857 (A. Bollen, ULB/CRI, Belgium) as an Asp7181 (blunt) 5' - HindIII (blunt) 3' restriction fragment (2188 bp) into the SmaI
site of pEE 14 (Celltech). A Kozak sequence in lieu of the FG start ATG was generated into the pNIV 2857 construction as follows:
pEE 14---ccc gtacc ATG GAG -----x-----CAG TAG aagct ggg ---pEE 14 (SmaI) Metl Gln(298)Stop Asp718I(klenow) HindIII(klenow) 3 0 5' F( 1-525)-------x--------G(69-298)3' WO 98l15287 PCT/EP97l05578 The F sequence in pEEl4-FG is from SS2 RSV strain, and was kindly made available by Dr. PRINGLE as a cDNA construct in a Vaccinia vector (Baybutt and Pringle, J.Gen.ViroL, 1987, 2789-2796). The G sequence is from A2 RSV strain and was generated from a recombinant G Vaccinia virus obtained from Dr. G. WERTZ
5 (Alabama, USA).
CHO Kl transfection and stable FG protein expression.
CHO Kl cells derived from MCB 024M (Celltech) were transfected with 20 ug of pEE 14-FG plasmid DNA twice CsCI purified using the Ca-phosphate -glycerol 10 transfection procedure. Cell clones were selected according to the procedure of the GS
(glutamine synthetase ) expression system (Crocett et al BioTechn., 1990, VolB, 662) and amplified in the presence of 25 micro molar methionine sulphoximine (MSX) in G MEM medium containing no glutamine and supplemented with 10% dialyzed FBS
(Foetal Bovine Serum). Following transfection, 39 MSX resistant clones were screened in 24-well plates and their supernatants were tested for secretion of the FG
fusion protein. A11 transfectants proved to be positive for F antigen expression using a specific 'Sandwich' ELISA assay (i.e. rabbit polyclonal anti FG serum /
Antigen /
mAB 19). Monoclonal antibody 19 recognises a conformational F 1 - epitope and is neutralising.
The 3 best FG-producer clones (n~ 7, 13 and 37) were single-cell subcloned in a limiting dilution assay using 0.07 cells per well in a 96-well plates. A
total of ~ 9 positive subclones were obtained and the 16 best FG-producers were further characterised by western blot and ELISA. Again, the 8 best FG-subclones were further amplified and their FG expression was evaluated in presence and absence of sodiumbutyrate (2mM) or DMSO ( 1 or 2%). Six subclones were amplified and cell vials were made and stored at -80~C and liquid N2. Finally, the 3 best FG-subclones were selected. These are CHOK1 FG ~ 7.18, ~ 13.1 , and ~ 37.2.
Westernblot analyses (non-reducing conditions) with monoclonal mAB 19 indicated a major band of FG at about I35 kDa. The purified FG protein from recombinant Baculovirus FG infected cells (UPJOHN) appeared as major broad bands at +/-1 OOkDa and other bands at +/-70kDa under similar blot conditions.
The addition of Sodium butyrate in CHO-FG cell culture medium increased the expression level of FG 3 to 12 fold depending on the subclone and cell culture growth conditions. In particular, subclone CHO-FG 13.1 expressed 8-10 fold more FG
protein in the presence of butyrate (WB/ELISA).
Expression level determination was performed by ELISA (mAB 19 or MoAb AK13) using purified FG baculo protein as standard, as well as by western blot analysis using serial dilution.
Depending of the ELISA assay and cell culture conditions, the expression level of CHO-FG 13.1 is 5-12 ug of FG/ml after treatment with butyrate. Under accumulation conditions and medium replacements (3 to S days ) yields of 16 to 28 ug of secreted FG protein /ml were obtained.
CHOK1 FG l3.1 cell line was adapted to grow in suspension and serumfree (S/SF) conditions using a proprietary growth medium. Cell line CHO-FG 13.1 S/SF
grown in a medium without butyrate expressed similar yields as the parental adherent cell line grown in medium with butyrate. The addition of butyrate to CHO-FG
13.l 5/SF media has little effect on production of FG ( 1.5 to 2 fold increase).
Long term expression evaluation and preliminary genetic characterisation showed that CHO-FG 13.1 and the S/SF adapted 13.1 cell line were stable, contained intact FG expression cassettes giving rise to one single~mRNA band of about nucleotides long (Southern and Northern analyses). The CHO-FG clone 13.1 S/SF
was further used for production of FG antigen.
The use of alum/MPL/QS21 /SUV for the enhancement of the immune response in African Green Monkeys towards the FG protein from RS V
(Respiratory Syncytial Virus).
The FG protein (fusion protein containing the F- and G- proteins from RSV) was expressed in CHO cells and purified. 20 ~g of the purified protein was adsorbed on alum (S00 pg) to which monophosphoryl lipid A (MPL: 50 pg) was added. After incubating 30 minutes at room temperature, phosphate buffered saline was added.
Then either SUV alone or a mixture of SUV and QS21 (50 ~g QS21, SUV containing 250 ~g cholesterol and 1 mg DOPC) were added. African green monkeys were injected three times with these formulations, or with FG on alum alone or FG
mixed with MPL, SUV and QS21 in the absence of alum.
Figure S below show the RSV neutralising titres and the FG-ELISA titres obtained for each formulation. It is clear that the group alum/MPL/QS21 /SUV
induces the highest titres.
Example ~ Comparison of QS2I / SUV containing formulations with Alum formulation of Hepatitis B vaccine containing SL* antigen Introduction SL* was produced in accordance with the procedure set out in European Patent application No. 4l4374.
An immunogenicity study was conducted in Balb/c mice to compare the humoral responses induced by QS2I-SUV containing formulations in presence or not of Al(OH)3. MPL dose was 5pg, QS21 5pg, SUV contained 25p8 cholesterol and 100pg DOPC.
The experimental protocol is described in Material and Methods.
Briefly, mice were immunised intramuscularly in the leg twice at 4 weeks interval with SL* vaccines containing vehicle, immunostimulants or combinations of both.
Anti-HBs humoral responses (IgG and isotypes) were analysed.
The following groups were included in the study:
1. SL* (2ug) Al(OH)3 (50 ug) 2. SL* (2ug) Al(OH)3 (50 ug) / MPL / QS21-SUV
3. SL* (2ug) Al(OH)3 (50 ug) i QS21-SUV
4. SL* (2ug) MPL / QS21-SUV
Results Humoral responses were measured by Elisa as described in Material and Methods. Two time points were analyses: 28 days after the first injection (28 post I) and 14 days following the booster injection {14 post II).
Post I and post II anti-HBs response analysed on pooled sera are presented in Figure 6.
These data show that in primary response, comparable antibody titers are induced by all formulations containing QS2I-SUV while a weaker response is observed when Al(OH)3 alone is used.
In secondary response, the lowest antibody response was also induced by AI(OH)3 containing vaccine. However, all formulations containing QS21-SUV did not behave the same way.
The two formulations containing Al(OH)3 QS21-SUV (+/- MPL) induced the strongest antibody response (2x higher than MPL / QS21-SUV).
Although no statistical analysis has been performed, results on individual sera confirm this observation.
The combination of AI(OH)3 and QS21-SUV (+/- MPL) also qualitatively affects the immune response as shown by the isotypic profile of the humoral response 1 ~ (Figure 7).
Al(OH)3 induces a clear TH2 type of immune response (only 3 % IgG2a) whereas Al(OH)3 / QS21-SUV (+/- MPL) formulations induce up to 46% IgG2a.
Conclusion The combination of Alum with QS21-SUV (+i-MPL) induces higher antibody titers than formulations containing vehicle or immunostimulants alone.
Material and Methods Immunisations 10 groups of ~ female Balb/c mice (6-8 weeks) were immunised intramuscularly in the leg (gastrocnemien) twice at 4 weeks interval with SOpI
vaccine containing 2 ~g SL* formulated in Al(OH)3(~0 ug equivalent A13+), Al(OH)3 /
QS21-SUV, Al(OH)3 / MPL / QS21-SUV, MPL / QS21-SUV. A dose of ~ ug of immunostimulants was used.
CHO Kl transfection and stable FG protein expression.
CHO Kl cells derived from MCB 024M (Celltech) were transfected with 20 ug of pEE 14-FG plasmid DNA twice CsCI purified using the Ca-phosphate -glycerol 10 transfection procedure. Cell clones were selected according to the procedure of the GS
(glutamine synthetase ) expression system (Crocett et al BioTechn., 1990, VolB, 662) and amplified in the presence of 25 micro molar methionine sulphoximine (MSX) in G MEM medium containing no glutamine and supplemented with 10% dialyzed FBS
(Foetal Bovine Serum). Following transfection, 39 MSX resistant clones were screened in 24-well plates and their supernatants were tested for secretion of the FG
fusion protein. A11 transfectants proved to be positive for F antigen expression using a specific 'Sandwich' ELISA assay (i.e. rabbit polyclonal anti FG serum /
Antigen /
mAB 19). Monoclonal antibody 19 recognises a conformational F 1 - epitope and is neutralising.
The 3 best FG-producer clones (n~ 7, 13 and 37) were single-cell subcloned in a limiting dilution assay using 0.07 cells per well in a 96-well plates. A
total of ~ 9 positive subclones were obtained and the 16 best FG-producers were further characterised by western blot and ELISA. Again, the 8 best FG-subclones were further amplified and their FG expression was evaluated in presence and absence of sodiumbutyrate (2mM) or DMSO ( 1 or 2%). Six subclones were amplified and cell vials were made and stored at -80~C and liquid N2. Finally, the 3 best FG-subclones were selected. These are CHOK1 FG ~ 7.18, ~ 13.1 , and ~ 37.2.
Westernblot analyses (non-reducing conditions) with monoclonal mAB 19 indicated a major band of FG at about I35 kDa. The purified FG protein from recombinant Baculovirus FG infected cells (UPJOHN) appeared as major broad bands at +/-1 OOkDa and other bands at +/-70kDa under similar blot conditions.
The addition of Sodium butyrate in CHO-FG cell culture medium increased the expression level of FG 3 to 12 fold depending on the subclone and cell culture growth conditions. In particular, subclone CHO-FG 13.1 expressed 8-10 fold more FG
protein in the presence of butyrate (WB/ELISA).
Expression level determination was performed by ELISA (mAB 19 or MoAb AK13) using purified FG baculo protein as standard, as well as by western blot analysis using serial dilution.
Depending of the ELISA assay and cell culture conditions, the expression level of CHO-FG 13.1 is 5-12 ug of FG/ml after treatment with butyrate. Under accumulation conditions and medium replacements (3 to S days ) yields of 16 to 28 ug of secreted FG protein /ml were obtained.
CHOK1 FG l3.1 cell line was adapted to grow in suspension and serumfree (S/SF) conditions using a proprietary growth medium. Cell line CHO-FG 13.1 S/SF
grown in a medium without butyrate expressed similar yields as the parental adherent cell line grown in medium with butyrate. The addition of butyrate to CHO-FG
13.l 5/SF media has little effect on production of FG ( 1.5 to 2 fold increase).
Long term expression evaluation and preliminary genetic characterisation showed that CHO-FG 13.1 and the S/SF adapted 13.1 cell line were stable, contained intact FG expression cassettes giving rise to one single~mRNA band of about nucleotides long (Southern and Northern analyses). The CHO-FG clone 13.1 S/SF
was further used for production of FG antigen.
The use of alum/MPL/QS21 /SUV for the enhancement of the immune response in African Green Monkeys towards the FG protein from RS V
(Respiratory Syncytial Virus).
The FG protein (fusion protein containing the F- and G- proteins from RSV) was expressed in CHO cells and purified. 20 ~g of the purified protein was adsorbed on alum (S00 pg) to which monophosphoryl lipid A (MPL: 50 pg) was added. After incubating 30 minutes at room temperature, phosphate buffered saline was added.
Then either SUV alone or a mixture of SUV and QS21 (50 ~g QS21, SUV containing 250 ~g cholesterol and 1 mg DOPC) were added. African green monkeys were injected three times with these formulations, or with FG on alum alone or FG
mixed with MPL, SUV and QS21 in the absence of alum.
Figure S below show the RSV neutralising titres and the FG-ELISA titres obtained for each formulation. It is clear that the group alum/MPL/QS21 /SUV
induces the highest titres.
Example ~ Comparison of QS2I / SUV containing formulations with Alum formulation of Hepatitis B vaccine containing SL* antigen Introduction SL* was produced in accordance with the procedure set out in European Patent application No. 4l4374.
An immunogenicity study was conducted in Balb/c mice to compare the humoral responses induced by QS2I-SUV containing formulations in presence or not of Al(OH)3. MPL dose was 5pg, QS21 5pg, SUV contained 25p8 cholesterol and 100pg DOPC.
The experimental protocol is described in Material and Methods.
Briefly, mice were immunised intramuscularly in the leg twice at 4 weeks interval with SL* vaccines containing vehicle, immunostimulants or combinations of both.
Anti-HBs humoral responses (IgG and isotypes) were analysed.
The following groups were included in the study:
1. SL* (2ug) Al(OH)3 (50 ug) 2. SL* (2ug) Al(OH)3 (50 ug) / MPL / QS21-SUV
3. SL* (2ug) Al(OH)3 (50 ug) i QS21-SUV
4. SL* (2ug) MPL / QS21-SUV
Results Humoral responses were measured by Elisa as described in Material and Methods. Two time points were analyses: 28 days after the first injection (28 post I) and 14 days following the booster injection {14 post II).
Post I and post II anti-HBs response analysed on pooled sera are presented in Figure 6.
These data show that in primary response, comparable antibody titers are induced by all formulations containing QS2I-SUV while a weaker response is observed when Al(OH)3 alone is used.
In secondary response, the lowest antibody response was also induced by AI(OH)3 containing vaccine. However, all formulations containing QS21-SUV did not behave the same way.
The two formulations containing Al(OH)3 QS21-SUV (+/- MPL) induced the strongest antibody response (2x higher than MPL / QS21-SUV).
Although no statistical analysis has been performed, results on individual sera confirm this observation.
The combination of AI(OH)3 and QS21-SUV (+/- MPL) also qualitatively affects the immune response as shown by the isotypic profile of the humoral response 1 ~ (Figure 7).
Al(OH)3 induces a clear TH2 type of immune response (only 3 % IgG2a) whereas Al(OH)3 / QS21-SUV (+/- MPL) formulations induce up to 46% IgG2a.
Conclusion The combination of Alum with QS21-SUV (+i-MPL) induces higher antibody titers than formulations containing vehicle or immunostimulants alone.
Material and Methods Immunisations 10 groups of ~ female Balb/c mice (6-8 weeks) were immunised intramuscularly in the leg (gastrocnemien) twice at 4 weeks interval with SOpI
vaccine containing 2 ~g SL* formulated in Al(OH)3(~0 ug equivalent A13+), Al(OH)3 /
QS21-SUV, Al(OH)3 / MPL / QS21-SUV, MPL / QS21-SUV. A dose of ~ ug of immunostimulants was used.
Animals were bled on day 28 (28 post I) and 42 (14 post II) for antibody determination by Elisa.
Formulations Components batches used.
Formulation process SL* (2ug) is adsorbed or not for 15 min on 50 ug of water diluted Al(OH)3.
If needed, 5 ug of MPL is added to the preparation as a suspension of 100 nm particles (MPL out) for 15 min. If needed, ten fold concentrated buffer is added before adding 5 ug of QS21 mixed with liposomes in a weight ratio QS21 /
Cholesterol of 1/5.
Thiomersal is added to the formulations 15 min after QS21/SUV addition.
Formulations containing QS21-SUV are buffered with PBS pH 7.4 and the others are prepared in PBS pH 6.8 Serology Quantitation of anti-HBs antibody was performed by Elisa using HBs (Hep286) as coating antigen. Antigen and antibody solutions were used at 50 ul per well. Antigen was diluted at a final concentration of 1 ug/ml in PBS and was adsorbed overnight at 4~c to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for 1 hr at 3 7~c with PB S
containing 1 % bovine serum albumin and 0.1 % Tween 20 (saturation buffer). Two-fold dilutions of sera (starting at 1/100 dilution) in the saturation buffer were added to the HBs-coated plates and incubated for 1 hr 30 min at 37~c. The plates were washed four times with PB S 0. l % Tween 20 and biotin-conjugated anti-mouse IgG 1, IgG2a, IgG2b or a mix of the three antibodies (Amersham, UK) diluted 1/l000 in saturation buffer was added to each well and incubated for 1 hr 30 min at 37~c. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted l/5000 in saturation buffer was added for an additional 30 min at 37~c. Plates were washed as above and incubated for 20 min with a solution of o-phenylenediamine (Sigma) 0.04% H202 0.03% in 0.1 % tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H2S04 2N and read at 492/620 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation ) and expressed in EU/ml.
Formulations Components batches used.
Formulation process SL* (2ug) is adsorbed or not for 15 min on 50 ug of water diluted Al(OH)3.
If needed, 5 ug of MPL is added to the preparation as a suspension of 100 nm particles (MPL out) for 15 min. If needed, ten fold concentrated buffer is added before adding 5 ug of QS21 mixed with liposomes in a weight ratio QS21 /
Cholesterol of 1/5.
Thiomersal is added to the formulations 15 min after QS21/SUV addition.
Formulations containing QS21-SUV are buffered with PBS pH 7.4 and the others are prepared in PBS pH 6.8 Serology Quantitation of anti-HBs antibody was performed by Elisa using HBs (Hep286) as coating antigen. Antigen and antibody solutions were used at 50 ul per well. Antigen was diluted at a final concentration of 1 ug/ml in PBS and was adsorbed overnight at 4~c to the wells of 96 wells microtiter plates (Maxisorb Immuno-plate, Nunc, Denmark). The plates were then incubated for 1 hr at 3 7~c with PB S
containing 1 % bovine serum albumin and 0.1 % Tween 20 (saturation buffer). Two-fold dilutions of sera (starting at 1/100 dilution) in the saturation buffer were added to the HBs-coated plates and incubated for 1 hr 30 min at 37~c. The plates were washed four times with PB S 0. l % Tween 20 and biotin-conjugated anti-mouse IgG 1, IgG2a, IgG2b or a mix of the three antibodies (Amersham, UK) diluted 1/l000 in saturation buffer was added to each well and incubated for 1 hr 30 min at 37~c. After a washing step, streptavidin-biotinylated peroxydase complex (Amersham, UK) diluted l/5000 in saturation buffer was added for an additional 30 min at 37~c. Plates were washed as above and incubated for 20 min with a solution of o-phenylenediamine (Sigma) 0.04% H202 0.03% in 0.1 % tween 20 0.05M citrate buffer pH4.5. The reaction was stopped with H2S04 2N and read at 492/620 nm. ELISA titers were calculated from a reference by SoftmaxPro (using a four parameters equation ) and expressed in EU/ml.
Claims (14)
1. An adjuvant composition comprising alum, QS21 and a sterol characterised in that alum is bound to the QS21.
2. An adjuvant composition according to claim 1 wherein the saponin is associated with liposomes or an Iscom comprising a phospholipid and a sterol.
3. An adjuvant composition according to claims 1 or 2 wherein the sterol is cholesterol.
4. An adjuvant composition according to any one of claim 1 to 3 wherein the ratio of QS21 aterol is from 1:100 to 1:1.
5. An adjuvant composition according to any of claims 1 to 5 which further contains 3-de-O-acylated monophosphoryl lipid A.
6. A vaccine comprising an adjuvant of claim 1 to 6 and an antigen.
7. A vaccine composition as claimed in claim 6 comprising an antigen or antigenic composition derived from any of Human Immunodeficiency Virus, Feline Immunodeficiency Virus, Varicella Zoster virus, Herpes Simplex Virus type 1, Herpes Simplex virus type 2, Human cytomegalovirus, Hepatitis A, B, C or E, Respiratory Syncytial virus, human papilloma virus, Influenza virus, Hib, Meningitis virus, Salmonella, Neisseria, Borrelia, Chlamydia, Bordetella, Plasmodium or Toxoplasma, adjuvanted with a composition of claim 1 to 6.
8. A vaccine as claimed in claim 6 wherein the antigen is a tumour antigen.
9. A vaccine as claimed in claim 6, wherein the antigen is selected from the group SL* derived from Hepatitis B, HSV gD2t, or an RSV FG chimeric protein.
10. Use of composition as defined in any of claims 1 to 5 for the manufacture of a vaccine for the prophylactic treatment of viral, bacterial, or parasitic infections.
11. Use of composition as defined in any of claims 1 to 5 for the manufacture of a vaccine for the immunotherapeutic treatment of viral, bacterial, parasitic infections or cancer.
12. A method of treating a mammal suffering from or susceptible to a pathogenic infection comprising the administration of a safe and effective amount of a composition according to any of claims 1 to 4.
13. A method of treating a mammal suffering from cancer comprising the administration of a safe and effective amount of a composition according to any of claims 1 to 4.
14. A process for making an adjuvant composition according to claim 1 comprising admixing QS21 fraction and cholesterol and binding the QS21 to alum.
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Application Number | Priority Date | Filing Date | Title |
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GB9620795.6 | 1996-10-05 | ||
GBGB9620795.6A GB9620795D0 (en) | 1996-10-05 | 1996-10-05 | Vaccines |
PCT/EP1997/005578 WO1998015287A1 (en) | 1996-10-05 | 1997-09-30 | Vaccines |
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ID=10800990
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CA002267191A Abandoned CA2267191A1 (en) | 1996-10-05 | 1997-09-30 | Vaccines |
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EP (1) | EP0939650A1 (en) |
JP (1) | JP2001501640A (en) |
KR (1) | KR20000048866A (en) |
CN (1) | CN1238696A (en) |
AR (1) | AR009958A1 (en) |
AU (1) | AU714930B2 (en) |
BR (1) | BR9711853A (en) |
CA (1) | CA2267191A1 (en) |
CO (1) | CO4910170A1 (en) |
CZ (1) | CZ116799A3 (en) |
GB (1) | GB9620795D0 (en) |
HU (1) | HUP9904549A3 (en) |
IL (1) | IL128985A0 (en) |
NO (1) | NO991524D0 (en) |
NZ (1) | NZ334734A (en) |
PL (1) | PL332633A1 (en) |
TR (1) | TR199900729T2 (en) |
WO (1) | WO1998015287A1 (en) |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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US20230109193A1 (en) * | 2021-10-02 | 2023-04-06 | Massachusetts Institute Of Technology | Synergistic Combination of Alum and Non-Liposome, Non-Micelle Particle Vaccine Adjuvants |
Families Citing this family (69)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE435661T1 (en) * | 1997-08-29 | 2009-07-15 | Antigenics Inc | COMPOSITIONS CONTAINING ADJUVANT QS-21 WITH POLYSORBATE OR CYCLODEXTRIN AS AID |
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EP2266604A3 (en) * | 1998-10-16 | 2011-05-11 | GlaxoSmithKline Biologicals S.A. | Adjuvant systems and vaccines |
AU769539B2 (en) | 1999-01-29 | 2004-01-29 | Zoetis Services Llc | Adjuvants for use in vaccines |
GB9909077D0 (en) * | 1999-04-20 | 1999-06-16 | Smithkline Beecham Biolog | Novel compositions |
US6558670B1 (en) | 1999-04-19 | 2003-05-06 | Smithkline Beechman Biologicals S.A. | Vaccine adjuvants |
DK1187629T3 (en) | 1999-04-19 | 2005-01-17 | Glaxosmithkline Biolog Sa | Adjuvant composition comprising saponin and an immunostimulatory oligonucleotide |
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DE60018769T2 (en) | 1999-09-24 | 2005-08-04 | Nihon Nohyaku Co., Ltd. | AROMATIC DIAMIDE DERIVATIVES OR ITS SALTS, CHEMICALS FOR AGRICULTURE / GARDENING AND METHOD OF THEIR USE |
AU1013601A (en) | 1999-10-22 | 2001-05-08 | Aventis Pasteur Limited | Method of inducing and/or enhancing an immune response to tumor antigens |
NZ518999A (en) * | 1999-11-19 | 2002-12-20 | Csl Ltd | Vaccine compositions |
WO2001041802A1 (en) * | 1999-12-08 | 2001-06-14 | Statens Veterinärmedicinska Anstalt | Vaccine adjuvants comprising ginseng plant extract and added aluminium salt |
US6905712B2 (en) | 1999-12-08 | 2005-06-14 | Statens Veterinarmedicinska Anstalt | Vaccine adjuvants comprising ginseng plant extract and added aluminum salt |
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PL211151B1 (en) | 2000-10-18 | 2012-04-30 | Glaxosmithkline Biolog Sa | Vaccines |
DE60239594D1 (en) * | 2001-02-23 | 2011-05-12 | Glaxosmithkline Biolog Sa | INFLUENZA VACCINE COMPOSITIONS FOR INTRADERMAL ADMINISTRATION |
WO2002067983A1 (en) * | 2001-02-23 | 2002-09-06 | Glaxosmithkline Biologicals S.A. | Novel vaccine |
GB0110431D0 (en) * | 2001-04-27 | 2001-06-20 | Glaxosmithkline Biolog Sa | Novel compounds |
GB0123580D0 (en) * | 2001-10-01 | 2001-11-21 | Glaxosmithkline Biolog Sa | Vaccine |
CA2484941A1 (en) | 2002-07-24 | 2004-02-05 | Intercell Ag | Antigens encoded by alternative reading frame from pathogenic viruses |
US7378234B2 (en) | 2002-09-13 | 2008-05-27 | Intercell Ag | Method for isolating hepatitis C virus peptides |
US8232255B2 (en) | 2002-10-23 | 2012-07-31 | Glaxosmithkline Biologicals S.A. | Methods for vaccinating against malaria |
AU2004224746B2 (en) | 2003-03-24 | 2009-04-23 | Valneva Austria Gmbh | Improved vaccines |
WO2004084937A1 (en) * | 2003-03-24 | 2004-10-07 | Intercell Ag | Use of alum and a th1 immune response inducing adjuvant for enhancing immune responses |
GB0417494D0 (en) | 2004-08-05 | 2004-09-08 | Glaxosmithkline Biolog Sa | Vaccine |
GB0503337D0 (en) | 2005-02-17 | 2005-03-23 | Glaxosmithkline Biolog Sa | Compositions |
GB0513421D0 (en) | 2005-06-30 | 2005-08-03 | Glaxosmithkline Biolog Sa | Vaccines |
GB0519871D0 (en) | 2005-09-30 | 2005-11-09 | Secr Defence | Immunogenic agents |
US11707520B2 (en) | 2005-11-03 | 2023-07-25 | Seqirus UK Limited | Adjuvanted vaccines with non-virion antigens prepared from influenza viruses grown in cell culture |
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TWI457133B (en) | 2005-12-13 | 2014-10-21 | Glaxosmithkline Biolog Sa | Novel composition |
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CN103550764A (en) | 2007-03-02 | 2014-02-05 | 葛兰素史密丝克莱恩生物有限公司 | Novel method and compositions |
KR101579947B1 (en) | 2007-06-26 | 2015-12-28 | 글락소스미스클라인 바이오로지칼즈 에스.에이. | Vaccine comprising streptococcus pneumoniae capsular polysaccharide conjugates |
JP5508266B2 (en) | 2007-08-13 | 2014-05-28 | 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 | vaccine |
ES2626634T3 (en) | 2007-12-19 | 2017-07-25 | The Henry M. Jackson Foundation For The Advancement Of Military Medicine, Inc. | Soluble forms of Hendra and Nipah virus F glycoprotein and uses thereof |
GB0815872D0 (en) | 2008-09-01 | 2008-10-08 | Pasteur Institut | Novel method and compositions |
GB0900455D0 (en) | 2009-01-13 | 2009-02-11 | Secr Defence | Vaccine |
GB0901423D0 (en) | 2009-01-29 | 2009-03-11 | Secr Defence | Treatment |
GB0901411D0 (en) | 2009-01-29 | 2009-03-11 | Secr Defence | Treatment |
CA2751379C (en) | 2009-02-10 | 2018-06-12 | Novartis Ag | Influenza vaccines with reduced amounts of squalene |
US20120058149A1 (en) * | 2009-03-05 | 2012-03-08 | Mccloskey Jenny Colleen | Treatment of infection |
GB0906234D0 (en) | 2009-04-14 | 2009-05-20 | Secr Defence | Vaccine |
GB0913680D0 (en) | 2009-08-05 | 2009-09-16 | Glaxosmithkline Biolog Sa | Immunogenic composition |
MX2012011189A (en) | 2010-03-26 | 2013-02-07 | Glaxosmithkline Biolog Sa | Hiv vaccine. |
WO2012038801A1 (en) | 2010-09-21 | 2012-03-29 | National Institute Of Immunology | A spray dried powder formulation for vaccines entrapping alum and the antigen in biodegradable polymer particles |
BR112013004582A2 (en) | 2010-09-27 | 2016-09-06 | Crucell Holland Bv | method for inducing an immune response in a subject against a parasite antigen that causes malaria |
WO2013038156A1 (en) | 2011-09-16 | 2013-03-21 | Ucb Pharma S.A. | Neutralising antibodies to the major exotoxins tcda and tcdb of clostridium difficile |
EP2780034A1 (en) | 2011-11-14 | 2014-09-24 | Crucell Holland B.V. | Heterologous prime-boost immunization using measles virus-based vaccines |
WO2015092710A1 (en) | 2013-12-19 | 2015-06-25 | Glaxosmithkline Biologicals, S.A. | Contralateral co-administration of vaccines |
US11498956B2 (en) | 2016-08-23 | 2022-11-15 | Glaxosmithkline Biologicals Sa | Fusion peptides with antigens linked to short fragments of invariant chain(CD74) |
GB201614799D0 (en) | 2016-09-01 | 2016-10-19 | Glaxosmithkline Biologicals Sa | Compositions |
BE1024774B1 (en) | 2016-09-29 | 2018-07-02 | Glaxosmithkline Biologicals Sa | COMPOSITIONS AND METHODS OF TREATMENT |
WO2018104313A1 (en) * | 2016-12-07 | 2018-06-14 | Glaxosmithkline Biologicals Sa | Novel process |
GB201620968D0 (en) | 2016-12-09 | 2017-01-25 | Glaxosmithkline Biologicals Sa | Adenovirus polynucleotides and polypeptides |
IE87414B1 (en) * | 2017-05-30 | 2023-07-19 | Glaxosmithkline Biologicals Sa | Novel methods for manufacturing an adjuvant |
GB201721069D0 (en) | 2017-12-15 | 2018-01-31 | Glaxosmithkline Biologicals Sa | Hepatitis B Immunisation regimen and compositions |
GB201721068D0 (en) | 2017-12-15 | 2018-01-31 | Glaxosmithkline Biologicals Sa | Hepatitis B immunisation regimen and compositions |
BR112020024285A2 (en) | 2018-06-12 | 2021-03-02 | Glaxosmithkline Biologicals S.A. | adenovirus polynucleotides and polypeptides |
MX2021001479A (en) | 2018-08-07 | 2021-04-28 | Glaxosmithkline Biologicals Sa | Processes and vaccines. |
US20220184158A1 (en) | 2018-12-21 | 2022-06-16 | Glaxosmithkline Biologicals Sa | Methods of inducing an immune response |
CN113573730A (en) | 2019-03-05 | 2021-10-29 | 葛兰素史密斯克莱生物公司 | Hepatitis B immunization protocols and compositions |
WO2024133160A1 (en) | 2022-12-19 | 2024-06-27 | Glaxosmithkline Biologicals Sa | Hepatitis b compositions |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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NL9002314A (en) * | 1990-10-23 | 1992-05-18 | Nederlanden Staat | IMMUNOGENE COMPLEXES, IN PARTICULAR ISCOMS. |
US5585103A (en) * | 1991-07-25 | 1996-12-17 | Idec Pharmaceutical Corporation | Induction of cytotoxic T-lymphocyte responses |
ATE240117T1 (en) * | 1993-03-29 | 2003-05-15 | Pfizer | MULTI-COMPONENT CLOSTRIDIAL VACCINE USING SAPONIN AS AN ADJUVANT |
UA56132C2 (en) * | 1995-04-25 | 2003-05-15 | Смітклайн Бічем Байолоджікалс С.А. | Vaccine composition (variants), method for stabilizing qs21 providing resistance against hydrolysis (variants), method for manufacturing vaccine |
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1996
- 1996-10-05 GB GBGB9620795.6A patent/GB9620795D0/en active Pending
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1997
- 1997-09-30 HU HU9904549A patent/HUP9904549A3/en unknown
- 1997-09-30 CZ CZ991167A patent/CZ116799A3/en unknown
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- 1997-09-30 AU AU47812/97A patent/AU714930B2/en not_active Ceased
- 1997-09-30 CA CA002267191A patent/CA2267191A1/en not_active Abandoned
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- 1997-09-30 KR KR1019990702874A patent/KR20000048866A/en not_active Application Discontinuation
- 1997-09-30 TR TR1999/00729T patent/TR199900729T2/en unknown
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US20230109193A1 (en) * | 2021-10-02 | 2023-04-06 | Massachusetts Institute Of Technology | Synergistic Combination of Alum and Non-Liposome, Non-Micelle Particle Vaccine Adjuvants |
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ZA978868B (en) | 1999-04-06 |
HU9904549A (en) | 2000-05-28 |
PL332633A1 (en) | 1999-09-27 |
HUP9904549A3 (en) | 2001-06-28 |
KR20000048866A (en) | 2000-07-25 |
CN1238696A (en) | 1999-12-15 |
NO991524L (en) | 1999-03-29 |
AR009958A1 (en) | 2000-05-17 |
JP2001501640A (en) | 2001-02-06 |
GB9620795D0 (en) | 1996-11-20 |
AU714930B2 (en) | 2000-01-13 |
NO991524D0 (en) | 1999-03-29 |
EP0939650A1 (en) | 1999-09-08 |
WO1998015287A1 (en) | 1998-04-16 |
NZ334734A (en) | 2000-05-26 |
CO4910170A1 (en) | 2000-04-24 |
TR199900729T2 (en) | 1999-07-21 |
CZ116799A3 (en) | 1999-08-11 |
BR9711853A (en) | 1999-08-24 |
IL128985A0 (en) | 2000-02-17 |
AU4781297A (en) | 1998-05-05 |
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