WO2006109186A2 - Composes de neutralisation d'endosome precoce servant d'adjuvants de vaccin - Google Patents

Composes de neutralisation d'endosome precoce servant d'adjuvants de vaccin Download PDF

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WO2006109186A2
WO2006109186A2 PCT/IB2006/001296 IB2006001296W WO2006109186A2 WO 2006109186 A2 WO2006109186 A2 WO 2006109186A2 IB 2006001296 W IB2006001296 W IB 2006001296W WO 2006109186 A2 WO2006109186 A2 WO 2006109186A2
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antigen
adjuvant
chloroquine
neutralising
immune response
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2

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  • This invention is in the field of vaccine adjuvants, particularly adjuvants which promote a cellular immune response and/or protective CD8 immunity.
  • Vaccination with purified antigens alone is typically insufficient to elicit a protective immune response, so vaccines almost always require formulation with adjuvants. Different adjuvants have different immunological profiles and there is an ongoing need for new adjuvants for inclusion in vaccines.
  • aluminium salts and the MF59TM are approved for human use.
  • aluminium salts are subject to safety concerns and are incompatible with some antigens. There is therefore a need to develop further adjuvants.
  • the invention provides an immunogenic composition comprising: (a) an adjuvant comprising an early endosome neutralising compound; and (b) an antigen.
  • composition is preferably suitable for inducing a cellular immune response against the antigen, more preferably a protective CD8 response.
  • class I major histocompatibility complex (MHC) molecules present peptides derived from endogenous antigens ⁇ i.e., viral, tumor-, or self-proteins), that have been synthesized and processed within cells. This selective pathway guarantees that effector CD8+ T cells kill unwanted cells, sparing cells which have taken up soluble pathogen or tumor-derived proteins via the endocytic route.
  • endogenous antigens i.e., viral, tumor-, or self-proteins
  • APCs specialized antigen-presenting cells
  • cross-presentation is primarily carried out by dendritic cells (DCs) in vivo, and seems to be crucial for inducing both cytotoxic T lymphocyte (CTL) immunity (cross-priming) against allografts, tumors and pathogens that do not infect or functionally impair APCs, and tolerance (cross-tolerance) against self-antigens.
  • CTL cytotoxic T lymphocyte
  • the inventor has found that the endosomal processing machinery of professional antigen presenting cells plays a critical role in the limitation of cross-presentation of soluble antigens. Treatment with early endosome neutralising compounds ⁇ e.g.
  • chloroquine abolishes endosomal antigen degradation which greatly improves cross-presentation efficiency and leads to a dramatic improvement of both antigen-accumulation in early endosomes, and translocation into cytosol. Inhibition of endocytic processing favours human cross-presentation in vivo.
  • the immune response
  • the adjuvant comprising an early endosome neutralising compound is preferably suitable for enhancing a cellular immune response against the antigen e.g. it can promote the activation of cytotoxic T cells and inflammatory T cells.
  • the cellular immune response is a protective CD8 response, such that the adjuvant promotes the formation and persistence of memory CD8+ T cells for long-term, vaccine-induced resistance against the antigen.
  • the adjuvant included in the compositions of the invention may be any suitable early endosome neutralising compound, including those which are isolated from a naturally occurring source and those which are chemically synthesised.
  • early endosome neutralising compound refers to compounds that exert a pH neutralising effect or reduction in acidity of the early endosome compartment. This effect can conveniently be measured in vitro using dendritic cells (DC).
  • the pH indicator LysoSensor Yellow/Blue DND-160 (L-7545 - Molecular Probe) increases in fluorescence intensity upon acidification, as indicated on the manufacturer's sheet. Briefly, DC are incubated with or without a given neutralizing compound for 30 min, and then pulsed with 5 ⁇ M LysoSensor for further 4 h. After washing, DC are fixed and then visualized by microscope. The dye emits yellow fluorescence when it accumulated in acidic vesicles, and green fluorescence in neutral vesicles.
  • the adjuvant may be selected from the group consisting of chloroquine, quinine, quinidine, cinchonine, mefloquine, halofantrine, DIDS (4,4'-diisothiocyanatostil- bene-2,2'-disulfonic acid), NPPB (5-nitro-2-(3-phenylpropylamino-benzoic acid), vacuolar-type H+-ATPase inhibitors such as bafilomycin Al or folimycin and protonophores such as monensin and nigericin.
  • the adjuvant is non toxic i.e. it has substantially no toxic effect when administered in pharmacologically useful amounts.
  • the early endosome neutralising compound is lysosomotrophic. More preferably the early endosome neutralising compound is chloroquine.
  • the adjuvant can be formulated in various ways within the composition.
  • the adjuvant is preferably present in a soluble aqueous form. If the composition is formulated as an oil-in-water emulsion then the adjuvant will usually be present in the aqueous phase.
  • the adjuvant will generally be used at a concentration of between 0.1 and 1 ⁇ g/ml
  • the early endosome neutralising compound adjuvant is chloroquine or a chloroquine related compound.
  • Chloroquine is a lysosomotropic amine which is used as an antimalarial and an amebicide. Chloroquine also has anti-inflammatory activity and is used in high doses to treat the auto-immune diseases rheumatoid arthritis, systemic lupus erythematosus, and discoid lupus erythematosus. Chloroquine reaches high concentration within the lysosomes of the malarial parasites. Chloroquine also inhibits the parasitic enzyme heme polymerase that converts the toxic heme into non-toxic hemazoin, thereby resulting in the accumulation of toxic heme within the parasite. It may also interfere with the biosynthesis of nucleic acids.
  • Chloroquine has been associated with the inhibition of antigen presentation to T-cells and was also found to reduce macrophage antigen catabolism [I]. Chloroquine has also been shown to suppress the T-cell response to minor histocompatibility antigens (MiHCs) and has been postulated to induce decreased T-cell viability at high concentrations [2]. Its activity as an adjuvant is therefore surprising.
  • Chloroquine offers many advantages when used as an adjuvant owing to its proven safety record in humans (including during pregnancy), low cost and ease of use. Furthermore, the chloroquine may have a further function within the compositions of the invention as a malarial treatment. As such, the immunogenic compositions of the invention may be particularly suitable in vaccines for use in malarial regions.
  • the chloroquine adjuvant has the formula (1):
  • any pharmaceutically acceptable salt or ester of chloroquine may be used as an adjuvant according to the invention.
  • suitable salts are chloroquine hydrochloride, chloroquine phosphate and chloroquine diphosphate.
  • the salt used in the immunogenic composition of the invention is chloroquine diphosphate.
  • the invention extends to all isomers of the compounds of formula (1), and to mixtures thereof, including racemates.
  • Quinine, quinidine (cinchona alkaloids), cinchonine, mefloquine and halofantrine are aryl amino alcohols with similar structures and are predicted to have similar mechanisms of action. Plasmodium cross resistance for more than one compound of this class has been reported. Quinine acts as a blood schizonticide although it also has gametocytocidal activity against P. vivax and P. malariae. Because it is a weak base, it is concentrated in the food vacuoles of P. falciparum. It is said to act by inhibiting heme polymerase, thereby allowing accumulation of its cytotoxic substrate, heme. Quinidine is an antiarrhythmic drug related to quinine.
  • Mefloquine has been found to produce swelling of P. falciparum food vacuoles. It may act by forming toxic complexes with free heme that damage membranes and interact with other plasmodial components. It is effective against the blood forms of falciparum malaria, including the chloroquine resistant types.
  • Halofantrine is a phenanthrene methanol structurally related to quinine. Its mechanism of action may be similar to that of chloroquine, quinine, and mefloquine; by forming toxic complexes with ferritoporphyrin IX that damage the membrane of the parasite. This synthetic anti malarial is effective against multi drug resistant (including mefloquine resistant) P. falciparum malaria.
  • compositions of the invention As with chloroquine, quinine, quinidine (cinchona alkaloids), cinchonine, mefloquine and halofantrine further function within the compositions of the invention as malarial treatments.
  • the immunogenic compositions of the invention may be particularly suitable in vaccines for use in malarial regions.
  • NPPB nitro-2-(3-phenylpropylamino) benzoic acid
  • DIDS 4,4'diisothio-cyanatostilbene- 2,2'-disulfonicacid
  • NPPB is a Cl- channel inhibitor and, like DIDS, reduces endosomal acidification due to the inhibition of chloride channel activity (Biochim Biophys Acta. 1996 Oct 23; 1284(2): 171 -80). Via the same mechanism, NPPB inhibits the transport of a wide range of solutes into human erythrocytes infected in vitro with P. falciparum. This generally increases human erythrocyte permeability to a variety of low molecular weight solutes (Biol Chem. 1994 Feb;269(5):3339-47).
  • DIDS is known to specifically bind to band 3-related adhesin and to inhibit the binding of Plasmodium falciparum-infected erythrocytes (PE) to endothelial cells (mediated by the band 3-related adhesin).
  • PE Plasmodium falciparum-infected erythrocytes
  • Monensin and nigericin which are protonophores, are known to neutralize the endosome lumen [3]. These compounds are also suitable for use in the present invention.
  • compositions of the invention are preferably immunogenic e.g. vaccines.
  • Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic ⁇ i.e. to treat disease after infection), but will typically be prophylactic.
  • compositions of the invention therefore comprise an immunologically effective amount of at least one antigen.
  • 'immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention.
  • This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • Antigens suitable for use in the compositions of the invention may be bacterial or viral antigens. Suitable antigens may be further classified as protein antigens, carbohydrate antigens or glycoconjugate antigens.
  • the compositions of the invention may include one or more antigens. Examples of suitable antigens are:
  • OMV outer-membrane vesicle
  • a saccharide antigen from N. meningitidis serogroup A, C, W 135 and/or Y such as the oligosaccharide disclosed in ref. 8 from serogroup C [see also ref. 9] or the oligosaccharides ofref. 10.
  • N. meningitidis serogroup B such as those disclosed in refs. 1 1-19, etc.
  • antigens from Helicobacter pylori such as CagA [20 to 23], VacA [24, 25], NAP [26, 27, 28],
  • HopX e.g. 29
  • HopY e.g. 29
  • urease
  • a protein antigen from S.pneumoniae ⁇ e.g. from PhtA, PhtD, PhtB, PhtE, SpsA, LytB, LytC, LytA, Sp 125, SpIOl, Sp 128, SpI 30 and Sp 133, as disclosed in reference 33.
  • an antigen from hepatitis A virus such as inactivated virus [e.g. 34, 35].
  • an antigen from hepatitis B virus such as the surface and/or core antigens [e.g. 35, 36].
  • diphtheria antigen such as a diphtheria toxoid [e.g. chapter 3 ofref. 38].
  • tetanus antigen such as a tetanus toxoid [e.g. chapter 4 ofref. 38].
  • tetanus toxoid e.g. chapter 4 ofref. 38.
  • an antigen from Bordetella pertussis such as pertussis holotoxin (PT) and filamentous hemagglutinin (FHA) from B.pertussis, optionally also in combination with pertactin and/or agglutinogens 2 and 3 [e.g. refs. 39 & 40].
  • polio antigen(s) e.g. 41, 42
  • IPV. an antigen from Neisseria gonorrhoeae [e.g. 1 1,13, 43].
  • Chlamydia pneumoniae an antigen from Chlamydia pneumoniae [e.g. refs. 44 to 50].
  • rabies antigen(s) such as lyophilised inactivated virus [e.g. 54, RabAvertTM].
  • - measles, mumps and/or rubella antigens e.g. chapters 9, 10 & 11 ofref. 38].
  • - influenza antigen(s) e.g. chapter 19 of ref. 38] such as the haemagglutinin and/or neuraminidase surface proteins.
  • the flu antigen may be selected from a pandemic strain.
  • antigen(s) from a paramyxovirus such as respiratory syncytial virus (RSV [55, 56]) and/or parainfluenza virus (PIV3 [57]).
  • a paramyxovirus such as respiratory syncytial virus (RSV [55, 56]) and/or parainfluenza virus (PIV3 [57]).
  • PIV3 parainfluenza virus
  • Moraxella catarrhalis e.g. 58].
  • Streptococcus pyogenes group A streptococcus [e.g. 59, 60, 61].
  • Bacillus anthracis e.g. 66, 67, 68].
  • Bacillus anthracis e.g. 66, 67, 68.
  • an antigen from a virus in the flaviviridae family such as from yellow fever virus, Japanese encephalitis virus, four serotypes of Dengue viruses, tick-borne encephalitis virus, West Nile virus.
  • a pestivirus antigen such as from classical porcine fever virus, bovine viral diarrhoea virus, and/or border disease virus.
  • a parvovirus antigen e.g. from parvovirus B 19.
  • a prion protein e.g. the CJD prion protein
  • amyloid protein such as a beta peptide [69]
  • a cancer antigen such as those listed in Table 1 of ref. 70 or in tables 3 & 4 of ref. 71.
  • a saccharide antigen is used, it is preferably conjugated to a carrier in order to enhance immunogenicity. Conjugation of H.influenzae B, meningococcal and pneumococcal saccharide antigens is well known in the art. Toxic protein antigens may be detoxified where necessary (e.g. detoxification of pertussis toxin by chemical and/or genetic means).
  • diphtheria antigen is included in the composition it is preferred also to include tetanus antigen and pertussis antigens. Similarly, where a tetanus antigen is included it is preferred also to include diphtheria and pertussis antigens. Similarly, where a pertussis antigen is included it is preferred also to include diphtheria and tetanus antigens. DTP combinations are thus preferred.
  • Saccharide antigens are preferably in the form of conjugates.
  • Preferred carrier proteins for conjugates are bacterial toxins or toxoids, such as diphtheria toxoid or tetanus toxoid.
  • the CRMl 97 mutant of diphtheria toxin [72-74] is a particularly preferred carrier for, as is a diphtheria toxoid.
  • Other suitable carrier proteins include the N.
  • Antigens in the composition will typically be present at a concentration of at least 1 ⁇ g/ml each. In general, the concentration of any given antigen will be sufficient to elicit an immune response against that antigen.
  • nucleic acid encoding the antigen may be used. Protein components of the mixture may thus be replaced by nucleic acid (preferably DNA e.g. in the form of a plasmid) that encodes the protein.
  • compositions of the invention may comprise proteins which mimic saccharide antigens e.g. mimotopes [91] or anti-idiotype antibodies.
  • composition of the invention will typically, in addition to the components mentioned above, comprise one or more 'pharmaceutically acceptable carriers', which include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition.
  • Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes).
  • lipid aggregates such as oil droplets or liposomes.
  • the vaccines may also contain diluents, such as water, saline, glycerol, etc.
  • compositions of the invention are generally presented in aqueous form (e.g. solutions or suspensions). In some embodiments of the invention the compositions are in aqueous form from the packaging stage to the administration stage ("full liquid vaccine"). In this way the composition can be administered direct from their packaged form, without the need for reconstitution in an aqueous medium.
  • compositions of the invention may be packaged in a lyophilised form, and a vaccine for actual administration may be reconstituted when necessary.
  • compositions of the invention may be prepared at a packaging stage, or may be prepared extemporaneously prior to use.
  • the immunogenic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect.
  • Direct delivery of the compositions will generally be parenteral (e.g. by injection, either subcutaneously, intraperitoneal Iy, intravenously or intramuscularly or delivered to the interstitial space of a tissue).
  • the compositions can also be administered into a lesion.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal or transcutaneous applications (e.g. see ref. 93), needles, and hyposprays.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule (e.g. including booster doses).
  • Compositions may be presented in vials or in ready-filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses.
  • compositions of the invention may be packaged in unit dose form or in multiple dose form. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of a composition for injection has a volume of about 0.5ml. Similar doses may be used for other delivery routes e.g. an intranasal vaccine for atomisation may have a volume of about 125 ⁇ l per spray, with four sprays administered to give a total dose of about 0.5ml.
  • the pH of the composition (including lyophilised compositions, after reconstitution) is preferably between 6 and 8, preferably about 7. Stable pH may be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen-free. Compositions of the invention may be isotonic with respect to humans.
  • compositions of the invention may include an antimicrobial and/or a preservative, particularly when packaged in multiple dose format.
  • compositions of the invention may comprise detergent e.g. a Tween (polysorbate), such as Tween 80.
  • Detergents are generally present at low levels e.g. ⁇ 0.01%.
  • compositions of the invention may include sodium salts (e.g. sodium chloride) to give tonicity.
  • sodium salts e.g. sodium chloride
  • compositions of the invention will generally include a buffer.
  • a phosphate or histidine buffer is typical.
  • the invention also provides the use of: (a) an early endosome neutralising compound adjuvant; and (b) at least one antigen, in the manufacture of a medicament for administration to a mammal to induce an immune response, preferably a cellular immune response, more preferably protective CD8 immunity.
  • the invention also provides a method for raising an immune response against at least one antigen, comprising the step of administering at least one antigen to a patient with an early endosome neutralising compound adjuvant.
  • the adjuvant and the antigen(s) may be administered simultaneously, sequentially or separately.
  • the adjuvant may be administered to prime the mammal before administration of the antigen or after the administration of the antigen to boost the mammal's immune response to that antigen.
  • the invention also provides the use of at least one antigen in the manufacture of a medicament for raising an immune response in a patient, wherein the medicament is administered with early endosome neutralising compound adjuvant.
  • the invention provides the use of an adjuvant comprising an early endosome neutralising compound in the manufacture of a medicament for raising an immune response in a patient, wherein the medicament is administered with at least one antigen.
  • the inclusion of chloroquine in the medicament preferably increases the immune response against the antigen.
  • the invention also provides the use of at least one antigen in the manufacture of a medicament for raising an immune response in a patient, where the patient has been pre-treated with an early endosome neutralising compound adjuvant.
  • the invention also provides the use of an adjuvant comprising an early endosome neutralising compound in the manufacture of a medicament for raising an immune response in a patient, where the patient has been pre-treated with at least one antigen.
  • an adjuvant comprising an early endosome neutralising compound in the manufacture of a medicament for raising an immune response in a patient, where the patient has been pre-treated with at least one antigen.
  • the inclusion of chloroquine in the medicament preferably increases the immune response against the antigen.
  • the invention also provides a composition of the invention for use in medicine.
  • the invention also provides (a) an early endosome neutralising compound adjuvant; and (b) at least one antigen, for simultaneous separate of sequential administration.
  • the mammal is preferably a human.
  • the human is preferably a child (e.g. a toddler or infant) or a teenager; where the vaccine is for therapeutic use, the human is preferably an adult.
  • a vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, immunogenicity, etc.
  • the invention may be used to elicit systemic and/or mucosal immunity.
  • the invention may be used to elicit the production of specific IgA, IgG and/or IgM antibodies.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunisation schedule and/or in a booster immunisation schedule. A primary dose schedule may be followed by a booster dose schedule. Suitable timing between priming doses (e.g. between 4-16 weeks), and between priming and boosting, can be routinely determined. Further adjuvants
  • Chloroquine can act as an adjuvant within the compositions of the invention. It is also possible to include one or more further adjuvants. Such adjuvants include, but are not limited to:
  • Mineral containing compositions suitable for use as adjuvants in the invention include mineral salts, such as aluminium salts and calcium salts.
  • the invention includes mineral salts such as hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. [e.g. see chapters 8 & 9 of ref. 94], or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred.
  • the mineral containing compositions may also be formulated as a particle of metal salt [95].
  • a typical aluminium phosphate adjuvant is amorphous aluminium hydroxyphosphate with PO 4 /A1 molar ratio between 0.84 and 0.92, included at 0.6mg Al 3+ /ml.
  • Adsorption with a low dose of aluminium phosphate may be used e.g. between 50 and lOO ⁇ g Al 3+ per conjugate per dose.
  • an aluminium phosphate it used and it is desired not to adsorb an antigen to the adjuvant, this is favoured by including free phosphate ions in solution (e.g. by the use of a phosphate buffer).
  • Oil emulsion compositions suitable for use as adjuvants in the invention include squalene-water emulsions, such as MF59 [Chapter 10 of ref. 94; see also ref. 96] (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer). Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA) may also be used.
  • CFA Complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • Saponin formulations may also be used as adjuvants in the invention.
  • Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponin from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponin can also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria qfficianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21 , as well as lipid formulations, such as ISCOMs. QS21 is marketed as StimulonTM.
  • Saponin compositions have been purified using HPLC and RP-HPLC. Specific purified fractions using these techniques have been identified, including QS7, QS 17, QS 18, QS21, QH-A, QH-B and QH-C.
  • the saponin is QS21.
  • a method of production of QS21 is disclosed in ref. 97.
  • Saponin formulations may also comprise a sterol, such as cholesterol [98].
  • ISCOMs immunostimulating complexs
  • phospholipid such as phosphatidylethanolamine or phosphatidylcholine.
  • Any known saponin can be used in ISCOMs.
  • the ISCOM includes one or more of QuilA, QHA and QHC. ISCOMs are further described in refs. 98-100.
  • the ISCOMS may be devoid of additional detergent [101].
  • Virosomes and virus-like particles can also be used as adjuvants in the invention.
  • These structures generally contain one or more proteins from a virus optionally combined or formulated with a phospholipid. They are generally non-pathogenic, non-replicating and generally do not contain any of the native viral genome.
  • the viral proteins may be recombinantly produced or isolated from whole viruses.
  • viral proteins suitable for use in virosomes or VLPs include proteins derived from influenza virus (such as HA or NA), Hepatitis B virus (such as core or capsid proteins), Hepatitis E virus, measles virus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus, Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages, Q ⁇ -phage (such as coat proteins), GA- phage, fr-phage, AP205 phage, and Ty (such as retrotransposon Ty protein pi).
  • VLPs are discussed further in refs. 104-109.
  • Virosomes are discussed further in, for example, ref. 110
  • Adjuvants suitable for use in the invention include bacterial or microbial derivatives such as non-toxic derivatives of enterobacterial lipopolysaccharide (LPS), Lipid A derivatives, immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • LPS enterobacterial lipopolysaccharide
  • Lipid A derivatives Lipid A derivatives
  • immunostimulatory oligonucleotides and ADP-ribosylating toxins and detoxified derivatives thereof.
  • Non-toxic derivatives of LPS include monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • a preferred "small particle" form of 3 De-O-acylated monophosphoryl lipid A is disclosed in ref. 1 11. Such "small particles" of 3dMPL are small enough to be sterile filtered through a 0.22 ⁇ m membrane [1 1 1].
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g. RC-529 [1 12, 1 13].
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM-174. OM-174 is described for example in refs. 114 & 115.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants in the invention include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine). Double-stranded RNAs and oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's can include nucleotide modifications/analogs such as phosphorothioate modifications and can be double-stranded or single-stranded.
  • References 1 16, 1 17 and 1 18 disclose possible analog substitutions e.g. replacement of guanosine with 2'-deoxy-7-deazaguanosine.
  • the adjuvant effect of CpG oligonucleotides is further discussed in refs. 1 19-124.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT [125].
  • the CpG sequence may be specific for inducing a ThI immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • CpG-A and CpG-B ODNs are discussed in refs. 126-128.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide is constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers". See, for example, refs. 125 & 129-131.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants in the invention.
  • the protein is derived from E. coli (E.coli heat labile enterotoxin "LT"), cholera ("CT"), or pertussis ("PT").
  • LT E.coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in ref. 132 and as parenteral adjuvants in ref. 133.
  • the toxin or toxoid is preferably in the form of a holotoxin, comprising both A and B subunits.
  • the A subunit contains a detoxifying mutation; preferably the B subunit is not mutated.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LT-Gl 92.
  • LT-K63 LT-K63
  • LT-R72 LT-R72
  • LT-Gl 92 LT-Gl 92.
  • ADP-ribosylating toxins and detoxified derivaties thereof, particularly LT-K63 and LT- R72, as adjuvants can be found in refs. 134-141.
  • Numerical reference for amino acid substitutions is preferably based on the alignments of the A and B subunits of ADP-ribosylating toxins set forth in ref. 142, specifically incorporated herein by reference in its entirety.
  • Human immunomodulators suitable for use as adjuvants in the invention include cytokines, such as interleuldns ⁇ e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [143], etc.) [144], interferons ⁇ e.g. interferon- ⁇ ), macrophage colony stimulating factor, and tumor necrosis factor.
  • cytokines such as interleuldns ⁇ e.g. IL-I, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 [143], etc.) [144]
  • interferons ⁇ e.g. interferon- ⁇
  • macrophage colony stimulating factor IL-12 [143], etc.
  • tumor necrosis factor tumor necrosis factor
  • Bioadhesives and mucoadhesives may also be used as adjuvants in the invention.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres [145] or mucoadhesives such as cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants in the invention [146].
  • Microparticles may also be used as adjuvants in the invention.
  • Microparticles ⁇ i.e. a particle of -lOOnm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ m in diameter) formed from materials that are biodegradable and non-toxic ⁇ e.g. a poly( ⁇ -hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, a polyanhydride, a polycaprolactone, etc.), with poly(lactide-co-glycolide) are preferred, optionally treated to have a negatively-charged surface ⁇ e.g. with SDS) or a positively-charged surface ⁇ e.g. with a cationic detergent, such as CTAB).
  • a negatively-charged surface ⁇ e.g. with SDS
  • a positively-charged surface ⁇ e.g. with a cationic detergent,
  • liposome formulations suitable for use as adjuvants are described in refs. 147-149.
  • Adjuvants suitable for use in the invention include polyoxyethylene ethers and polyoxyethylene esters [150]. Such formulations further include polyoxyethylene sorbitan ester surfactants in combination with an octoxynol [151] as well as polyoxyethylene alkyl ethers or ester surfactants in combination with at least one additional non-ionic surfactant such as an octoxynol [152].
  • Preferred polyoxyethylene ethers are selected from the following group: polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steoryl ether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4- lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.
  • PCPP Polyphosphazene
  • PCPP formulations are described, for example, in refs. 153 and 154.
  • muramyl peptides suitable for use as adjuvants in the invention include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), and N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-aIanine-2-(l '-2'-d ipalm ttoy 1- ⁇ «- glycero-3-hydiOxyphosphoryloxy) ⁇ ethylarnine MTP-PE).
  • thr-MDP N-acetyl- muramyl-L-threonyl-D-isoglutamine
  • nor-MDP N-acetyl-normuramyl-L-alanyl-D-isoglutamine
  • imidazoquinolone compounds suitable for use adjuvants in the invention include Imiquamod and its homologues (e,g, "Resiquimod 3M"), described further in refs. 155 and 156.
  • the invention may also comprise combinations of aspects of one or more of the adjuvants identified above.
  • the following adjuvant compositions may be used in the invention: (1) a saponin and an oil-in-water emulsion [157]; (2) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) [158]; (3) a saponin (e.g. QS21) + a non-toxic LPS derivative (e.g. 3dMPL) + a cholesterol; (4) a saponin (e.g.
  • RibiTM adjuvant system (RAS), (Ribi Immunochem) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (DetoxTM); and (8) one or more mineral salts (such as an aluminum salt) + a non-toxic derivative of LPS (such as 3dMPL).
  • MPL monophosphorylipid A
  • TDM trehalose dimycolate
  • CWS cell wall skeleton
  • LPS such as 3dMPL
  • compositions of the invention The early endosome neutralising compound adjuvant of the invention is particularly suited to inclusion in immunogenic compositions and vaccines.
  • a process of the invention may therefore include the step of mixing the adjuvant with an antigen.
  • the invention provides a composition or vaccine obtainable in this way. Where a composition of the invention includes antigens from more than one organism, the antigens are preferably prepared separately and then admixed with the early endosome neutralising compound adjuvant to give a composition of the invention.
  • a composition of the invention may thus be prepared from a kit comprising: (a) early endosome neutralising compound adjuvant and (b) at least one antigen.
  • the early endosome neutralising compounds and/or the at least one antigen may be present in lyophilised form.
  • the invention also provides a method for preparing a composition of the invention, comprising mixing an early endosome neutralising compound adjuvant with one or more antigens (e.g. 1, 2, 3), wherein said one or more antigens are in liquid form.
  • Compositions of the invention may be formed by adding antigen to bulk adjuvant, or adding adjuvant to bulk antigen. Where the composition includes more than one antigen and/or more than one adjuvant, antigen(s) and adjuvant(s) may be mixed in any suitable order.
  • composition comprising
  • X may consist exclusively of X or may include something additional e.g. X + Y.
  • Figure 1 Cross-presentation efficiency is dependent on inhibition of antigen degradation.
  • a-d Representative flow cytometry analysis of HLA-A2-restricted NS3i 406 -i 4 i 5 -specific CD8 + T cell clone producing IFN- ⁇ in response to NS3Ag cross-presented by HLA-A2 + DCs, in the presence or absence of the indicated compounds. Dot plots show double CD8 and IFN- ⁇ staining. Results are expressed as percentage of cells, indicated in each quadrant, e.
  • NS3Ag cross-presentation after 12 hr pulsing by six independent HLA-A2 + DC populations to NS3i 4 0 6 -i 4 is-specific CD8 clone (mean SD), in the absence (diamond) or presence of chloroquine (triangle) (presence versus absence of chloroquine: *p ⁇ 0.0001), leupeptin (square), or lactacystin (circle), f. Chase time of NS3Ag (50 ⁇ g/ml) cross-presentation after 30 min of pulsing by six independent DC populations (mean ⁇ SD), in the presence (triangle) or absence (diamond) of chloroquine.
  • NS3Ag filled diamond
  • NS3Ag + chloroquine triangle
  • peptide empty diamond
  • presentation of NS3Ag by four independent DC populations to NS3i 24 i-i 26 o-specific CD4 T cell clone (mean ⁇ SD), in the presence (triangle) or absence (diamond) of chloroquine.
  • i-j Presentation of NS3Ag (presence versus absence of chloroquine: *p ⁇ 0.0001) by four independent DC populations to NS3i 24 i-i 26 o-specific CD4 T cell clone (mean ⁇ SD), in the presence (triangle) or absence (diamond) of chloroquine. i-j.
  • FIG. 1 Chloroquine-boosted cross-presentation elicits antigen-specific CD8* T cells in vivo.
  • a-b Representative flow cytometry experiments on PBMCs from either a subject, submitted to both chloroquine-treatment (300 mg for two consecutive days) and anti-hepatitis B virus (HBV) vaccine boost (a), or a subject only submitted to the anti-HBV vaccine boost (b).
  • Fresh cells were tested for their capacity to promptly produce IFN- ⁇ within 4-6 hrs of contact with the indicated antigen or peptide. Dot plot are gated on CD8 cells and show IFN- ⁇ staining. Results are expressed as percentage of IFN- ⁇ + cells, c-d. Histograms reporting percentages of functional HBenvAg-specific
  • the responses both to increasing HBenvAg concentrations and to a single dose of the indicated HLA-A2-binder peptide are reported.
  • DCs Dendritic cells
  • EBV-transformed (EBV-)B cells EBV-transformed (EBV-)B cells, as antigen-presenting cells (APCs)
  • DCs Dendritic cells
  • EBV-transformed (EBV-)B cells as antigen-presenting cells (APCs)
  • DCs Dendritic cells
  • EBV-transformed (EBV-)B cells as antigen-presenting cells (APCs)
  • APCs antigen-presenting cells
  • mice were immunized intraperitoneal ⁇ with syngeneic dendritic cells (DCs) that had previously been pulsed with high concentrations of soluble ovalbumin (0.5-1 mg) in the presence or absence of 10-20 ⁇ g/ml of chloroquine.
  • DCs syngeneic dendritic cells
  • mice were sacrificed and their spleen or lymph node cells were tested in a cytotoxicity assay using syngeneic target cells pulsed with OVA 2S7-264 peptide. Results of one representative experiment are below:
  • chloroquine increases the ability of DCs to lyse target cells.
  • Chloroquine-boosted cross-presentation elicits antigen-specific CD8 + T cells in vivo
  • HLA-A2+ hepatitis B virus
  • PBMCs were collected before and after 15 days from the vaccination booster, and each sample was stored in at least three independent vials in liquid nitrogen, in order to perform the functional analyses at least twice for each time point.
  • PBMC samples were stimulated for 6 hr with autologous irradiated PBMCs, which had been previously pulsed with different concentrations of recombinant HBenvAg (GlaxoSmithKline Biologicals Rixensart, Belgium) or peptide in the presence of chloroquine, and stained with FITC- labelled anti-CD8 mAb. Finally, the induction IFN-g production was determined, as described above. The results for this experiment are presented in figure 2.
  • OT-I cells nonaive ovalbumin (OVA)-specific CD8 T cells
  • OVA ovalbumin
  • Mice are then vaccinated with soluble OVA with or without intraperitoneal administration of a chloroquine adjuvant.
  • Tumour progression size/growth is monitored to determine the effect of the chloqoquine adjuvant in relation to priming, expansion and effector differentiation of the OT-I cells.

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Abstract

L'invention concerne des composés de neutralisation d'endosome précoce, notamment de la chloroquine, pouvant être utilisés comme des adjuvants de vaccin. L'invention concerne une composition immunogène comprenant: (a) un adjuvant comprenant un composé de neutralisation d'endosome précoce; et (b) un antigène. L'invention concerne également des méthodes de traitement employant ces compositions.
PCT/IB2006/001296 2005-04-15 2006-04-13 Composes de neutralisation d'endosome precoce servant d'adjuvants de vaccin Ceased WO2006109186A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2203065A4 (fr) * 2007-10-31 2012-02-29 Functional Genetics Inc Procédés d'inhibition d'une infection virale
US20180298388A1 (en) * 2013-08-27 2018-10-18 Deutsches Krebsforschungszentrum Cytotoxic t cell response modifiers

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* Cited by examiner, † Cited by third party
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FR2522968B1 (fr) * 1982-03-10 1986-03-28 Sanofi Sa Medicament cytotoxique forme de l'association d'a u moins une immunotoxine et de la chloroquine
FR2536997B1 (fr) * 1982-03-11 1987-08-07 Sanofi Sa Medicaments cytotoxiques comportant au moins une immunotoxine et de la methylamine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2203065A4 (fr) * 2007-10-31 2012-02-29 Functional Genetics Inc Procédés d'inhibition d'une infection virale
US20180298388A1 (en) * 2013-08-27 2018-10-18 Deutsches Krebsforschungszentrum Cytotoxic t cell response modifiers
US11802285B2 (en) 2013-08-27 2023-10-31 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Cytotoxic T cell response modifiers

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